CN109406786A - A kind of immune chromatography test paper detecting acephatemet - Google Patents
A kind of immune chromatography test paper detecting acephatemet Download PDFInfo
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- CN109406786A CN109406786A CN201810992492.5A CN201810992492A CN109406786A CN 109406786 A CN109406786 A CN 109406786A CN 201810992492 A CN201810992492 A CN 201810992492A CN 109406786 A CN109406786 A CN 109406786A
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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
- G01N33/577—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
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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
A kind of immune chromatography test paper detecting acephatemet, including supporter and the adsorption layer being fixed on supporter, adsorption layer is followed successively by sample pad, bonding pad, chromatographic film and absorption pad since test lead, and the anti-MTP monoclonal antibody of nano material label is adsorbed on the bonding pad；The stealthy control trace that the chromatographic film is equipped with the stealthy detection trace printed with MTP artificial antigen solution and is printed with goat-anti or rabbit anti-mouse IgG antibody solution；The nano material is nitrogen-doped carbon nano material, carbon nanomaterial, carbon quantum dot fluorescent nano particle.Test strips of the invention have the characteristics that high specificity, high sensitivity, stability are high, safety is good, easy, quick, vivid, intuitive, applied widely, easy to carry as the result is shown and can quantify.It is extremely important in terms of ensuring food safety, protecting consumer health, will have apparent economic benefit and social benefit.
Description
Technical field
The present invention relates to a kind of immune chromatography test papers, more particularly to a kind of immunity-chromatography test for detecting acephatemet (MTP)
Paper.
Background technique
Acephatemet (Methamidopho, MTP) is a kind of organic phosphorus compound, be it is a kind of efficiently, broad spectrum organic phosphorus class kills
Worm, acaricide, have tag, stomach toxicity and certain fumigation action are not easily decomposed but since its toxicity is higher, residual life, is longer, one
The acephatemet for determining dosage can cause the symptoms such as human body giddy, nausea, pulmonary edema, brain edema and respiratory paralysis, to human health and
Environmental pollution all brings huge negative effect.China has prohibited its use in water fruits and vegetables in 2008,
But due to acephatemet wide spectrum, efficient, cheap feature, there is considerable plant personnel in violated use, still so as to cause methylamine
Phosphorus is exceeded and food poisoning happens occasionally.Therefore, how to improve and methamidophos pesticide is effectively supervised, especially fastly
The residual quantity of organophosphorus pesticide in fast quantitative detection veterinary antibiotics, has become a urgent problem to be solved.
Mainly there are biological detection method, chemical analysis, instrumental method for the detection method of MTP both at home and abroad at present and exempt from
4 major class of epidemic disease analytic approach.It is very pure that the advantages of biological detection method, is that measuring samples are not required to, the disadvantage is that sensitivity is low, experimental period is longer.
The advantages of chemical analysis is economical and practical, but is unable to accurate quantitative analysis, and the repeatable and reproducibility for analyzing result is poor.Instrument
Analytic approach has high score from advantages such as, high detection efficiency and quick analysis ability, but pre-treatment to sample and operator
Technical requirements are high, and instrument and equipment is expensive, is unsuitable for field quick detection.Immunoassay is easy to operate, at low cost, knot
Nitrogen-doped carbon nano material marker is closed with excitation-emission length flexible is adjustable, fluorescent stability is high and flicker free phenomenon etc. is excellent
Gesture, final that high sensitive, high stable live rapid quantitative detection can be achieved, society and economic significance are great.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of immune chromatography test paper for detecting acephatemet (MTP), the examinations
Paper has the characteristics that special, sensitive, quick, easy.
To achieve the goals above, the technical scheme adopted by the invention is that:
A kind of immune chromatography test paper detecting acephatemet, including supporter and the adsorption layer being fixed on supporter, absorption
Layer is followed successively by sample pad, bonding pad, chromatographic film and absorption pad since test lead, is adsorbed with nano material on the bonding pad
The anti-MTP monoclonal antibody of label；The chromatographic film be equipped with the stealthy detection trace printed with MTP artificial antigen solution and
The stealthy control trace printed with goat-anti or rabbit anti-mouse IgG antibody solution；The nano material is nitrogen-doped carbon nanometer material
Material, carbon nanomaterial, carbon quantum dot fluorescent nano particle.
Supporter includes the bottom that adsorption layer bottom surface is arranged in and the surface layer that adsorption layer top surface is arranged in.
Nitrogen-doped carbon nano material N-CDs is using chitosan as carbon source, and ethylenediamine is nitrogen dopant, is prepared using hydro-thermal method
It forms, method particularly includes: chitosan 2.5g is dissolved in 5mL ultrapure water, 5mL ethylenediamine is added, mixing is placed on reaction under high pressure
In the polytetrafluoroethylliner liner of kettle, 180 DEG C of reaction 2h after reaction filter product, and distilled water washs 2 times, 60 DEG C of baking ovens
It is dried to obtain N-CDs.
Nitrogen-doped carbon nano material label anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface carboxylation SiO2The preparation of nano particle
SiO is synthesized using reverse microemulsion method2Nano particle: by volume 10:30:10:1 ratio take Ttiton X-100,
Hexamethylene, n-hexyl alcohol, ultrapure water stir to form 5.1mL microemulsion, after 200 μ L ammonium hydroxide of addition stir evenly, add the 80 positive silicic acid of μ L
Ethyl ester, room temperature are protected from light for 24 hours；6000rpm is centrifuged 10min after reaction, is redissolved after ethanol washing 4 times with 1mL ethyl alcohol, shape
At solution A；0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution B；By solution A plus
Enter into solution B, reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, and obtained precipitating is steamed with double
After water washing 4 times, nitrogen drying is dry, and 4 DEG C are sealed；
(2)N-CDs-SiO2The preparation of fluorescence probe
By the SiO of 2mg surface carboxylation2Nano particle and 2mg N, N'- carbonyl dimidazoles are added to 400 μ L N, N- bis-
In methylformamide, magnetic agitation reacts 3h at room temperature；Add it to 1mL concentration be l mg/mL N-CDs solution in,
15min is added, room temperature is protected from light be stirred to react 4h after；20 μ L ethyl orthosilicates, 0.12g monoxone, 0.25mg N- are added again
CDs particle, room temperature, which is protected from light, is stirred to react 2h progress involucrum, and involucrum 3 times repeatedly, nitrogen drying is dry, and 4 DEG C are sealed；
(3) label of MTP-mAb
The fluorescence probe 15mg for taking step (2) to prepare be dissolved in 1.5mL dioxane, 1.5mL DMF, 60 μ L triethylamines it is mixed
It closes in solution, ice bath 30min, 20 μ L isobutyl chlorocarbonates are added in stirring, and ice bath 2h obtains label solution；Label solution is dripped
It is added in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, reaction is stirred at room temperature overnight；Under the conditions of 4 DEG C, reactant is used
The PBS buffer solution dialysis 3d of 0.01mol/L, pH 7.4, obtains the MTP-mAb solution of N-CDs label, 4 DEG C of preservations.
It is carbon source, Mercaptamine for passivator that carbon nanomaterial, which is using citric acid, is prepared by hydrothermal synthesis method
At: 1.5g citric acid and 1.62g Mercaptamine are dissolved in 7.5mL ultrapure water, shift solution after completely dissolution to 50mL
Among polytetrafluoroethylliner liner, then liner being placed in autoclave, 200 DEG C of reaction 3h after reaction filter product,
Ethanol washing 2 times, 65 DEG C of oven dryings obtain carbon nanomaterial TPCA.
Carbon nanomaterial label anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface siliconization of carbon nanomaterial TPCA
Carbon nanomaterial TPCA is dispersed in the ethanol solution that volumetric concentration is 10%, being configured to concentration is 1mg/mL's
TPCA solution；2mL ammonium hydroxide is added dropwise in 2mL TPCA solution under stirring, 150rpm react at room temperature 25min, then plus
Enter 80 μ L ethyl orthosilicates, room temperature is protected from light 3h；6000rpm is centrifuged 10min after reaction, and after ethanol washing 4 times, nitrogen is blown
Dry, 4 DEG C are sealed；
(2) surface carboxylation of carbon nanomaterial TPCA
0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution A；Take 200mg table
The TPCA of face silication is added in the ethyl alcohol that volume is 2.5mL, forms solution B；Solution B is added in solution A, room temperature is stirred
Mix reaction 70min；After reaction, 6000rpm is centrifuged 10min, after obtained precipitating is washed 4 times with distilled water, nitrogen drying
Dry, 4 DEG C are sealed；
(3) label of MTP-mAb
TPCA and 2mg N, the N'- carbonyl dimidazoles of 2mg surface carboxylation are added to 400 μ L N, N- dimethyl formyls
In amine, magnetic agitation reacts 3h at room temperature, obtains label solution；Label solution is added drop-wise to the Dan Ke that 1mL concentration is l mg/mL
In grand antibody-solutions, 15min is added, 4 DEG C be protected from light be stirred to react overnight；With the 3d that dialyses under the conditions of 4 DEG C of PBS, TPCA label is obtained
MTP-mAb solution, 4 DEG C preservation.
Carbon nanomaterial is, by water and phosphorus pentoxide exothermic heat of reaction, to promote tryptophan carbonization using tryptophan as carbon source
And molecule aggregation is prepared: weighing 0.3g tryptophan and is dissolved in 500-1000 μ L ultrapure water；Until completely dissolved, it imports rapidly
In vial equipped with 3.0g phosphorus pentoxide；Restore after reactive material heat release to room temperature, distilled water be added and washs 2 times,
Centrifuging and taking supernatant, 65 DEG C of oven dryings, obtains carbon nanomaterial.
Carbon nanomaterial label anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface siliconization of carbon nanomaterial
Carbon nanomaterial is dispersed in the ethanol solution that volumetric concentration is 10%, is configured to the carbon that concentration is 1mg/mL and receives
Rice solution；2mL ammonium hydroxide is added dropwise in 2mL carbon nano-solution under stirring, 150rpm react at room temperature 25min, then plus
Enter 80 μ L ethyl orthosilicates, room temperature is protected from light 3h；6000rpm is centrifuged 10min after reaction, and after ethanol washing 4 times, nitrogen is blown
Dry, 4 DEG C are sealed；
(2) surface carboxylation of carbon nanomaterial
0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution A；Take 200mg table
The carbon nanomaterial of face silication is added in the ethyl alcohol that volume is 2.5mL, forms solution B；Solution B is added in solution A,
Reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, after obtained precipitating is washed 4 times with distilled water,
Nitrogen drying is dry, and 4 DEG C are sealed；
(3) label of MTP-mAb
The carbon nanomaterial for weighing 12.78mg surface carboxylation is dissolved in 500 μ L n,N-Dimethylformamide, is added
9.34mg DCC sufficiently dissolves；The DMF solution that 200 μ L are dissolved with 4.17mg n-hydroxysuccinimide is added dropwise again, room temperature is stirred
Reaction 8h is mixed, label solution is obtained；Label solution is added drop-wise in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, is stirred
Mix reaction overnight；With the PBS buffering dialysis 3d of 0.01mol/L, pH 7.4, carbon quantum dot fluorescent nano particle label is obtained
MTP-mAb solution, 4 DEG C of preservations.
Carbon quantum dot fluorescent nano particle is to be prepared using citric acid and methylamine salt as raw material by microwave assisting method:
0.5g citric acid and 0.176g methylamine hydrochloride are weighed, is dissolved in 5mL water, ultrasound sufficiently dissolution after mixing, is placed in function
Rate is in the micro-wave oven of 700W, and after microwave 5min, reaction terminates, natural cooling, and distilled water washs 2 times, and 65 DEG C of oven dryings obtain
To black solid, as carbon quantum dot fluorescent nano particle.
Carbon quantum dot fluorescent nano particle label anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface carboxylation SiO2The preparation of nano particle
SiO is synthesized using reverse microemulsion method2Nano particle: by volume 10:30:10:1 ratio take Ttiton X-100,
Hexamethylene, n-hexyl alcohol, ultrapure water stir to form microemulsion 5.1mL, after 200 μ L ammonium hydroxide of addition stir evenly, add the 80 positive silicic acid of μ L
Ethyl ester, room temperature are protected from light for 24 hours；6000rpm is centrifuged 10min after reaction, is redissolved after ethanol washing 4 times with 1mL ethyl alcohol, shape
At solution A；0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution B；By solution A plus
Enter into solution B, reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, and obtained precipitating is steamed with double
After water washing 4 times, nitrogen drying is dry, and 4 DEG C are sealed；
(2) preparation of fluorescence probe
By the SiO of 2mg surface carboxylation2Nano particle and 2mg N, N'- carbonyl dimidazoles are added to 400 μ L N, N- bis-
In methylformamide, magnetic agitation reacts 3h at room temperature；Adding it to 1mL concentration is l mg/mL carbon quantum dot fluorescence nano
In particle solution, 15min is added, room temperature is protected from light be stirred to react 4h after；It is added 20 μ L ethyl orthosilicates again, 0.12g monoxone,
0.25mg carbon quantum dot fluorescent nano particle, room temperature, which is protected from light, is stirred to react 2h progress involucrum, and involucrum 3 times repeatedly, nitrogen drying is dry, and 4
It DEG C is sealed；
(3) label of MTP-mAb
The fluorescence probe for weighing 12.78mg step (2) preparation is dissolved in 500 μ L n,N-Dimethylformamide, is added
9.34mg DCC sufficiently dissolves；The DMF solution that 200 μ L are dissolved with 4.17mg n-hydroxysuccinimide is added dropwise again, room temperature is stirred
Reaction 8h is mixed, label solution is obtained；Label solution is added drop-wise in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, is stirred
Mix reaction overnight；With the PBS buffering dialysis 3d of 0.01mol/L, pH 7.4, carbon quantum dot fluorescent nano particle label is obtained
MTP-mAb solution, 4 DEG C of preservations.
Test strips of the invention have that high specificity, high sensitivity, stability are high, safety is good, easy, quick, result
Show feature that is vivid, intuitive, applied widely, easy to carry and can quantifying.The needs of different levels personnel are able to satisfy, including
Professional chemical examination, customs quarantine control, health quarantine, quality-monitoring, livestock products processing, raiser and consumer individual etc..The present invention
It is extremely important in terms of ensuring food safety, protecting consumer health, will have apparent economic benefit and society
It can benefit.
Detailed description of the invention
Fig. 1 is the main view of test paper of the invention, in figure, 1 is sample pad, 2 is bonding pad, 3 is chromatographic film, 4 is absorption
Pad, 7 be bottom, 8 be surface layer.
Fig. 2 is the top view of test paper of the invention, in figure, 4 be absorption pad, 5 be it is stealthy detect trace, 6 be stealthy control
Trace, 8 are surface layer.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Embodiment 1
The immune chromatography test paper of detection MTP of the invention including supporter and is fixed on supporter referring to Fig.1-2
Adsorption layer, adsorption layer are followed successively by sample pad 1, bonding pad 2, chromatographic film 3 and absorption pad 4 since test lead, which is characterized in that institute
The anti-MTP monoclonal antibody MTP-mAb of nano material label is adsorbed on the bonding pad stated；The chromatographic film is equipped with even
Join the stealthy detection trace 5 of carrier protein (MTP artificial antigen) the solution printing of MTP and with goat-anti or rabbit anti-mouse IgG antibody
The stealthy control trace 6 of solution printing；The nano material is nitrogen-doped carbon nano material, carbon nanomaterial, carbon quantum dot
Fluorescent nano particle.
The supporter includes the bottom 7 that adsorption layer bottom surface is arranged in and the surface layer 8 that adsorption layer top surface is arranged in.
The supporting plate material is the toughness PVC material not absorbed water.
The sample cushion material can also be nylon membrane, PVDF membrane or polyester film in addition to glass fibre cotton.
The combination cushion material is glass fibre cotton.
The chromatography membrane material can also be pure cellulose film or carboxylated cellulose film in addition to nitrocellulose filter.
The water suction cushion material is strong water absorption function filter paper.
The carrier protein of the described coupling MTP except bovine serum albumin(BSA) (BSA) in addition to, can also be chicken egg white (OVA) or
Hemocyanin (KLH).
The described stealthy detection trace and stealthy control trace are Chu " ∣ ∣ " it can also be " 10 " font in addition to linear trace
Arrangement trace,Font arrangement trace,Font arrangement trace,Font arrange trace orFont row
It prints mark.
It is printed with red samples label warning line in sample pad surface layer corresponding with bonding pad intersection, and is printed on max
Printed words, the label alert at the side 1.1-1.2cm of linear distance sample pad top.
It is white that the surface layer, which is covered on above sample pad and bonding pad, and being covered on is blue above water absorption pad,
It can be also other colors (such as green).
Embodiment 2
The present embodiment detects MTP test paper, specifically includes that preparation, the MTP monoclonal antibody (MTP- of MTP artificial antigen
MAb), the preparation of nitrogen-doped carbon nano material (N-CDs) label MTP antibody and the immune chromatography test paper based on N-CDs label
Preparation, wherein each product the preparation method is as follows:
1, the preparation of MTP artificial antigen (MTP-BSA)
The bovine serum albumin(BSA) (BSA) for weighing 10mg is placed in 10mL screw socket bottle, with the NaHCO of 0.13mol/L3Solution system
At mass fraction 10%BSA activated solution, adjusting pH is 7.6.1mg MTP, 1.073mg N are weighed, N- dicyclohexyl phosphinylidyne is sub-
Amine and 0.598mg n-hydroxysuccinimide, are dissolved in anhydrous tetrahydro furan, 30 DEG C of oscillation 4h, then 4000rpm from
Heart l5min, and wash precipitating with anhydrous tetrahydro furan, it is to be precipitated in tetrahydrofuran volatilization completely after, dissolve the residue in
0.2mL dimethylformamide.And this solution is slowly dropped in BSA activated solution, it is placed on magnetic stirring apparatus, is protected from light room temperature
Reaction is overnight.Reaction product, with PBS dialysis 3d, is changed liquid 3-6 times, the MTP- purified after dialysis daily under 4 DEG C of stirrings
BSA。
2, the preparation of MTP-mAb
Animal immune: the dosage with the artificial antigen MTP-BSA of preparation with 20-25 μ g/ only, it is immune using 4 points of back
Method is immunized 6-8 week old Balb/C female rat 4 times, and head exempts to be emulsified with Freund's complete adjuvant, remaining is emulsified with incomplete Freund's adjuvant,
Each 3 weeks immunization interval time selects the mouse that antibody titer is high, inhibiting rate is good for 4 weeks after last time is immune, carries out superpower exempt from
Epidemic disease.
Cell fusion: it is superpower immune 3 days latter, sinus under immune mouse socket of the eye is taken into blood, neck is taken off and puts to death, take spleen；With 75%
Alcohol impregnates mouse 5-10min and sterilizes body surface, sterile to take its spleen, and spleen is shredded and ground, through 120 mesh nylon gauze mistakes
Filter, 1000rpm are centrifuged 10min, collect splenocyte.By splenocyte and NS0Myeloma cell is in the ratio of 10:1 in centrifuge tube
Mixing, places it in 40 DEG C of water-baths；1mL PEG-1500 is added in centrifuge tube in 60s along tube wall, is continued in water-bath light
After shaking reaction 90s, according to 1mL/30s, centrifuge tube is added in 37 DEG C of 15mL of GNK solution by the speed of 3mL/30s, 11mL/30s
In, 5min is then reacted in 37 DEG C of water-baths, 1000rpm is centrifuged 10min, abandons supernatant；After breaing up cell mass, 40mL is added
HAT culture medium is added on feeder cells culture plate after mixing, and 100 holes μ L/ set 37 DEG C, 5%CO2In incubator.
The screening of monoclonal antibody: culture 10-14 days carries out positive hole with indirect elisa method and screens, selection strong positive,
Inhibiting rate is high, (until cell clone is monoclonal, detection is each for 3-6 limited dilution cloning of cell eugonic hole progress
A clone hole potency inhibits valence almost the same), then expand culture, establishes hybridoma cell strain.Prepared hybridoma
The monoclonal antibody of secretion can specifically be reacted with MTP, and affinity constant reaches 1010-1012L/mol, light chain subtype are κ or λ,
Heavy chain subgroup is IgG1、IgG2a、IgG2b、IgG3。
3, the preparation of the immune chromatography test paper based on N-CDs label
(1) nitrogen-doped carbon nano material N-CDs is prepared using hydro-thermal method
Chitosan 2.5g is dissolved in 5mL ultrapure water, 5mL ethylenediamine is added, mixing is placed on poly- the four of autoclave
In vinyl fluoride liner, 180 DEG C of reaction 2h after reaction filter product, and distilled water washs 2 times, and 60 DEG C of oven dryings obtain
N-CDs。
(2) surface carboxylation SiO2The preparation of nano particle
SiO is synthesized using reverse microemulsion method2Nano particle: by volume 10:30:10:1 ratio take Ttiton X-100,
Hexamethylene, n-hexyl alcohol, ultrapure water stir to form microemulsion 5.1mL, after 200 μ L ammonium hydroxide of addition stir evenly, add the 80 positive silicic acid of μ L
Ethyl ester, room temperature are protected from light for 24 hours；6000rpm is centrifuged 10min after reaction, is redissolved after ethanol washing 4 times with 1mL ethyl alcohol, shape
At solution A；0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution B；By solution A plus
Enter into solution B, reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, and obtained precipitating is steamed with double
After water washing 4 times, nitrogen drying is dry, and 4 DEG C are sealed.
(3)N-CDs-SiO2The preparation of fluorescence probe
By the SiO of 2mg surface carboxylation2Nano particle and 2mg N, N'- carbonyl dimidazoles are added to 400 μ L N, N- bis-
In methylformamide (DMF), magnetic agitation reacts 3h at room temperature；Adding it to 1mL concentration is l mg/mL N-CDs solution
In (dissolution of 0.1mol/L NaOH solution), 15min is added, room temperature is protected from light be stirred to react 4h after；The positive silicic acid second of 20 μ L is added again
Ester, 0.12g monoxone, 0.25mg N-CDs particle, room temperature, which is protected from light, is stirred to react 2h progress involucrum, repeatedly involucrum 3 times, nitrogen drying
Dry, 4 DEG C are sealed.
(4) label of MTP-mAb
The fluorescence probe 15mg for taking step (2) to prepare be dissolved in 1.5mL dioxane, 1.5mL DMF, 60 μ L triethylamines it is mixed
It closes in solution, ice bath 30min, 20 μ L isobutyl chlorocarbonates are added in stirring, and ice bath 2h obtains label solution；Label solution is dripped
It is added in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, reaction is stirred at room temperature overnight；Under the conditions of 4 DEG C, reactant is used
PBS buffer solution the dialysis 3d, the MTP-mAb (N-CDs-MTP-mAb) for obtaining N-CDs label of 0.01mol/L, pH 7.4 is molten
Liquid, 4 DEG C of preservations.
(5) preparation of the immune chromatography test paper based on N-CDs label
By N-CDs-MTP-mAb solution spraying on glass fibre membrane, 37 DEG C of freeze-day with constant temperature 4h form bonding pad；By MTP
Artificial antigen is drawn in chromatographic film respectively together with goat-anti or rabbit-anti IgG solution, and form two markings: one is stealthy detection print
Mark (T line), one be stealthy control trace (C line), 37 DEG C of freeze-day with constant temperature are stayed overnight, and chromatographic film is made；By sample pad, bonding pad,
After chromatographic film, absorption pad are pasted on bottom in order, then surface layer is pasted, cuts into suitable dimension and obtain test paper product.
Detect reaction principle
After test paper test lead is inserted into testing sample solution, under siphonage drive, solution to be measured can be from the survey of test paper
Examination end is diffused into handle end.
In diffusion process, the acephatemet in solution to be measured can be combined with the N-CDs-MTP-mAb on bonding pad, in turn
The antigen-combining site of acephatemet on N-CDs-MTP-mAb is closed, the detection trace knot in N-CDs-MTP-mAb and chromatographic film is prevented
It closes, and compareing goat-anti or rabbit anti-mouse IgG antibody on trace then can read an instrument by fluorescence in conjunction with N-CDs-MTP-mAb,
Under ultraviolet light excitation, would not occur absorption peak at T line, will appear absorption peak at C line.If otherwise without acephatemet in sample solution
When, then it cannot prevent N-CDs-MTP-mAb in conjunction with the detection trace in chromatographic film, and goat-anti or rabbit anti-mouse IgG antibody
Instrument can be read by fluorescence in conjunction with N-CDs-MTP-mAb, under ultraviolet light excitation, will appear absorption peak at T line and C line.
If there is no C line absorption peak in chromatographic film, show that test strips have failed.
Sensitivity, specific detection of the present embodiment to the test paper of the quantitative detection MTP marked based on N-CDs.
The detection of sensitivity: with phosphate buffer PBS (pH 7.4) or distilled water difference compound concentration be 10ng/mL,
The MTP standard items of 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, the loading 80- on immune chromatography test paper of the invention
After reacting 5min, the relative optical density number (relative of T line scan area optical density is read by reading an instrument by 100 μ L
Optical density, ROD).It is vertical sit with the percentage of various concentration standard items and zero standard product relative optical density number
Mark draws standard curve using the common logarithm value of various criterion product concentration as abscissa, carries out correlation regression analysis, and calculating should
IC of the test paper to MTP50And minimum detection limit.After measured, the Regression Equations of MTP pairs of the test paper are as follows: y=-0.7117x+
1.7295 related coefficient R2=0.9965, which is gone out to the IC of MTP according to regression equation calculation50For 53.40ng/mL, most
Low detection is limited to 20.23ng/mL.Show immune chromatography test paper to MTP sensitivity with higher.
Specificity detection: using other organophosphorus pesticides as competitor, prepare above-mentioned standard product various concentration, be immunized
Chromatographic test paper detects its inhibiting rate, with the test paper to MTP's and IC50Each competitor IC50Percentage as its cross reaction
Rate.Measurement result is shown in Table 1.As seen from Table 1, the specificity of the immune chromatography test paper is preferable, with the equal nothing of other organophosphorus pesticides
Cross reaction.
The cross reactivity of immune chromatography test paper of the table 1 based on the N-CDs quantitative detection acephatemet marked
| Compound | Half-inhibitory concentration (ng/mL) | Cross reacting rate (%) |
| Acephatemet | 53.40 | 100 |
| Parathion | > 1.0 × 105 | <0.028 |
| Glufosinate | > 1.0 × 105 | <0.028 |
| Fenamiphos | > 1.0 × 105 | <0.028 |
| Glyphosate | > 1.0 × 105 | <0.028 |
| Pirimiphos-methyl | > 1.0 × 105 | <0.028 |
Embodiment 3
The present embodiment detects MTP test paper, specifically includes that preparation, the MTP monoclonal antibody (MTP- of MTP artificial antigen
MAb), the preparation of carbon nanomaterial label MTP antibody and the preparation etc. of the immune chromatography test paper based on carbon nanomaterial label
Step, wherein each product the preparation method is as follows:
1, the preparation of MTP artificial antigen (MTP-BSA)
With embodiment 2.
2, the preparation of MTP-mAb
With embodiment 2.
3, the preparation of the immune chromatography test paper based on carbon nanomaterial label
(1) preparation of carbon nanomaterial TPCA
1.5g citric acid and 1.62g Mercaptamine are dissolved in 7.5mL ultrapure water, shift solution extremely after completely dissolution
Among 50mL polytetrafluoroethylliner liner, then liner is placed in autoclave, 200 DEG C of reaction 3h, after reaction by product
It filters, ethanol washing 2 times, 65 DEG C of oven dryings obtain carbon nanomaterial TPCA.
(2) surface siliconization of carbon nanomaterial TPCA
Carbon nanomaterial TPCA is dispersed in the ethanol solution that volumetric concentration is 10%, being configured to concentration is 1mg/mL's
TPCA solution；2mL ammonium hydroxide is added dropwise in 2mL TPCA solution under stirring, 150rpm react at room temperature 25min, then plus
Enter 80 μ L ethyl orthosilicates, room temperature is protected from light 3h；6000rpm is centrifuged 10min after reaction, and after ethanol washing 4 times, nitrogen is blown
Dry, 4 DEG C are sealed
(3) surface carboxylation of carbon nanomaterial TPCA
0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution A；Take 200mg table
The TPCA of face silication is added in the ethyl alcohol that volume is 2.5mL, forms solution B；Solution B is added in solution A, room temperature is stirred
Mix reaction 70min；After reaction, 6000rpm is centrifuged 10min, after obtained precipitating is washed 4 times with distilled water, nitrogen drying
Dry, 4 DEG C are sealed.
(4) label of MTP-mAb
TPCA and 2mg N, the N'- carbonyl dimidazoles of 2mg surface carboxylation are added to 400 μ L N, N- dimethyl formyls
In amine, magnetic agitation reacts 3h at room temperature, obtains label solution；Label solution is added drop-wise to the Dan Ke that 1mL concentration is l mg/mL
In grand antibody-solutions, 15min is added, 4 DEG C be protected from light be stirred to react overnight；With the 3d that dialyses under the conditions of 4 DEG C of PBS, TPCA label is obtained
MTP-mAb solution, 4 DEG C preservation.
Reaction principle is detected with embodiment 2.
Sensitivity, specific detection of the present embodiment to the immune chromatography test paper of the quantitative detection MTP marked based on TPCA.
The detection of sensitivity: method is the same as embodiment 2.After measured, the Regression Equations of MTP pairs of the test paper are as follows: y=-
0.7684x+1.9333, related coefficient R2=0.9910, which is gone out to the IC of MTP according to regression equation calculation50For
73.28ng/mL, lowest detection are limited to 29.85ng/mL.Show immune chromatography test paper to MTP sensitivity with higher.
The detection of specificity: method is the same as embodiment 2.Measurement result is shown in Table 2.As seen from Table 2, the immune chromatography test paper
It is specific preferable, with the equal no cross reaction of other organophosphorus pesticides.
The cross reactivity of immune chromatography test paper of the table 2 based on the TPCA quantitative detection acephatemet marked
Embodiment 4
The present embodiment detects MTP test paper, specifically includes that preparation, the MTP monoclonal antibody (MTP- of MTP artificial antigen
MAb), the preparation of carbon nanomaterial label MTP antibody and the preparation etc. of the immune chromatography test paper based on carbon nanomaterial label
Step, wherein each product the preparation method is as follows:
1, the preparation of MTP artificial antigen (MTP-BSA)
With embodiment 2.
2, the preparation of MTP-mAb
With embodiment 2.
3, the preparation of the immune chromatography test paper based on carbon nanomaterial label
(1) preparation of carbon nanomaterial
0.3g tryptophan is weighed to be dissolved in 500-1000 μ L ultrapure water；Until completely dissolved, it imports rapidly and 3.0g five is housed
In the vial for aoxidizing two phosphorus；Restore after reactive material heat release to room temperature, appropriate distilled water is added and washs 2 times, centrifuging and taking
Supernatant, 65 DEG C of oven dryings, obtains carbon nanomaterial.
(2) surface siliconization of carbon nanomaterial
Carbon nanomaterial is dispersed in the ethanol solution that volumetric concentration is 10%, is configured to the carbon that concentration is 1mg/mL and receives
Rice solution；2mL ammonium hydroxide is added dropwise in 2mL carbon nano-solution under stirring, 150rpm react at room temperature 25min, then plus
Enter 80 μ L ethyl orthosilicates, room temperature is protected from light 3h；6000rpm is centrifuged 10min after reaction, and after ethanol washing 4 times, nitrogen is blown
Dry, 4 DEG C are sealed；
(3) surface carboxylation of carbon nanomaterial
0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution A；Take 200mg table
The carbon nanomaterial of face silication is added in the ethyl alcohol that volume is 2.5mL, forms solution B；Solution B is added in solution A,
Reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, after obtained precipitating is washed 4 times with distilled water,
Nitrogen drying is dry, and 4 DEG C are sealed；
(4) label of MTP-mAb
The carbon nanomaterial for weighing 12.78mg surface carboxylation is dissolved in 500 μ L n,N-Dimethylformamide, is added
9.34mg DCC sufficiently dissolves；The DMF solution that 200 μ L are dissolved with 4.17mg n-hydroxysuccinimide is added dropwise again, room temperature is stirred
Reaction 8h is mixed, label solution is obtained；Label solution is added drop-wise in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, is stirred
Mix reaction overnight；With the PBS buffering dialysis 3d of 0.01mol/L, pH 7.4, carbon quantum dot fluorescent nano particle label is obtained
MTP-mAb solution, 4 DEG C of preservations.
Reaction principle is detected with embodiment 2.
The present embodiment is to the sensitivity of the immune chromatography test paper of the quantitative detection MTP marked based on carbon nanomaterial, special
Property detection.
The detection of sensitivity: method is the same as embodiment 2.After measured, the Regression Equations of MTP pairs of the test paper are as follows: y=-
0.6250x+1.5749, related coefficient R2=0.9924, which is gone out to the IC of MTP according to regression equation calculation50For
52.48ng/mL, lowest detection are limited to 17.38ng/mL.Show immune chromatography test paper to MTP sensitivity with higher.
The detection of specificity: method is the same as embodiment 2.Measurement result see the table below 3, the immune chromatography test paper specificity compared with
It is good, with the equal no cross reaction of other organophosphorus pesticides.
The cross reactivity of the immune chromatography test paper for the quantitative detection acephatemet that table 3 is marked based on carbon nanomaterial
| Compound | Half-inhibitory concentration (ng/mL) | Cross reacting rate (%) |
| Acephatemet | 52.48 | 100 |
| Parathion | > 1.0 × 105 | <0.028 |
| Glufosinate | > 1.0 × 105 | <0.028 |
| Fenamiphos | > 1.0 × 105 | <0.028 |
| Glyphosate | > 1.0 × 105 | <0.028 |
| Pirimiphos-methyl | > 1.0 × 105 | <0.028 |
Embodiment 5
The present embodiment detects MTP test paper, specifically includes that preparation, the MTP monoclonal antibody (MTP- of MTP artificial antigen
MAb), carbon quantum dot fluorescent nano particle marks the preparation of MTP antibody and based on carbon quantum dot fluorescent nano particle label
The preparation of immune chromatography test paper, wherein each product the preparation method is as follows:
1, the preparation of MTP artificial antigen (MTP-BSA)
With embodiment 2.
2, the preparation of MTP-mAb
With embodiment 2.
3, the preparation of the immune chromatography test paper based on carbon quantum dot fluorescent nano particle label
(1) preparation of carbon quantum dot fluorescent nano particle
0.5g citric acid and 0.176g methylamine hydrochloride are weighed, is dissolved in 5mL water, ultrasound sufficiently dissolves after mixing,
It is placed in the micro-wave oven that power is 700W, after microwave 5min, reaction terminates, natural cooling, and distilled water washs 2 times, 65 DEG C of bakings
Case is dry, obtains black solid, as carbon quantum dot fluorescent nano particle.
(2) surface carboxylation SiO2The preparation of nano particle
SiO is synthesized using reverse microemulsion method2Nano particle: by volume 10:30:10:1 ratio take Ttiton X-100,
Hexamethylene, n-hexyl alcohol, ultrapure water stir to form microemulsion 5.1mL, after 200 μ L ammonium hydroxide of addition stir evenly, add the 80 positive silicic acid of μ L
Ethyl ester, room temperature are protected from light for 24 hours；6000rpm is centrifuged 10min after reaction, is redissolved after ethanol washing 4 times with 1mL ethyl alcohol, shape
At solution A；0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution B；By solution A plus
Enter into solution B, reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, and obtained precipitating is steamed with double
After water washing 4 times, nitrogen drying is dry, and 4 DEG C are sealed；
(3) preparation of fluorescence probe
By the SiO of 2mg surface carboxylation2Nano particle and 2mg N, N'- carbonyl dimidazoles are added to 400 μ L N, N- bis-
In methylformamide, magnetic agitation reacts 3h at room temperature；Adding it to 1mL concentration is l mg/mL carbon quantum dot fluorescence nano
In particle solution (dissolution of 0.1mol/L NaOH solution), 15min is added, room temperature is protected from light be stirred to react 4h after；20 μ L are added again
Ethyl orthosilicate, 0.12g monoxone, 0.25mg carbon quantum dot fluorescent nano particle, room temperature, which is protected from light, is stirred to react 2h progress involucrum,
Involucrum 3 times repeatedly, nitrogen drying is dry, and 4 DEG C are sealed；
(3) label of MTP-mAb
The fluorescence probe for weighing 12.78mg step (2) preparation is dissolved in 500 μ L n,N-Dimethylformamide, is added
9.34mg DCC sufficiently dissolves；The DMF solution that 200 μ L are dissolved with 4.17mg n-hydroxysuccinimide is added dropwise again, room temperature is stirred
Reaction 8h is mixed, label solution is obtained；Label solution is added drop-wise in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, is stirred
Mix reaction overnight；With the PBS buffering dialysis 3d of 0.01mol/L, pH 7.4, carbon quantum dot fluorescent nano particle label is obtained
MTP-mAb solution, 4 DEG C of preservations.
Reaction principle is detected with embodiment 2.
Spirit of the present embodiment to the immune chromatography test paper of the quantitative detection MTP marked based on carbon quantum dot fluorescent nano particle
Quick property, specific detection.
The detection of sensitivity: method is the same as embodiment 2.After measured, the Regression Equations of MTP pairs of the test paper are as follows: y=-
0.6228x+1.6209, related coefficient R2=0.9983, which is gone out to the IC of MTP according to regression equation calculation50For
63.10ng/mL, lowest detection are limited to 20.80ng/mL.Show immune chromatography test paper to MTP sensitivity with higher.
The detection of specificity: method is the same as embodiment 2.Measurement result see the table below 4, the immune chromatography test paper specificity compared with
It is good, with the equal no cross reaction of other organophosphorus pesticides.
The intersection of the immune chromatography test paper for the quantitative detection acephatemet that table 4 is marked based on carbon quantum dot fluorescent nano particle is anti-
Ying Xing
| Compound | Half-inhibitory concentration (ng/mL) | Cross reacting rate (%) |
| Acephatemet | 63.10 | 100 |
| Parathion | > 1.0 × 105 | <0.028 |
| Glufosinate | > 1.0 × 105 | <0.028 |
| Fenamiphos | > 1.0 × 105 | <0.028 |
| Glyphosate | > 1.0 × 105 | <0.028 |
| Pirimiphos-methyl | > 1.0 × 105 | <0.028 |
The foregoing is merely the optimal embodiments of the present invention, and for those skilled in the art, the present invention can have
Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all
It is included within protection scope of the present invention.
Claims (10)
1. a kind of immune chromatography test paper for detecting acephatemet, including supporter and the adsorption layer being fixed on supporter, adsorption layer
Sample pad (1), bonding pad (2), chromatographic film (3) and absorption pad (4) are followed successively by since test lead, which is characterized in that described
The anti-MTP monoclonal antibody of nano material label is adsorbed on bonding pad；The chromatographic film is equipped with molten with MTP artificial antigen
Stealthy detection trace (5) of liquid printing and stealthy control trace (6) printed with goat-anti or rabbit anti-mouse IgG antibody solution；Institute
The nano material stated is nitrogen-doped carbon nano material, carbon nanomaterial, carbon quantum dot fluorescent nano particle.
2. the immune chromatography test paper of detection acephatemet according to claim 1, which is characterized in that the supporter includes
The bottom (7) that adsorption layer bottom surface is set and the surface layer (8) that adsorption layer top surface is set.
3. the immune chromatography test paper of detection acephatemet according to claim 1, which is characterized in that the nitrogen-doped carbon is received
Rice material N-CDs is using chitosan as carbon source, and ethylenediamine is nitrogen dopant, is prepared using hydro-thermal method, method particularly includes: it will
Chitosan 2.5g is dissolved in 5mL ultrapure water, and 5mL ethylenediamine is added, and mixes the polytetrafluoroethylliner liner for being placed on autoclave
In, 180 DEG C of reaction 2h after reaction filter product, and distilled water washs 2 times, and 60 DEG C of oven dryings obtain N-CDs.
4. the immune chromatography test paper of detection acephatemet according to claim 1-3, which is characterized in that the nitrogen
Adulterate carbon nanomaterial label anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface carboxylation SiO2The preparation of nano particle
SiO is synthesized using reverse microemulsion method2Nano particle: 10:30:10:1 ratio takes Ttiton X-100, hexamethylene by volume
Alkane, n-hexyl alcohol, ultrapure water stir to form 5.1mL microemulsion, after 200 μ L ammonium hydroxide of addition stir evenly, add 80 μ L ethyl orthosilicates,
Room temperature is protected from light for 24 hours；6000rpm is centrifuged 10min after reaction, is redissolved, is formed molten with 1mL ethyl alcohol after ethanol washing 4 times
Liquid A；0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution B；Solution A is added to
In solution B, reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, and obtained precipitating is washed with distilled water
After washing 4 times, nitrogen drying is dry, and 4 DEG C are sealed；
(2)N-CDs-SiO2The preparation of fluorescence probe
By the SiO of 2mg surface carboxylation2Nano particle and 2mg N, N'- carbonyl dimidazoles are added to 400 μ L N, N- dimethyl methyls
In amide, magnetic agitation reacts 3h at room temperature；Adding it to 1mL concentration is in l mg/mL N-CDs solution, and 15min is added,
Room temperature is protected from light be stirred to react 4h after；20 μ L ethyl orthosilicates, 0.12g monoxone, 0.25mg N-CDs particle, room temperature are added again
It is protected from light and is stirred to react 2h progress involucrum, involucrum 3 times repeatedly, nitrogen drying is dry, and 4 DEG C are sealed；
(3) label of MTP-mAb
It is molten that the fluorescence probe 15mg for taking step (2) to prepare is dissolved in 1.5mL dioxane, 1.5mL DMF, the mixing of 60 μ L triethylamines
In liquid, 20 μ L isobutyl chlorocarbonates are added in ice bath 30min, stirring, and ice bath 2h obtains label solution；Label solution is added drop-wise to
500 μ L concentration are that reaction is stirred at room temperature overnight in the monoclonal antibody solution of l mg/mL；Under the conditions of 4 DEG C, reactant is used
The PBS buffer solution dialysis 3d of 0.01mol/L, pH 7.4, obtains the MTP-mAb solution of N-CDs label, 4 DEG C of preservations.
5. the immune chromatography test paper of detection acephatemet according to claim 1, which is characterized in that the carbon nanomaterial
Be using citric acid be carbon source, Mercaptamine for passivator, be prepared by hydrothermal synthesis method: by 1.5g citric acid and
1.62g Mercaptamine is dissolved in 7.5mL ultrapure water, after completely dissolution shift solution to 50mL polytetrafluoroethylliner liner it
In, then liner is placed in autoclave, 200 DEG C of reaction 3h after reaction filter product, ethanol washing 2 times, 65 DEG C
Oven drying obtains carbon nanomaterial TPCA.
6. according to claim 1, detecting the immune chromatography test paper of acephatemet described in 2 or 5, which is characterized in that the carbon is received
Rice material marking anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface siliconization of carbon nanomaterial TPCA
Carbon nanomaterial TPCA is dispersed in the ethanol solution that volumetric concentration is 10%, is configured to the TPCA that concentration is 1mg/mL
Solution；2mL ammonium hydroxide is added dropwise in 2mL TPCA solution under stirring, 150rpm reacts at room temperature 25min, is then added 80
μ L ethyl orthosilicate, room temperature are protected from light 3h；6000rpm is centrifuged 10min after reaction, after ethanol washing 4 times, nitrogen drying
Dry, 4 DEG C are sealed；
(2) surface carboxylation of carbon nanomaterial TPCA
0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution A；Take 200mg surface silicon
The TPCA of change is added in the ethyl alcohol that volume is 2.5mL, forms solution B；Solution B is added in solution A, is stirred at room temperature anti-
Answer 70min；After reaction, 6000rpm is centrifuged 10min, and after obtained precipitating is washed 4 times with distilled water, nitrogen drying is dry, and 4
It DEG C is sealed；
(3) label of MTP-mAb
TPCA and 2mg N, the N'- carbonyl dimidazoles of 2mg surface carboxylation are added in 400 μ L n,N-Dimethylformamide,
Magnetic agitation reacts 3h at room temperature, obtains label solution；Label solution is added drop-wise to the monoclonal that 1mL concentration is l mg/mL to resist
In liquid solution, 15min is added, 4 DEG C be protected from light be stirred to react overnight；With the 3d that dialyses under the conditions of 4 DEG C of PBS, TPCA label is obtained
MTP-mAb solution, 4 DEG C of preservations.
7. the immune chromatography test paper of detection acephatemet according to claim 1, which is characterized in that the carbon nanomaterial
It is, by water and phosphorus pentoxide exothermic heat of reaction, tryptophan carbonization and molecule aggregation to be promoted to be prepared using tryptophan as carbon source:
0.3g tryptophan is weighed to be dissolved in 500-1000 μ L ultrapure water；Until completely dissolved, it imports rapidly and 3.0g phosphorus pentoxide is housed
Vial in；Restore after reactive material heat release to room temperature, distilled water is added and washs 2 times, centrifuging and taking supernatant, 65 DEG C of baking ovens
It is dry, obtain carbon nanomaterial.
8. according to claim 1, detecting the immune chromatography test paper of acephatemet described in 2 or 7, which is characterized in that the carbon is received
Rice material marking anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface siliconization of carbon nanomaterial
Carbon nanomaterial is dispersed in the ethanol solution that volumetric concentration is 10%, it is molten to be configured to the carbon nanometer that concentration is 1mg/mL
Liquid；2mL ammonium hydroxide is added dropwise in 2mL carbon nano-solution under stirring, 150rpm reacts at room temperature 25min, and 80 μ are then added
L ethyl orthosilicate, room temperature are protected from light 3h；6000rpm is centrifuged 10min after reaction, and after ethanol washing 4 times, nitrogen drying is dry,
4 DEG C are sealed；
(2) surface carboxylation of carbon nanomaterial
0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution A；Take 200mg surface silicon
The carbon nanomaterial of change is added in the ethyl alcohol that volume is 2.5mL, forms solution B；Solution B is added in solution A, room temperature
It is stirred to react 70min；After reaction, 6000rpm is centrifuged 10min, and after obtained precipitating is washed 4 times with distilled water, nitrogen is blown
Dry, 4 DEG C are sealed；
(3) label of MTP-mAb
The carbon nanomaterial for weighing 12.78mg surface carboxylation is dissolved in 500 μ L n,N-Dimethylformamide, is added
9.34mg DCC sufficiently dissolves；The DMF solution that 200 μ L are dissolved with 4.17mg n-hydroxysuccinimide is added dropwise again, room temperature is stirred
Reaction 8h is mixed, label solution is obtained；Label solution is added drop-wise in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, is stirred
Mix reaction overnight；With the PBS buffering dialysis 3d of 0.01mol/L, pH 7.4, carbon quantum dot fluorescent nano particle label is obtained
MTP-mAb solution, 4 DEG C of preservations.
9. the immune chromatography test paper of detection acephatemet according to claim 1, which is characterized in that the carbon quantum dot is glimmering
Light nano particle is to be prepared using citric acid and methylamine salt as raw material by microwave assisting method: weigh 0.5g citric acid and
0.176g methylamine hydrochloride is dissolved in 5mL water, and ultrasound sufficiently dissolution after mixing, is placed in the micro-wave oven that power is 700W
In, after microwave 5min, reaction terminates, natural cooling, and distilled water washs 2 times, and 65 DEG C of oven dryings obtain black solid, as
Carbon quantum dot fluorescent nano particle.
10. according to claim 1, detecting the immune chromatography test paper of acephatemet described in 2 or 9, which is characterized in that the carbon amounts
Son point fluorescent nano particle label anti-MTP monoclonal antibody the preparation method comprises the following steps:
(1) surface carboxylation SiO2The preparation of nano particle
SiO is synthesized using reverse microemulsion method2Nano particle: 10:30:10:1 ratio takes Ttiton X-100, hexamethylene by volume
Alkane, n-hexyl alcohol, ultrapure water stir to form microemulsion 5.1mL, after 200 μ L ammonium hydroxide of addition stir evenly, add 80 μ L ethyl orthosilicates,
Room temperature is protected from light for 24 hours；6000rpm is centrifuged 10min after reaction, is redissolved, is formed molten with 1mL ethyl alcohol after ethanol washing 4 times
Liquid A；0.47g monoxone is added in the NaOH solution that 2.5mL concentration is 6mol/L, forms solution B；Solution A is added to
In solution B, reaction 70min is stirred at room temperature；After reaction, 6000rpm is centrifuged 10min, and obtained precipitating is washed with distilled water
After washing 4 times, nitrogen drying is dry, and 4 DEG C are sealed；
(2) preparation of fluorescence probe
By the SiO of 2mg surface carboxylation2Nano particle and 2mg N, N'- carbonyl dimidazoles are added to 400 μ LN, N- dimethyl methyls
In amide, magnetic agitation reacts 3h at room temperature；Adding it to 1mL concentration is that l mg/mL carbon quantum dot fluorescent nano particle is molten
In liquid, 15min is added, room temperature is protected from light be stirred to react 4h after；20 μ L ethyl orthosilicates, 0.12g monoxone, 0.25mg are added again
Carbon quantum dot fluorescent nano particle, room temperature, which is protected from light, is stirred to react 2h progress involucrum, and involucrum 3 times repeatedly, nitrogen drying is dry, 4 DEG C of sealings
It saves；
(3) label of MTP-mAb
The fluorescence probe for weighing 12.78mg step (2) preparation is dissolved in 500 μ LN, in dinethylformamide, adds 9.34mg
DCC sufficiently dissolves；The DMF solution that 200 μ L are dissolved with 4.17mg n-hydroxysuccinimide is added dropwise again, reaction is stirred at room temperature
8h obtains label solution；Label solution is added drop-wise in the monoclonal antibody solution that 500 μ L concentration are l mg/mL, is stirred to react
Overnight；With the PBS buffering dialysis 3d of 0.01mol/L, pH 7.4, the MTP-mAb of carbon quantum dot fluorescent nano particle label is obtained
Solution, 4 DEG C of preservations.
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