CN108918854A - A method of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label - Google Patents
A method of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label Download PDFInfo
- Publication number
- CN108918854A CN108918854A CN201810459487.8A CN201810459487A CN108918854A CN 108918854 A CN108918854 A CN 108918854A CN 201810459487 A CN201810459487 A CN 201810459487A CN 108918854 A CN108918854 A CN 108918854A
- Authority
- CN
- China
- Prior art keywords
- psi
- oam
- antibody
- solution
- fluorescence probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- 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
- G01N33/54346—Nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention provides a kind of methods of fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label, are easily isolated based on magnetic fluorescence probe and its superior fluorescence property, the method improve the sensitivity of detection.Pass through preparation PSIOAmThe anti-PSI of envelope antigen, magnetic fluorescence probe labelOAmAntibody, in conjunction with the specific to oleyl amine scion grafting polysuccinimide (PSI of antigen-antibody reactionOAm) high molecular nanometer particles have carried out ultra trace detection and analysis.Compared with prior art, method provided by the invention has the features such as easy to operate, low background, high sensitivity, high specificity, achievable high throughput, accurate targeting and non-destructive testing.
Description
Technical field
The present invention relates to the quantitative detections of the functionalization of magnetic fluorescence probe and nano material, and in particular to one kind is based on magnetic
Property fluorescence probe label fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier method.
Background technique
The Chinese tumour hospital's market research in 2017 and prediction of the development trend of China's Industry investigation net publication are reported
Think, currently, China's tumor incidence is improved every year with the speed of 3%-5%, average annual medical oncology expense is all at 150,000,000,000 yuan
More than, the continuous rising of Cancer Mortality and dead busy rate necessarily brings the demand for services to oncotherapy, therefore develop
The development of efficient anti-tumor drug and supply/demand also seem more and more necessary.
Drug fiting chemical therapy, radiotherapy, but current change are mainly used for the treatment of cancer in clinic at present
Drug poorly water-soluble is treated, does not have targeting, normal cell can be also killed while killing cancer cell, so as to cause serious
Toxic side effect hinders the development and application of chemotherapeutics.
Realize targeting administration, researcher proposes antibody drug carrier system, and antibody drug carrier system will exactly resist
Tumour medicine and monoclonal antibody carry out the system that coupling is got up by chemical bond, will be resisted using the specific reaction of antigen-antibody
Tumour medicine is carried to specified position.Although antibody drug carrier system improves part easily modification anticancer to a certain extent
The targeting and water solubility of drug, but since it is carried, medicine quantity is few, and the high toxicity of anti-tumor drug has seriously affected antigen-antibody
Between specific reaction, be not enough to kill tumour reaching tumor locus.Therefore, drug therapy efficiency is improved, medicine is reduced
Object toxic side effect, improvement drug distribution etc. have become field of biomedicine a major challenge.
Nano-medicament carrier is a kind of novel carriers, is usually made of natural or synthetic high molecular material, major advantage
It is the trap and stability for improving drug, improves pharmaceutical properties and targeting, drug treating time extends, and curative effect increases, poison
Side reaction is small, reduces the toxic etc. to normal cell, and therefore, more and more researchers have turned one's attention to nanometer medicine
Object carrier.Mainly there are metal organic frame class, inorganic non-metallic class, high molecular polymerization species, magnetic fluid and liposome etc. at present.
Drug molecule can be wrapped up wherein or be adsorbed on its surface by nano-carrier particle size about 10~500nm, by targeted molecular with
Cell surface specific receptor combines or magnetic targeted, enters under cellular uptake effect into the cell, realization safely and effectively targets
Drug conveying, therefore there is special value and significance in drug delivery.
Important member one of of the oleyl amine scion grafting polysuccinimide nano material as polymer carrier, due to its system
Standby simplicity, property is stable, drugloading rate type and quantity are more, good biocompatibility and surface are easily modified, and sends out in field of biomedicine
Important function is waved.It will be carried in target polypeptide RGD modification to polysuccinimide nano material as a kind of novel targeted drug
Body is a kind of nano-medicament carrier more preferably with intelligent effect, provides new thinking and hand for the treatment of cancer
Section, solve mankind's major disease diagnosis, in terms of have more quantum jump.For example, Wang Leyu has been developed
Oleyl amine scion grafting polysuccinimide polymer nano micelle is used as the delivery vehicles of the anticancer drugs such as adriamycin, camptothecine,
The efficiency and type for carrying medicine are improved on the basis of certain.
Currently, polymer micelle has numerous studies report as pharmaceutical carrier, but sufficiently safely, effectively entering clinic
Before, how to realize that real-time dynamically targeting, more accurate targeting substance, the more effective therapeutic agent of nano-carrier are cleverer
Quick, operational more easily sensor, biocompatibility and degradability, encapsulation rate and release time carry large biological molecule
Stability and integrality and internal carrier function mechanism dynamic test with a series of problems, such as analysis method still to further
It researchs and solves.From the point of view of existing result of study, it is typically limited to investigate it in vivo in medical domain for Application of micron
Distribution, degradation, drug release efficiency etc..Due to nano material specific surface energy with higher, transported in human body
The ingredient of various complexity interacts to form one layer of albumen clothing in Shi Huiyu blood, and has to the targeting of nano-medicament carrier
Certain influence, in addition, different dosage may generate different effect or toxic side effect to body.Therefore, Nano medication carries
Whether the dose measurement of body, which can be applied to clinic for investigating the material, is particularly important.And Nano medication is carried at present
The positioning characterizing method of body is mainly fluorescent spectrometry.Imaging agent of the fluorescent reagent as nano-medicament carrier is usually selected,
But due between the anti-light Bleachability difference of the various fluorescent reagents encapsulated in pharmaceutical carrier or even some fluorescent imaging agent meetings and drug
Having an effect causes drug effect to change.Therefore, detect unification is to be unable to reach with fluorescent imaging agent marking nano pharmaceutical carrier
Prolonged Real-time and Dynamic Detection.
However, the quantitative analysis method of currently used nano material is mainly by means of expensive analysis instrument.And
Test reagent used has certain toxicity, to the destructive big of sample, and stability and sensitivity often all do not reach requirement, inspection
Survey technology is still not mature enough.Therefore, these methods can not all realize the real-time and precise quantitative detection point to nano material well
Analysis.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of fluorescence immunoassay absorption survey based on magnetic fluorescence probe label
The method for determining ultra trace polymeric nano medicine carrier, is easily isolated and its superior fluorescence property based on magnetic fluorescence probe,
The method improves the sensitivity of detection.Pass through preparation PSIOAmThe anti-PSI of envelope antigen, magnetic fluorescence probe labelOdmIt is anti-
Body, in conjunction with the specific to oleyl amine scion grafting polysuccinimide (PSI of antigen-antibody reactionOAm) high molecular nanometer particles carry out
Ultra trace tests and analyzes.This method has easy to operate, low background, high sensitivity, high specificity, high throughput can be achieved, precisely
The features such as targeting and non-destructive testing.
Specific technical solution of the present invention is as follows：
A kind of fluorescence immunoassay determining adsorption ultra trace macromolecule based on magnetic fluorescence probe label provided by the invention is received
The method of rice pharmaceutical carrier, includes the following steps：
A, PSI is preparedOAmHydrating solution；
B, PSI is preparedOAmEnvelope antigen and immunogene；
C, anti-PSIOAmThe preparation of antibody；
D, the preparation of magnetic fluorescence probe；
E, the anti-PSI of magnetic fluorescence probe labelOAmThe preparation of antibody；
F, by PSIOdmEnvelope antigen is coated in 96 orifice plates after being coated liquid dilution, and various concentration is added in closing
PSIOAmStandard items mark anti-PSI with functionalization magnetic fluorescence probeOAmAntibody establishes direct competitive fluorescence immunoassay determining adsorption
PSIOAm；
G, with PSIOAmThe logarithm of standard concentration is abscissa, and fluorescence intensity level is ordinate drafting standard curve, thus
Quantitative detection goes out PSIOAmConcentration.
Specifically, step a specifically includes following steps：By the PSI of 20~60mgOAmIt is molten to be dissolved into 1~5mL chloroform
In liquid, above-mentioned solution is added in the sodium hydroxide solution that 10~16mL concentration is 0.001~0.008mg/mL after dissolution, is surpassed
10~30min of sound, 300~600W of power, then solution, is evaporated removing three by 20~50min of magnetic agitation at 40~60 DEG C
Chloromethanes is centrifuged 10~15min in 12000~20000r/min, is washed 3 times with the PBS of pH=7.4, precipitating is taken to be dispersed in 0.5
In the PBS buffer solution of~2mL pH=7.4, the PSI after being hydrolyzedOAm-COO-Solution.
Step b specifically includes following steps：
B-1,1mgOVA is dissolved in 1mLPBS solution, the PSI after step a hydrolysis is then addedOAm-COO-Solution 1mL,
Be eventually adding 1mLPBS buffer solution, in the buffer comprising 0.1~1mg n-hydroxysuccinimide (NHS) and 0.1~
1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate of 1mg, mixed 10~30min of reaction, standing 30~
It is centrifugated after 60min, is washed 3 times with the PBS of pH=7.4,1~10mg bovine serum albumin(BSA) is then added, it is warm at 25 DEG C
2~4h is educated, 10~15min is centrifuged in 12000~20000r/min of centrifuge later, precipitating is taken to be distributed to 1mLPBS (pH=
7.4) in buffer, solution is fitted into bag filter, is put into PBS buffer solution and dialyses 12 hours or more, can be prepared by
PSIOAmThe envelope antigen of-OVA；
B-2, to the PSI after hydrolysisOAmIn solution, HOSu NHS, 1- ethyl-(3- dimethylamino third is added
Base) phosphinylidyne diimmonium salt hydrochlorate and bovine serum albumin(BSA), after 20-30 DEG C of 3~6h of incubation, centrifuge separation takes precipitating to be scattered in pH
PSI is obtained in=7.4 PBS buffer solutionOAmImmunogen solution；
In the step b-1, the molecular cut off of bag filter is 8000~25000Da；
Step c specifically includes following steps：
C-1, first immunisation：By PSIOAmImmunogene and Freund's complete adjuvant in equal volume than mixing after, it is more using dorsal sc
The mode of point injection is injected into White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；First immunisation after three weeks into
Row booster immunization；
C-2, booster immunization：By PSIOAmImmunogene and incomplete Freund's adjuvant in equal volume than mixing after, take same side
Formula is injected into White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；Hereafter reinforced exempting from again every two weeks
Epidemic disease, serum titer is surveyed in intermediate Zhou Jinhang ear vein blood sampling, until potency reaches 1:64000, then last time booster immunization is carried out,
And take a blood sample after a week from animal arteria carotis immune, it stands and antiserum is precipitated and is purified, obtain anti-PSIOAmAntibody.
Step d specifically includes following steps：
D-1, first by the FeCl of 10.8g3·6H2The enuatrol of O and 36.5g is dissolved in 80mL ethyl alcohol, sequentially adds 60mL
Deionized water and 140mL hexamethylene, then in 70 DEG C of heating 4h, it is to be cooled to room temperature when organic layer deionized water extract 3 times,
Acquired solution mixing is extracted, 4h is heated at 70 DEG C and evaporates hexamethylene, is cooled to room temperature to get iron oleate, it is spare；
D-2, iron oleate 9g prepared by step d-1 is added in the octadecene of 50g, adds 1.4g after mixing evenly
Oleic acid, mixed solution vacuumizes after being heated to 100 DEG C of holding 30min, and mixture is heated to 320 DEG C of holdings under nitrogen atmosphere
3h, the dehydrated alcohol that 20mL is added when reacting to room temperature precipitate ferriferrous oxide nano crystalline substance, dry in the air after washing 3 times repeatedly
It is dry brilliant to get ferriferrous oxide nano；
D-3, it step d-2 is prepared into ferriferrous oxide nano crystalline substance is dissolved with hexamethylene, obtained 100mg/mL ferroso-ferric oxide and receive
The brilliant solution of rice, by 14.4mL hexamethylene, 3.3mLTX-100,0.5mLCO-520,3.3mL n-hexyl alcohol, 1.5mL deionized water and
0.25mL ammonium hydroxide stirs and evenly mixs, and the ferriferrous oxide nano crystalline substance solution of 1mL 100mg/mL is then added, adds the silicon of 50 μ L
Sour tetra-ethyl ester is stirred to react 2 hours, and centrifugation after having reacted is washed 3 times with dehydrated alcohol, is freeze-dried to get silica function
The ferriferrous oxide nano of energyization is brilliant, spare；
The ferriferrous oxide nano crystalline substance of silica functionalization prepared by d-4, the step d-3 for weighing 6.7mg is scattered in
In the deionized water of 50mL, with the pH=9.0 of the sodium hydrate regulator solution of 2M, system is heated to 70 DEG C, is then successively added
Enter the tetraethyl orthosilicate of 0.5mL, the 3- aminopropyl triethoxysilane of the rhodamine isothiocyanates label of 0.05mL and 3mL
Ethyl acetate adds the 3- aminopropyl triethoxysilane of 0.05mL after being protected from light stirring 10min, stirs 3h, is cooled to room temperature
Ethanol washing 3 times afterwards are freeze-dried to get magnetic fluorescence probe, spare.
It is brilliant according to the square ferriferrous oxide nano of high temperature Co deposited synthesis first in the step d, size distribution
Uniformly, layer of silicon dioxide has been wrapped up with surface of the reverse micelle of microemulsion to ferriferrous oxide nano crystalline substance, it is single under mild conditions
The ferriferrous oxide nano crystalline substance of dispersion is assembled into the spherical structure of high-crystallinity and has gem-pure lattice fringe, sufficiently says
It is as made of identical ferroso-ferric oxide nanometer monocrystalline particle aggregation that the magnetic fluorescence combined probe, which is illustrated,；Compared to traditional
Magnetic fluorescence combined probe, the magnetic fluorescence probe are still assembled in the case where loading the rhodamine isothiocyanates of same amount
At the spherical structure of high-crystallinity, this sufficiently extends magnetic composite in the application of biomarker.
Step e specifically includes following steps：
E-1, anti-PSI after purification is takenOAmAntibody 0.4mg is added after the acetate buffer solution of 0.07mL, is added 0.14mL's
Sodium periodate solution, room temperature are protected from light stirring 2h；
E-2, the magnetic fluorescence probe for taking 2mg step d-4 to prepare, are washed 3 times with PBS, are sufficiently suspended in the PBS of 0.6mL,
Antibody after being slowly added to step e-1 treated oxidation stirs 20h at 4 DEG C；Add the hydroboration of 2mg/mL 0.02mL
Sodium solution is protected from light 2h, to get the anti-PSI of magnetic fluorescence probe label after washing 3 times with PBSOAmAntibody, it is spare.
Step f specifically includes following steps：
F-1, coating：With coating buffer by PSIOAmEnvelope antigen is diluted to 25 μ g/mL, is coated with 96 orifice plates, every 100 μ of hole
L, 4 DEG C of refrigerator overnights；
F-2, closing：PBST solution washs 3 times, and drying, 3~5min, washes away unbonded PSI every timeOAmEnvelope antigen,
1wt% casein is added, every 200 μ L of hole is closed, and 37 DEG C of baking ovens incubate 1~2h；
F-3, sample-adding competition：PBST solution washs 3 times, and drying, 3~5min, washes away extra confining liquid every time, then will
The anti-PSI of 50 μ L magnetic fluorescence probes label after optimizationOdmThe PSI of antibody and 50 μ L various concentrationsOAmStandard items point gradient adds
Enter in each hole, is allowed to that competitive reaction occurs, 37 DEG C of baking ovens incubate 1~2h；
F-4, detection：PBST solution washs 3 times, and drying, 3~5min, removes the PSI of free state every timeOAmStandard items are anti-
Body conjugate, measuring each hole in excitation wavelength with multi-function microplate reader is 530nm, and launch wavelength is the fluorescence intensity at 585nm
Value.
The linear equation of standard curve described in step g is F=11477.59-1537.63lgC, and wherein F is fluorescence intensity
Value, C PSIOAmConcentration.Its coefficient R2=0.994, the range of linearity is 5 × 10-5-5×103Ng/mL, detection are limited to 3
×10-7ng/mL。
The present invention provides a kind of poly- ambers of fluorescence immunoassay determining adsorption oleyl amine scion grafting based on magnetic fluorescence probe label
Acid imide (PSIOAm) polymeric nano medicine carrier method, in conjunction with antigen, antibody response specificity it is glimmering with multifunction magnetic
Light probe realizes the immunization method to PSI as markerOAmUltra trace detection.
Compared with prior art, the present invention has the characteristics that following：
(1) magnetic fluorescence probe has been prepared as fluorescence signal molecule, to establish Gao Ling using simple, green method
Quick Immunoassay quantification detects PSIOAmIt lays a good foundation；
(2) it is established using the specificity of magnetic fluorescence probe and antigen, antibody response a kind of based on magnetic fluorescence probe
The fluorescence immunoassay determining adsorption PSI of labelOAmNew method, for nano material from now on provide it is a kind of simple, quickly, it is accurate fixed
The method for measuring detection；
(3) magnetic fluorescence probe marking nano pharmaceutical carrier antibody is utilized, that is, enhances the aqueous solution of magnetic fluorescence probe
Dispersibility, while also improving the material and the targeting of nano material is detected.
(4) the fluorescence probe particle diameter distribution is uniform, and carried dye is more, and meso-hole structure effectively improves the anti-of organic dyestuff
Photobleaching, and multiple ferriferrous oxide nanos crystalline substances are wrapped up, Magnetic Isolation effect is further enhanced, the spirit of experiment is improved
Sensitivity；
(6) this method is easy to operate, quick, high sensitivity, high specificity, high-throughput non-destructive testing can be achieved.
Detailed description of the invention
Fig. 1 is with PSIOAmThe logarithm of standard concentration is abscissa, and fluorescence intensity level is the standard curve that ordinate is established
Figure.
Specific embodiment
Freund's complete adjuvant, bovine serum albumin(BSA) (BSA) and chicken ovalbumin (OVA) purchase are in raw work bioengineering
(Shanghai) limited liability company.The 3- aminopropyl triethoxysilane of rhodamine isothiocyanates label is bought in Hefei Baeyer
Enlightening, other reagents can sale producer from the market be commercially available.
The present invention relates to the preparation method of each solution be：
PBS solution (0.01mol/L pH=7.4)：Weigh NaCl 8.0g, KCl 0.1g, NaH2PO4·2H2O
0.106g、Na2HPO4·12H2O 3.34g is dissolved in distilled water and is settled to 1000mL.
Carbonate buffer solution CD (0.5mol/L pH=9.6)：Weigh Na2CO3 1.59g、NaHCO32.94g being dissolved in steaming
In distilled water and it is settled to 100mL.
PBST solution (0.01mol/L pH=7.4)：500 μ L Tween-20 are added in 1000mL PBS, mixing is equal
It is even.
It is coated with buffer fe (0.05mol/L pH=9.6)：Weigh Na2CO3 1.59g、NaHCO32.94g being dissolved in steaming
In distilled water and it is settled to 1000mL.
Acetate buffer solution：84.25g sodium acetate is weighed, is dissolved with water, 100ml acetic acid is added, is diluted with water to 2500ml i.e.
Obtain (PH=4.2) acetic acid sodium acetate buffer.
1wt% casein solution：It is confining liquid, weighs 0.01g casein and is dissolved in 1mL PBS, is uniformly mixed.
Embodiment 1
A kind of fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label
Method includes the following steps：
A, PSI is preparedOAmHydrating solution:
By the PSI of 20~60mgOAmIt is dissolved into 1~5mL chloroform soln, above-mentioned solution is added to 10 after dissolution
~16mL concentration be 0.001~0.008mg/mL sodium hydroxide solution in, ultrasound 10~30min, 300~600W of power, so
20~50min of magnetic agitation afterwards, solution is evaporated at 40~60 DEG C remove chloroform, in 12000~20000r/min from
10~15min of the heart is washed 3 times with the PBS of pH=7.4, precipitating is taken to be dispersed in the PBS buffer solution of 0.5~2mL pH=7.4,
PSI after being hydrolyzedOAm-COO-Solution.
B, PSI is preparedOAmEnvelope antigen and immunogene:
B-1,1mgOVA is dissolved in 1mLPBS solution, the PSI after step a hydrolysis is then addedOAm-COO-Solution 1mL,
Be eventually adding 1mLPBS buffer solution, in the buffer comprising 0.1~1mg n-hydroxysuccinimide (NHS) and 0.1~
1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate of 1mg, mixed 10~30min of reaction, standing 30~
It is centrifugated after 60min, is washed 3 times with the PBS of pH=7.4,1~10mg bovine serum albumin(BSA) is then added, it is warm at 25 DEG C
2~4h is educated, 10~15min is centrifuged in 12000~20000r/min of centrifuge later, precipitating is taken to be distributed to 1mLPBS (pH=
7.4) in buffer, solution is fitted into bag filter, is put into PBS buffer solution and dialyses 12 hours or more, can be prepared by
PSIOAmThe envelope antigen of-OVA；
B-2, to the PSI after hydrolysisOAmIn solution, HOSu NHS, 1- ethyl-(3- dimethylamino third is added
Base) phosphinylidyne diimmonium salt hydrochlorate and bovine serum albumin(BSA), after 20-30 DEG C of 3~6h of incubation, centrifuge separation takes precipitating to be scattered in pH
PSI is obtained in=7.4 PBS buffer solutionOAmImmunogen solution；
The molecular cut off of bag filter is 8000~25000Da in the step b-1；
C, anti-PSIOAmThe preparation of antibody:
C-1, first immunisation：By PSIOAmImmunogene and Freund's complete adjuvant in equal volume than mixing after, it is more using dorsal sc
The mode of point injection is injected into White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；First immunisation after three weeks into
Row booster immunization；
C-2, booster immunization：By PSIOAmImmunogene and incomplete Freund's adjuvant in equal volume than mixing after, take same side
Formula is injected into White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；Hereafter reinforced exempting from again every two weeks
Epidemic disease, serum titer is surveyed in intermediate Zhou Jinhang ear vein blood sampling, until potency reaches 1:64000, then last time booster immunization is carried out,
And take a blood sample after a week from animal arteria carotis immune, it stands and antiserum is precipitated and is purified, obtain anti-PSIOAmAntibody.
D, the preparation of magnetic fluorescence probe:
D-1, first by the FeCl of 10.8g3·6H2The enuatrol of O and 36.5g is dissolved in 80mL ethyl alcohol, sequentially adds 60mL
Deionized water and 140mL hexamethylene, 70 DEG C of heating 4h.It is to be cooled to room temperature when, organic layer is extracted 3 times with deionized water, extraction
Solution mixing, at 70 DEG C heat 4h evaporate hexamethylene, occur thick substance russet when being cooled to room temperature, i.e.,
Iron oleate is successfully synthesized；
D-2, the 9g step d-1 iron oleate prepared is added in the octadecene of 50g, adds 1.4g after mixing evenly
Oleic acid, mixed solution vacuumizes after being heated to 100 DEG C of holding 30min, and mixture is heated to 320 DEG C of holdings under nitrogen atmosphere
3h, 20mL dehydrated alcohol, which is added, when room temperature will be arrived by reacting makes the nanocrystalline precipitating of iron, dries after washing 3 times repeatedly to get four oxygen
It is nanocrystalline to change three-iron, it is spare；
D-3, it step d-2 is prepared into ferriferrous oxide nano crystalline substance is dissolved with hexamethylene, obtained 100mg/mL ferroso-ferric oxide and receive
The brilliant solution of rice, takes 14.4mL hexamethylene, 3.3mL TX-100,0.5mL CO-520,3.3mL n-hexyl alcohol, 1.5mL deionization respectively
Water, 0.25mL ammonium hydroxide stir evenly in 100mL beaker, and the ferriferrous oxide nano crystalline substance that 1mL 100mg/mL is then added is molten
Liquid, the tetraethyl orthosilicate for adding 50 μ L stir 2 hours, are centrifuged after having reacted, are washed 3 times with dehydrated alcohol, are freeze-dried,
It is brilliant up to the ferriferrous oxide nano of silica functionalization, it is spare；
D-4, the sample dispersion prepared in the d-3 of 6.7mg is accurately weighed in the deionized water of 50mL, with the hydroxide of 2M
Sodium adjusts the pH=9.0 of solution, and system is heated to 70 DEG C, sequentially adds the tetraethyl orthosilicate of 0.5mL, the rhodamine of 0.05mL
0.05mL is added after being protected from light stirring 10min in the 3- aminopropyl triethoxysilane of isothiocyanates label, the ethyl acetate of 3mL
3- aminopropyl triethoxysilane, stir 3h, be cooled to room temperature rear ethanol washing 3 times, freeze-drying to get magnetic fluorescence spy
Needle, it is spare.
E, the anti-PSI of magnetic fluorescence probe labelOAmThe preparation of antibody:
E-1, anti-PSI after purification is takenOAmAcetate buffer solution (0.05mol/L, the pH=of antibody 0.4mg addition 0.07mL
4.2) the sodium periodate solution (1.5mg/mL, pH=4.2) that 0.14mL is added after is protected from light stirring 2h；
E-2, it takes 2mg multifunction magnetic fluorescence probe to be washed 3 times with PBS, is sufficiently suspended in the PBS of 0.6mL, is slowly added to
Antibody after oxidation stirs 20h at 4 DEG C；The sodium borohydride solution of 0.02mL 2mg/mL is added, is protected from light 2h, is washed with PBS
It is spare after washing 3 times.
F, by PSIOdmEnvelope antigen is coated in 96 orifice plates after being coated liquid dilution, and various concentration is added in closing
PSIOAmStandard items mark anti-PSI with functionalization magnetic fluorescence probeOAmAntibody establishes direct competitive fluorescence immunoassay determining adsorption
PSIOAm:
F-1, coating：It is diluted with the carbonate buffer solution of 0.05M, pH=9.6 by PSIOAmEnvelope antigen solution is diluted to 25
μ g/mL is coated in 96 orifice plates, every 100 μ L of hole, and 4 DEG C of refrigerator overnight；
F-2, closing：96 orifice plates are taken out, PBST is washed 3 times, and each 3min washes away unbonded PSIOAmEnvelope antigen adds
Enter 1wt% casein to be closed, every hole 200 μ L, 37 DEG C of closing 1.5h；
F-3, sample-adding competition：PBST is washed 3 times, and drying, 50 μ L concentration are respectively 5 × 10 by each 3min-5ng/mL、1
×10-4ng/mL、5×10-4ng/mL、10-3ng/mL、5×10-3ng/mL、10-2ng/mL、5×10-2ng/mL、10-1ng/mL、
0.5ng/mL、1ng/mL、5ng/mL、10ng/mL、50ng/mL、1×102ng/mL、5×102ng/mL、103The PSI of ng/mLOAm
Titer is added sequentially in each row of 96 orifice plates, i.e., in triplicate, 50 μ L function are then added in each concentration gradient into each hole
The anti-PSI of magnetic fluorescence probe label can be changedOAmAntibody, 37 DEG C of competition 1.5h；
F-4,3 times are washed with cleaning solution later, 3 minutes every time, drying；The PSI of various concentrationOAmThe preparation side of titer
Method is：With the PBS buffer solution of 0.01mol/L pH=7.4 by PSIOAmIt is diluted to defined normal concentration；It is surveyed in microplate reader
Fixed each hole is 530nm in excitation wavelength, fluorescence intensity when launch wavelength is 585nm.
By PSIOAmEnvelope antigen is coated in 96 orifice plates after being coated liquid dilution, closing, the PSI that various concentration is addedOAm
Standard items, the PSI marked with functional magnetic fluorescence probeOAmIt is fixed to establish direct competitive fluoroimmunoassay as primary antibody for antibody
Amount detection PSIOAm.The PSI marked using functional magnetic fluorescence probeOAmAntibody and PSIOdmEnvelope antigen carries out direct competitive
Fluoroimmunoassay, since direct competitive fluoroimmunoassay is to utilize anti-PSIOAmAntibody and PSIOAmEnvelope antigen is special
Property combine, achieve the purpose that quantitative detection antigen by detecting the fluorescence signal of compound of antigen-antibody, this method is than direct
The optical signal detecting antigen of competitive enzyme-linked immune method has lower detection limit, higher sensitivity.
G, with PSIOAmThe logarithm of concentration of standard solution is abscissa, and fluorescence intensity level is that ordinate establishes standard curve, is made
Standard curve as shown in Figure 1, the linear equation of standard curve is：F=11477.59-1537.63lgC, wherein F is that fluorescence is strong
Angle value, f PSIOAmConcentration, coefficient R2=0.994, the range of linearity is 5 × 10-5-1×103Ng/mL, detection are limited to
3×10-7ng/mL。
Above each step is repeated, only by the PSI of the various concentration in step f-3OAmTiter replaces with unknown concentration
PSIOAmPrepare liquid, then measured in microplate reader each hole excitation wavelength be 530nm, launch wavelength be 585nm when fluorescence
Intensity seeks average fluorescent strength value, can calculate PSI according to above-mentioned standard curveOAmThe concentration of prepare liquid.
Above method be many experiments verifying after optimal experimental method, the obtained standard curve of the method it is linear
Relationship is best, and the range of linearity is most wide.
It is above-mentioned oily to the fluorescence immunoassay absorption method quantitative detection marked based on functional magnetic fluorescence probe referring to embodiment
Amine is grafted polysuccinimide (PSIOAm) polymeric nano medicine carrier method carry out detailed description, be it is illustrative and
It is not restrictive, several embodiments can be enumerated according to limited range, therefore in the case where not departing from present general inventive concept
Change and modification, should belong within protection scope of the present invention.
Claims (6)
1. a kind of side of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label
Method, which is characterized in that the preparation method comprises the following steps：
A, PSI is preparedOAmHydrating solution；
B, PSI is preparedOAmEnvelope antigen and immunogene；
C, anti-PSIOAmThe preparation of antibody；
D, the preparation of magnetic fluorescence probe；
E, the anti-PSI of magnetic fluorescence probe labelOAmThe preparation of antibody；
F, by PSIOdmEnvelope antigen is coated in 96 orifice plates after being coated liquid dilution, closing, the PSI that various concentration is addedOAmMark
Quasi- product mark anti-PSI with functionalization magnetic fluorescence probeOAmAntibody establishes direct competitive fluorescence immunoassay determining adsorption PSIOAm；
G, with PSIOAmThe logarithm of standard concentration is abscissa, and fluorescence intensity level is that ordinate draws standard curve, thus quantitative
Detect PSIOAmConcentration.
2. preparation method according to claim 1, which is characterized in that step d specifically includes following steps：
D-1, first by the FeCl of 10.8g3·6H2The enuatrol of O and 36.5g is dissolved in 80mL ethyl alcohol, sequentially add 60mL go from
Sub- water and 140mL hexamethylene, then in 70 DEG C of heating 4h, it is to be cooled to room temperature when organic layer deionized water extract 3 times, extraction
Acquired solution mixing heats 4h at 70 DEG C and evaporates hexamethylene, is cooled to room temperature to get iron oleate, spare；
D-2, iron oleate 9g prepared by step d-1 is added in the octadecene of 50g, adds the oil of 1.4g after mixing evenly
Acid, mixed solution vacuumize after being heated to 100 DEG C of holding 30min, and mixture is heated to 320 DEG C of holding 3h under nitrogen atmosphere,
The dehydrated alcohol that 20mL is added when reacting to room temperature precipitates ferriferrous oxide nano crystalline substance, dries after washing 3 times repeatedly, i.e.,
It is brilliant to obtain ferriferrous oxide nano；
D-3, it step d-2 is prepared into ferriferrous oxide nano crystalline substance is dissolved with hexamethylene, it is brilliant to obtain 100mg/mL ferriferrous oxide nano
Solution, by 14.4mL hexamethylene, 3.3mLTX-100,0.5mLCO-520,3.3mL n-hexyl alcohol, 1.5mL deionized water and 0.25mL
Ammonium hydroxide stirs and evenly mixs, and the ferriferrous oxide nano crystalline substance solution of 1mL 100mg/mL is then added, adds the silicic acid tetrem of 50 μ L
Ester is stirred to react 2 hours, and centrifugation after having reacted is washed 3 times with dehydrated alcohol, is freeze-dried to get silica functionalization
Ferriferrous oxide nano is brilliant, spare；
The ferriferrous oxide nano crystalline substance of silica functionalization prepared by d-4, the step d-3 for weighing 6.7mg is scattered in 50mL's
In deionized water, with the pH=9.0 of the sodium hydrate regulator solution of 2M, system is heated to 70 DEG C, then sequentially adds 0.5mL
Tetraethyl orthosilicate, 0.05mL rhodamine isothiocyanates label 3- aminopropyl triethoxysilane and 3mL acetic acid second
Ester adds the 3- aminopropyl triethoxysilane of 0.05mL after being protected from light stirring 10min, stirs 3h, is cooled to room temperature rear ethyl alcohol
Washing 3 times is freeze-dried to get magnetic fluorescence probe, spare.
3. preparation method according to claim 1 or 2, which is characterized in that step e specifically includes following steps：
E-1, anti-PSI after purification is takenOAmAntibody 0.4mg is added after the acetate buffer solution of 0.07mL, and 0.14mL is added crosses iodine
Acid sodium solution, room temperature are protected from light stirring 2h；
E-2, the magnetic fluorescence probe for taking 2mg step d-4 to prepare, are washed 3 times with PBS, are sufficiently suspended in the PBS of 0.6mL, slowly
Antibody after being added step e-1 treated oxidation, stirs 20h at 4 DEG C；The sodium borohydride for adding 2mg/mL 0.02mL is molten
Liquid is protected from light 2h, to get the anti-PSI of magnetic fluorescence probe label after washing 3 times with PBSOAmAntibody, it is spare.
4. preparation method according to claim 1 or 2, which is characterized in that step f specifically includes following steps：
F-1, coating：With coating buffer by PSIOAmEnvelope antigen is diluted to 25 μ g/mL, is coated with 96 orifice plates, 100 μ L of every hole, and 4
DEG C refrigerator overnight；
F-2, closing：PBST solution washs 3 times, and drying, 3~5min, washes away unbonded PSI every timeOAmEnvelope antigen is added
1wt% casein, every 200 μ L of hole are closed, and 37 DEG C of baking ovens incubate 1~2h；
F-3, sample-adding competition：PBST solution washs 3 times, and drying, 3~5min, washes away extra confining liquid every time, then will optimization
The anti-PSI of 50 μ L magnetic fluorescence probes label afterwardsOdmThe PSI of antibody and 50 μ L various concentrationsOAmIt is each that standard items divide gradient to be added
Kong Zhong is allowed to that competitive reaction occurs, and 37 DEG C of baking ovens incubate 1~2h；
F-4, detection：PBST solution washs 3 times, and drying, 3~5min, removes the PSI of free state every timeOAmStandard items or antibody knot
Object is closed, measuring each hole in excitation wavelength with multi-function microplate reader is 530nm, and launch wavelength is the fluorescence intensity level at 585nm.
5. preparation method according to claim 1-4, which is characterized in that the line of standard curve described in step g
Property equation be F=11477.59-1537.63lgC, wherein F be fluorescence intensity level, C PSIOAmConcentration.
6. preparation method according to claim 5, which is characterized in that its coefficient R2=0.994, the range of linearity be 5 ×
10-5-5×103Ng/mL, detection are limited to 3 × 10-7ng/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810459487.8A CN108918854B (en) | 2018-05-15 | 2018-05-15 | Method for determining ultra-trace macromolecular nano-drug carrier based on fluorescence immunoadsorption marked by magnetic fluorescent probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810459487.8A CN108918854B (en) | 2018-05-15 | 2018-05-15 | Method for determining ultra-trace macromolecular nano-drug carrier based on fluorescence immunoadsorption marked by magnetic fluorescent probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108918854A true CN108918854A (en) | 2018-11-30 |
| CN108918854B CN108918854B (en) | 2022-04-19 |
Family
ID=64403337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810459487.8A Active CN108918854B (en) | 2018-05-15 | 2018-05-15 | Method for determining ultra-trace macromolecular nano-drug carrier based on fluorescence immunoadsorption marked by magnetic fluorescent probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108918854B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568184A (en) * | 2019-08-21 | 2019-12-13 | 安徽师范大学 | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier |
| CN115433464A (en) * | 2022-08-31 | 2022-12-06 | 上海旦元新材料科技有限公司 | Preparation method of interconnected non-close-packed binary superstructure |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106706894A (en) * | 2017-01-20 | 2017-05-24 | 安徽师范大学 | Method for quantitatively detecting oleylamine grafted polysuccinimide macromolecule nanometer drug carrier based on indirect competitive enzyme-linked immunosorbent assay |
| CN107091826A (en) * | 2017-06-24 | 2017-08-25 | 安徽师范大学 | A kind of method that PSI OAm NAPI amphiphilic polymer/nanometer pharmaceutical carriers are quantitatively detected based on trace fluorescence immunoassay |
| CN107228938A (en) * | 2017-06-24 | 2017-10-03 | 安徽师范大学 | A kind of quantitative detecting method of folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
| CN107238710A (en) * | 2017-06-24 | 2017-10-10 | 安徽师范大学 | A kind of method that folic acid functionalized SiO 2 targeted nano pharmaceutical carrier is detected based on direct competitive fluorescence immunoassay standard measure |
| CN107727844A (en) * | 2017-11-18 | 2018-02-23 | 安徽师范大学 | A kind of method that fluorescence immunoassay quantitatively detects MFAP nano-medicament carriers |
| CN107782893A (en) * | 2017-11-18 | 2018-03-09 | 安徽师范大学 | A kind of method for detecting folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
| CN107957492A (en) * | 2017-11-18 | 2018-04-24 | 安徽师范大学 | A kind of method of the fluorescence immunoassay determining adsorption ultra trace oleyl amine scion grafting polysuccinimide based on carbon dots mark |
| CN107976538A (en) * | 2017-11-18 | 2018-05-01 | 安徽师范大学 | A kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, preparation method and application |
-
2018
- 2018-05-15 CN CN201810459487.8A patent/CN108918854B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106706894A (en) * | 2017-01-20 | 2017-05-24 | 安徽师范大学 | Method for quantitatively detecting oleylamine grafted polysuccinimide macromolecule nanometer drug carrier based on indirect competitive enzyme-linked immunosorbent assay |
| CN107091826A (en) * | 2017-06-24 | 2017-08-25 | 安徽师范大学 | A kind of method that PSI OAm NAPI amphiphilic polymer/nanometer pharmaceutical carriers are quantitatively detected based on trace fluorescence immunoassay |
| CN107228938A (en) * | 2017-06-24 | 2017-10-03 | 安徽师范大学 | A kind of quantitative detecting method of folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
| CN107238710A (en) * | 2017-06-24 | 2017-10-10 | 安徽师范大学 | A kind of method that folic acid functionalized SiO 2 targeted nano pharmaceutical carrier is detected based on direct competitive fluorescence immunoassay standard measure |
| CN107727844A (en) * | 2017-11-18 | 2018-02-23 | 安徽师范大学 | A kind of method that fluorescence immunoassay quantitatively detects MFAP nano-medicament carriers |
| CN107782893A (en) * | 2017-11-18 | 2018-03-09 | 安徽师范大学 | A kind of method for detecting folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
| CN107957492A (en) * | 2017-11-18 | 2018-04-24 | 安徽师范大学 | A kind of method of the fluorescence immunoassay determining adsorption ultra trace oleyl amine scion grafting polysuccinimide based on carbon dots mark |
| CN107976538A (en) * | 2017-11-18 | 2018-05-01 | 安徽师范大学 | A kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| GUO ZHANG，ET AL，: "Fluorescent magnetic nanoprobes：Design and application for cell imaging", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》 * |
| JONGNAM PARK,ET AL,: "Ultra-large-scale syntheses of monodisperse nanocrystals", 《NATURE MATERIALS》 * |
| 谭贵良，赖心田: "《现代分子生物学及组学技术在食品安全检测中的应用》", 31 December 2014 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568184A (en) * | 2019-08-21 | 2019-12-13 | 安徽师范大学 | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier |
| CN110568184B (en) * | 2019-08-21 | 2022-11-22 | 安徽师范大学 | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier |
| CN115433464A (en) * | 2022-08-31 | 2022-12-06 | 上海旦元新材料科技有限公司 | Preparation method of interconnected non-close-packed binary superstructure |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108918854B (en) | 2022-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| CN106706894B (en) | A kind of method that oleyl amine scion grafting polysuccinimide polymeric nano medicine carrier is quantitatively detected based on Indirect cELISA | |
| CN110095608A (en) | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly | |
| CN108508215A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application of detection tetracycline medication | |
| CN107091826B (en) | A method of based on trace fluorescence immunoassay quantitative detection PSI-OAm-NAPI amphiphilic polymer/nanometer pharmaceutical carrier | |
| CN107976538A (en) | A kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, preparation method and application | |
| CN108918854A (en) | A method of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label | |
| CN105181958B (en) | A kind of method that 80nmAuNPs is quantitatively detected based on Indirect cELISA | |
| CN107238710B (en) | A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier | |
| CN107782893B (en) | A method of detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier | |
| CN107957492A (en) | A kind of method of the fluorescence immunoassay determining adsorption ultra trace oleyl amine scion grafting polysuccinimide based on carbon dots mark | |
| CN105131121A (en) | Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit | |
| CN101173007B (en) | 1-amido-glycolyurea immunogen, producing method and application of the same | |
| CN107727844A (en) | A kind of method that fluorescence immunoassay quantitatively detects MFAP nano-medicament carriers | |
| CN107228938B (en) | A kind of quantitative detecting method of folic acid functionalized SiO 2 targeted nano pharmaceutical carrier | |
| CN110568184B (en) | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier | |
| CN108982863B (en) | Immunochromatographic test paper for detecting imidacloprid | |
| CN105181956B (en) | Application of the fluorescence detection specifically responded based on metal ion in immune detection | |
| CN104777317B (en) | The preparation of a kind of gold nanoparticle probe and the application in tachysynthesis detects thereof | |
| CN106841595B (en) | Quantitative detection method for oxidized glucan coated ferroferric oxide magnetic nano material | |
| CN109839500A (en) | A kind of label-free fluorescence enzyme-linked immune analytic method based on fluorescence gold nanoclusters | |
| CN104459119B (en) | A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof | |
| CN107102132A (en) | Quantum dot fluorescence probe and preparation method thereof, immune chromatography test paper | |
| CN112240930B (en) | Homogeneous chemiluminescence analysis method and application thereof | |
| CN107328929B (en) | A kind of quantitative detecting method of PSI-OAm-NAPI amphiphilic polymer/nanometer material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |