CN107238710A - A kind of method that folic acid functionalized SiO 2 targeted nano pharmaceutical carrier is detected based on direct competitive fluorescence immunoassay standard measure - Google Patents
A kind of method that folic acid functionalized SiO 2 targeted nano pharmaceutical carrier is detected based on direct competitive fluorescence immunoassay standard measure Download PDFInfo
- Publication number
- CN107238710A CN107238710A CN201710489337.7A CN201710489337A CN107238710A CN 107238710 A CN107238710 A CN 107238710A CN 201710489337 A CN201710489337 A CN 201710489337A CN 107238710 A CN107238710 A CN 107238710A
- Authority
- CN
- China
- Prior art keywords
- sio
- solution
- antibody
- pbs
- dissolved
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
- G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Optics & Photonics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of method that folic acid functionalized SiO 2 targeted nano pharmaceutical carrier is detected based on direct competitive fluorescence immunoassay standard measure, with reference to antigen, antibody response specificity with the sensitiveness of fluorescence to SiO2FO nano-particles have carried out quantitative detecting analysis.Step includes：Prepare SiO2FO envelope antigens and immunogene, will obtain antibody with high specificity in immunogen injection to animal body, then antibody on FITC marks is made into fluorescence antibody, then set up direct competitive fluorescent immune method Accurate Determining SiO2FO content.Detection method is simple to operate, sensitivity is high, high specificity, achievable high flux are detected.
Description
Technical field
The present invention relates to the quantitative detection of nano material, and in particular to one kind is based on the inspection of direct competitive fluorescence immunoassay standard measure
Survey folic acid functionalized SiO 2 (SiO2- FO) targeted nano pharmaceutical carrier method.
Background technology
It is more particularly to a kind of based on the inspection of direct competitive fluorescence immunoassay standard measure the present invention relates to the quantitative detection of nano material
Survey folic acid functionalized SiO 2 (SiO2- FO) targeted nano pharmaceutical carrier method.
Background technology
Cancer is to seriously endanger one of disease of human health, and treatment of cancer is for improving the life quality of patient, mitigating
The pain of patient, reduction death rate tool are of great significance.Treatment in clinical at present for cancer mainly uses chemotherapy
Method, but current chemotherapeutics does not have targeting, and normal cell can be also killed while cancer cell is killed, so as to cause tight
The toxic side effect of weight, hinders the development and application of chemotherapeutics, patient's body and mind is caused greatly to damage.Therefore, medicine is reduced
Thing toxic side effect, improvement drug distribution and metabolism, raising drug therapy efficiency etc. have become grinding for biomedical sector
Study carefully one of focus.
With the fast development and application of nano material, increasing researchers have turned one's attention to Nano medication load
Body.Drug molecule parcel wherein or can be adsorbed on its surface in 10~500nm, pass through targeting point by nano-carrier particle size
Son is combined or magnetic targeted with cell surface specific receptor, is entered intracellular under cellular uptake effect, is realized safely and effectively
Targeted drug is conveyed, therefore with special value and significance in drug delivery.
Functionalized nano material is showed in terms of targeting administration, slow releasing pharmaceutical, the toxic side effect of reduction medicine is realized
Go out good application prospect, it has also become the focus of newtype drug induction system research in recent years.Silica nano material conduct
One of important member of inorganic nanoparticles material, because its preparation is easy, property is stable, good biocompatibility and surface are easily repaiied
Decorations, important function has been played in biomedical sector.One will be used as on targeting ligand modified with folic acid to silica nano material
Novel targeted pharmaceutical carrier is planted, is that the treatment of cancer is filled with new vitality, is provided to improve antineoplastic medicine-carried system
New thinking and means, important function has been played in biomedical sector.
Nanometer biotechnology is forward position and the hot issue of biological technical field, from the point of view of existing result of study, for receiving
Rice materials application is typically limited to investigate its distribution, degraded, insoluble drug release efficiency and toxicity in vivo and ground in medical domain
In terms of studying carefully.It is well known that nano material may have a certain impact because of its special effect to human body, different dosage may
Effect different to body generation or side effect.Therefore, whether the dose measurement of nano-medicament carrier may be used for investigating the material
To be particularly important applied to clinic.And it is concentrated mainly on its structure, composition, grain for the characterizing method of nano material at present
The analysis such as degree, pattern and interface.And the quantitative analysis for nano material is not also a lot, conventional method has inductance at present
Coupled plasma mass (ICP-MS), High performance liquid chromatography-inductively coupled plasma mass spectrometry method (HPLC-ICP-MS) with
And gas-chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS), but these methods are not only cumbersome, instrument is held high
It is expensive, and stability and sensitivity is not high, and detection technique is still not mature enough.Therefore, for nano-carrier in itself in organism
Interior quantitative detection is still a very necessary and challenging job.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of based on direct competitive fluorescence immunoassay standard measure detection folic acid work(
Silica (SiO can be changed2- FO) targeted nano pharmaceutical carrier method.With reference to antigen, the specificity of antibody response and fluorescence
Sensitiveness is to SiO2- FO nano-particles have carried out quantitative detecting analysis.This method has that simple to operate, sensitivity is high, specificity
By force, the features such as high flux is detected can be achieved.
The technical scheme that the present invention takes is：
One kind is based on direct competitive fluorescence immunoassay standard measure detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano
The method of pharmaceutical carrier, the described method comprises the following steps：
a、SiO2The preparation of-FO envelope antigens and immunogene；
B, anti-SiO2The preparation of-FO antibody；
C, fluorescence antibody preparation；
D, by SiO2It is coated in after the coated liquid dilution of-FO envelope antigens in ELISA Plate, closes, adds various concentrations
SiO2- FO standard items, the anti-SiO marked with FITC2- FO antibody is set up direct competitive fluoroimmunoassay and quantitatively examined as primary antibody
Survey SiO2-FO；
E, with SiO2The logarithm of-FO standard concentrations is abscissa, and fluorescence intensity level is that ordinate draws standard curve, from
And quantitatively detect SiO2- FO concentration.
The linear equation of the standard curve is A=12434.4-1136.7logC, and wherein A is fluorescence intensity level, and C is
SiO2- FO concentration.Its coefficient R=- 0.9992, the range of linearity is 10-1-105Ng/mL, detection is limited to 0.035ng/mL.
The step a specifically includes following steps：
A-1, by SiO2- FO is dissolved in PBS solution, obtains water miscible SiO2- FO solution；
A-2, OVA is dissolved in PBS solution, then adds the SiO that step a-1 is obtained2- FO solution and glutaraldehyde solution,
Lucifuge stirs 2~5h at 25~28 DEG C；Place reaction liquid into bag filter the 24h that dialyses and can obtain SiO2- FO envelope antigens；
A-3, BSA is dissolved in PBS solution, then adds the SiO that step a-1 is obtained2- FO solution and glutaraldehyde solution,
Lucifuge stirs 2~5h at 25~28 DEG C；Place reaction liquid into bag filter the 24h that dialyses and can obtain SiO2- FO immunogenes.
Further, the step a specifically includes following steps：
A-1, weigh 10~20mg SiO2- FO is dissolved in 1~2mL PBS solutions, and 1~2h of magnetic agitation is to obtain water-soluble
The SiO of property2- FO solution；
A-2, weigh 10~20mg OVA and be dissolved in 2~4mL PBS solutions, and add what step a-1 was obtained under agitation
SiO2- FO solution, is added dropwise lucifuge at the glutaraldehyde solutions of 80~200 μ L 25%, 25~28 DEG C and stirs 2~5h again after being well mixed；
Place reaction liquid into bag filter, SiO is can obtain with being collected after PBS 24h2- FO envelope antigens, its concentration be 2~
4mg/mL；
A-3, weigh 10~20mg BSA and be dissolved in 2~4mL PBS solutions, and add what step a-1 was obtained under agitation
SiO2- FO solution, is added dropwise lucifuge at the glutaraldehyde solutions of 80~200 μ L 25%, 25~28 DEG C and stirs 2~5h again after being well mixed；
Place reaction liquid into bag filter, SiO is can obtain with being collected after PBS 24h2- FO immunogenes, its concentration is 2~4mg/
mL。
In the step a-2 and a-3, the molecular cut off of bag filter is 8000~14000Da.
The water miscible SiO2- FO solution concentrations are 5~15mg/mL.
The step b specifically includes following steps：
B-1, first immunisation：By SiO2- FO immunogenes and Freund's complete adjuvant in equal volume than mixing after, using dorsal sc
The mode of multi-point injection is expelled in White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；After first immunisation three weeks
Carry out booster immunization；
B-2, booster immunization：By SiO2- FO immunogenes and incomplete Freund's adjuvant in equal volume than mixing after, take same
Mode is expelled in White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；Hereafter strengthened exempting from again every two weeks
Serum titer is surveyed in epidemic disease, middle Zhou Jinhang ear veins blood sampling, until potency reaches 1:64000, then carry out last time booster immunization,
And from the blood sampling of animal arteria carotis after immune one week, stand and separate out antiserum and purified, obtain anti-SiO2- FO antibody, its
Concentration is 15~20mg/mL.
The step c specifically includes following steps：
C-1, take anti-SiO after purification2- FO antibody, be diluted to carbonate buffer solution every milliliter of inner protein 8~
15mg, is made antibody-solutions；
C-2, fluorescein isothiocynate (FITC) is dissolved in carbonate buffer solution, the FITC that concentration is 2mg/mL is made
Solution；
C-3, at 25~28 DEG C FITC solution is slowly added in the antibody-solutions that step c-1 is obtained, magnetic agitation 4~
6h；
C-4, the solution for obtaining step c-3 carry out Purification by filtration with sephadex, obtain the anti-SiO of FITC marks2-
FO antibody.
In the step c-3, the ratio between volume of FITC solution and antibody-solutions is 1:10.
The step d specifically includes following steps：
D-1, coating：With coating buffer solution by SiO2- FO envelope antigens dilute 80 times, are coated with 96 hole elisa Plates, per hole 100
μ L, 4 DEG C of refrigerator overnights；
D-2, closing：Dry, PBST solution is washed 3 times, and 3~5min, washes away the SiO not being combined every time2- FO is coated with
Antigen, adds 1wt% caseins, is closed per the μ L of hole 200, and 37 DEG C of baking ovens incubate 1~2h；
D-3, sample-adding competition：Dry, PBST solution is washed 3 times, and 3~5min, washes away unnecessary confining liquid every time, then will
The SiO of 50 μ L various concentrations2The anti-SiO of-FO standard items and 50 μ L FITC marks2- FO antibody point gradients are added in each hole, are allowed to
Generation competitive reaction, 37 DEG C of baking ovens incubate 2~4h；
D-4, detection：Dry, PBST solution is washed 3 times, and 3~5min, removes the SiO of free state every time2- FO standard items or
Antibody conjugates, it is 485nm that each hole is determined with multi-function microplate reader in excitation wavelength, and launch wavelength is that the fluorescence at 528nm is strong
Angle value.
Folic acid functionalized SiO 2 is detected based on direct competitive fluorescence immunoassay standard measure the invention provides one kind
(SiO2- FO) targeted nano pharmaceutical carrier method, realized with reference to antigen, the specificity of antibody response and the sensitiveness of fluorescence
The immunization method is to SiO2- FO quantitative detection.
Compared with prior art, the present invention has following feature：
(1) using the immune response effect of body, successfully prepare and filtered out the anti-SiO of high-titer2- FO antibody, be
Set up the Immunoassay quantification detection SiO of high specific2- FO lays a good foundation；
(2) established using the sensitiveness of antigen, the specificity of antibody response and fluorescence a kind of based on direct competitive fluorescence
Immunoassay standard measure detects SiO2- FO new method, is provided a kind of quick, accurate quantitative analysis detection for nano material from now on
Method；
(3) this method is simple to operate, sensitivity is high, high specificity, achievable high flux are detected.
Brief description of the drawings
Fig. 1 is with SiO2The logarithm of-FO standard concentrations is abscissa, and fluorescence intensity level is that the standard that ordinate is set up is bent
Line chart.
Embodiment
Freund's complete adjuvant, bovine serum albumin(BSA) (BSA) and chicken ovalbumin (OVA) buy spontaneous work bioengineering
(Shanghai) limited company.
Other reagents can be from the market sale producer be commercially available.
The present invention relates to the preparation method of each solution be：
PBS solution (0.01mol/L pH=8.0)：Weigh NaCl 8.0g, KCl 0.1g, NaH2PO4·2H200.106g、
Na2HPO4·12H2O 3.34g are dissolved in distilled water and are settled to 1000mL.
Carbonate buffer solution CB (0.5mol/L pH=9.6)：Weigh Na2CO3 1.59g、NaHCO32.94g it is dissolved in steaming
In distilled water and it is settled to 100mL.
PBST solution (0.01mol/L pH=8.0)：500 μ L Tween-20 are added in 1000mL PBS, mixing is equal
It is even.
It is coated with buffer solution CB (0.05mol/L pH=9.6)：Weigh Na2CO3 1.59g、NaHCO32.94g it is dissolved in steaming
In distilled water and it is settled to 1000mL.
1wt% casein solutions：It is confining liquid, weighs 0.01g caseins and is dissolved in 1mL PBS, is well mixed.
MES buffer solutions (0.1M, pH=5.5)：Claim 0.1921g MES to be dissolved in 10mL distilled water, adjusted with NaOH solution
Save its pH to 5.5.
Embodiment 1
One kind is based on direct competitive fluorescence immunoassay standard measure detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano
The method of pharmaceutical carrier, the described method comprises the following steps：
a、SiO2The preparation of-FO envelope antigens and immunogene
A-1, weigh 10mg SiO2- FO is dissolved in 1mL PBS solutions, and magnetic agitation 1h is to obtain concentration for 10mg/mL
SiO2- FO solution；
A-2, weigh 10mg OVA and be dissolved in 2mL PBS, and the SiO that step a-1 is obtained is added in stirring2- FO solution, is mixed
Lucifuge at the glutaraldehyde solutions of 90 μ L 25%, 25 DEG C is added dropwise after closing uniformly again and stirs 4h；Placing reaction liquid into molecular cut off is
In 8000-12000Da bag filter, SiO is can obtain with being collected after PBS 24h2- FO envelope antigens, its concentration is
3.33mg/mL, 4 DEG C of storages are stand-by；
A-3, weigh 10mg BSA and be dissolved in 2mL PBS, and the SiO that step a-1 is obtained is added in stirring2- FO solution, is mixed
Lucifuge at the glutaraldehyde solutions of 90 μ L 25%, 25 DEG C is added dropwise after closing uniformly again and stirs 4h；Placing reaction liquid into molecular cut off is
In 8000-12000Da bag filters, SiO is can obtain with being collected after PBS 24h2- FO immunogenes, its concentration is 3.33mg/
ML, 4 DEG C of storages are stand-by.
SiO in the step a-12- FO (folic acid functionalized SiO 2) preparation method is：
A-1-1, amino functional silica (SiO2–NH2) preparation
380 μ L tetraethyl orthosilicates and 12mL absolute ethyl alcohols are mixed into 0.5h；Add at the ammoniacal liquor of 570 μ L 26%, 25 DEG C
Stir 24h；Add at 400 μ L 3- aminopropyl triethoxysilanes, 25 DEG C and persistently stir 24h；By reaction solution 10000rpm/
Min centrifuges 20min, takes precipitation to be washed with absolute ethyl alcohol, and it is that can obtain amino functional dioxy to dry 24h at 25 DEG C
SiClx；
A-1-2, folic acid functionalized SiO 2 (SiO2- FO) preparation
Weigh 57.5mg folic acid and be dissolved in 2mL MES buffer solutions, add 40.3mg 1- ethyls-(3- dimethylaminos third
Base) phosphinylidyne diimmonium salt hydrochlorate (EDC) and 17.25mg n-hydroxysuccinimides, stir 1h at room temperature；
Weigh 100mg SiO2–NH2It is added to after being dissolved in 5mL MES buffer solutions in above-mentioned mixed liquor, lucifuge is stirred at 25 DEG C
24h；Reaction solution 10000rpm/min is centrifuged into 10min, takes precipitation to be washed with distilled water and absolute ethyl alcohol, and at 25 DEG C
It is that can obtain folic acid functionalized SiO 2 to dry 24h.
B, anti-SiO2The preparation of-FO antibody
The Male New Zealand White Rabbit for being 2~2.5kg from 4 body weight is immunization, first will purchase before experiment
New zealand white rabbit raise 2 weeks or so, maintain its health status, wherein 3 are only used as immunization, the 4th is only used as blank pair
According to blank control group is without any immune.
B-1, first immunisation：By SiO2- FO immunogenes and Freund's complete adjuvant in equal volume than mixing after, using dorsal sc
The mode of multi-point injection is expelled in 3 experimental rabbit bodies, injects 8~10 points, and injection volume is 1mL/；The mode of immunity inoculation
There are injecting immune, oral immunity, aerosol immunization etc., wherein injecting immune has hypodermic injection, intracutaneous injection, intramuscular injection vein again
The modes such as injection, dorsal sc injection is easy to operate, and drug diffusion is slower, is conducive to stimulating body to produce immune response, then
Produce antibody；And male rabbit can avoid its physiological period from producing influence to experiment as immunization.
B-2, booster immunization：First immunisation carries out booster immunization after three weeks.By SiO2- FO immunogenes are not exclusively helped with Freund
Agent in equal volume than mixing after, take same mode to be expelled in 3 experimental rabbit bodies, inject 8~10 points, injection volume is 1mL/
Only；Hereafter booster immunization again is carried out every two weeks, and serum titer is surveyed in middle Zhou Jinhang ear veins blood sampling, until potency reaches 1:
64000, then last time booster immunization is carried out, and stand from the blood sampling of animal arteria carotis after immune one week and separate out antiserum simultaneously
Purified, obtain anti-SiO2- FO antibody, its concentration is 16.55mg/mL.
The preparation method of incomplete Freund's adjuvant is：50g wool grease is weighed, the atoleine for measuring 100mL is mixed
Close, Ultrasound Instrument repeated ultrasonic, every time no more than 20min, prevents that temperature is too high in ultrasonic procedure, it is impossible to which radiating in time, ultrasound makes
Well mixed, until the mixing liquid is dripped in water and in half a minute untill indiffusion, obtained oily liquids is
Incomplete Freund's adjuvant, 4 DEG C of refrigerator storages are stand-by.
The specific method that antibody titer is identified is：Envelope antigen is diluted 400 times with coating buffer solution CB, 96 holes are coated on
On ELISA Plate, 4 DEG C of refrigerator overnights；Dry, washed with PBST solution 3 times, each 3min, add 200 μ L 1wt% caseins to seal per hole
Close liquid, 37 DEG C of closing 1h；Dry, PBST is washed 3 times, each 3min, 100 μ L are added per hole the anti-of various concentrations is diluted to PBS
Serum, its thinner ratio is 1/1000~1/128000,37 DEG C of incubation 2h；Dry, PBST is washed 3 times, each 3min, added per hole
The thinner ratio that 100 μ L PBS dilute is the goat anti-rabbit igg of 1/5000 HRP marks, 37 DEG C of incubation 2h；Dry, PBST is washed 3 times,
Each 3min, 100 μ L o-phenylene diamine substrates liquid are added per hole and carry out chromogenic reaction, 37 DEG C of incubation 0.5h；50 μ are added per hole
L2mol/L H2SO4Terminating reaction；Finally absorbance A of each hole at 490nm is determined with multi-function microplate reader.Comparative experiments
Light absorption value A of the group with blank control group under same antiserum extension rate, works as AExperimental group>=2 times of ABlank groupWhen corresponding maximum dilution
Multiple is sero-fast potency.
C, fluorescence antibody preparation
C-1, take anti-SiO after purification2- FO antibody 1mL, every milliliter of inner protein is diluted to carbonate buffer solution
8.5mg, is made antibody-solutions；
C-2, weigh 2mg fluorescein isothiocynates (FITC) and be dissolved in 1mL carbonate buffer solutions, FITC solution is made；
C-3, at 25 DEG C 0.1mLFITC solution is slowly added in 1mL antibody-solutions, magnetic agitation 5h；
C-4, the good solution of above-mentioned mark carries out to Purification by filtration with sephadex, collect post lower end solution, obtain
The anti-SiO of FITC marks2- FO antibody, -20 DEG C of storages are stand-by.
D, utilize the anti-SiO of the fluorescence marked2- FO antibody and SiO2- FO envelope antigens carry out direct competitive fluorescence immunoassay point
Analysis experiment, sets up standard curve to reach quantitative detection SiO in optimal conditions2- FO purpose
D-1, coating：With coating buffer solution by SiO2- FO envelope antigens dilute 80 times, are coated with 96 hole elisa Plates.Per hole 100
μ L, 4 DEG C of refrigerator overnights；
D-2, closing：Dry, PBST solution is washed 3 times, and each 3min washes away the SiO not being combined2- FO coatings are anti-
It is former.1wt% caseins are added, are closed (reduction non-specific adsorption) per the μ L of hole 200,37 DEG C of baking ovens incubate 1h；
D-3, sample-adding competition：Dry, PBST solution is washed 3 times, and each 3min washes away unnecessary confining liquid.50 μ L are dense
Degree is respectively 0.1ng/mL, 1ng/mL, 10ng/mL, 102ng/mL、103ng/mL、104ng/mL、105Ng/mL SiO2- FO is marked
Quasi- product are added sequentially in each row of ELISA Plate, i.e., each concentration gradient adds 50 μ L FITC into each hole in triplicate, then
The anti-SiO of mark2- FO antibody, is allowed to occur competitive reaction, and 37 DEG C of baking ovens incubate 3h；
The SiO of various concentrations2The preparation method of-FO standard items is：With 0.01mol/L pH=8.0 PBS cushioning liquid
By SiO2- FO is diluted to the concentration specified.
D-4, detection：Dry, PBST solution is washed 3 times, each 3min, remove the SiO of free state2- FO standard items are anti-
Body conjugate.It is 485nm that each hole is determined with multi-function microplate reader in excitation wavelength, and launch wavelength is the fluorescence intensity at 528nm
Value, the calculating fluorescence intensity level of averaging of same concentration gradient.
E, with SiO2The logarithm of-FO standard concentrations is abscissa, and fluorescence intensity level is that ordinate draws standard curve.System
The standard curve obtained is as shown in figure 1, the linear equation of standard curve is A=12434.4-1136.7logC, and wherein A is that fluorescence is strong
Angle value, C is SiO2- FO concentration.Its coefficient R=- 0.9992, the range of linearity is 10-1-105Ng/mL, detection is limited to
0.035ng/mL。
More than f, repetition each step, simply by the SiO of the various concentrations in step (3)2- FO standard items replace with unknown dense
The SiO of degree2- FO prepare liquids, it is 485nm in excitation wavelength then to determine each hole with multi-function microplate reader, and launch wavelength is 528nm
The fluorescence intensity level at place, asks for average fluorescent strength value.According to the linear equation A=12434.4- of above-mentioned standard curve
1136.7logC, you can calculate SiO2The concentration of-FO prepare liquids.
Above method is the optimal experimental method after many experiments are verified, the standard curve obtained by the method it is linear
Preferably, the range of linearity is most wide for relation.
It is above-mentioned that folic acid functionalized SiO 2 is detected based on direct competitive fluorescence immunoassay standard measure to one kind with reference to embodiment
The detailed description that the method for targeted nano pharmaceutical carrier is carried out, be it is illustrative rather than limited, can be according to limited model
Enclose and include several embodiments, therefore changing and modifications in the case where not departing from present general inventive concept, the guarantor of the present invention should be belonged to
Within the scope of shield.
Claims (10)
1. one kind is based on direct competitive fluorescence immunoassay standard measure detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano medicine
The method of thing carrier, it is characterised in that the described method comprises the following steps：
a、SiO2The preparation of-FO envelope antigens and immunogene；
B, anti-SiO2The preparation of-FO antibody；
C, fluorescence antibody preparation；
D, by SiO2It is coated in after the coated liquid dilution of-FO envelope antigens in ELISA Plate, closing, the SiO for adding various concentrations2-FO
Standard items, the anti-SiO marked with FITC2- FO antibody sets up direct competitive fluoroimmunoassay and quantitatively detects SiO as primary antibody2-
FO；
E, with SiO2The logarithm of-FO standard concentrations is abscissa, and fluorescence intensity level is that ordinate draws standard curve, depending on
Amount detects SiO2- FO concentration.
2. according to the method described in claim 1, it is characterised in that the linear equation of the standard curve is A=12434.4-
1136.7logC, wherein A are fluorescence intensity level, and C is SiO2- FO concentration.
3. according to the method described in claim 1, it is characterised in that the step a specifically includes following steps：
A-1, by SiO2- FO is dissolved in PBS solution, obtains water miscible SiO2- FO solution；
A-2, OVA is dissolved in PBS solution, then adds the SiO that step a-1 is obtained2- FO solution and glutaraldehyde solution, 25~28
Lucifuge stirs 2~5h at DEG C；Place reaction liquid into bag filter the 24h that dialyses and can obtain SiO2- FO envelope antigens；
A-3, BSA is dissolved in PBS solution, then adds the SiO that step a-1 is obtained2- FO solution and glutaraldehyde solution, 25~28
Lucifuge stirs 2~5h at DEG C；Place reaction liquid into bag filter the 24h that dialyses and can obtain SiO2- FO immunogenes.
4. the method according to claim 1 or 3, it is characterised in that the step a specifically includes following steps：
A-1, weigh 10~20mg SiO2- FO is dissolved in 1~2mL PBS solutions, and 1~2h of magnetic agitation is to obtain water miscible
SiO2- FO solution；
A-2, weigh 10~20mg OVA and be dissolved in 2~4mL PBS solutions, and add the SiO that step a-1 is obtained under agitation2-
FO solution, is added dropwise lucifuge at the glutaraldehyde solutions of 80~200 μ L 25%, 25~28 DEG C and stirs 2~5h again after being well mixed；Will be anti-
Answer liquid to be placed in bag filter, SiO is can obtain with being collected after PBS 24h2- FO envelope antigens, its concentration is 2~4mg/mL；
A-3, weigh 10~20mg BSA and be dissolved in 2~4mL PBS solutions, and add the SiO that step a-1 is obtained under agitation2-
FO solution, is added dropwise lucifuge at the glutaraldehyde solutions of 80~200 μ L 25%, 25~28 DEG C and stirs 2~5h again after being well mixed；Will be anti-
Answer liquid to be placed in bag filter, SiO is can obtain with being collected after PBS 24h2- FO immunogenes, its concentration is 2~4mg/mL.
5. the method according to claim 3 or 4, it is characterised in that the molecular cut off of bag filter is 8000~
14000Da。
6. method according to claim 4, it is characterised in that water miscible SiO2- FO solution concentrations are 5~15mg/mL.
7. according to the method described in claim 1, it is characterised in that the step b specifically includes following steps：
B-1, first immunisation：By SiO2- FO immunogenes and Freund's complete adjuvant in equal volume than mixing after, using dorsal sc multiple spot
The mode of injection is expelled in White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；First immunisation is carried out after three weeks
Booster immunization；
B-2, booster immunization：By SiO2- FO immunogenes and incomplete Freund's adjuvant in equal volume than mixing after, take same mode
It is expelled in White Rabbit body, injects 8~10 points, injection volume is 1~2mL/；Hereafter booster immunization again is carried out every two weeks,
Serum titer is surveyed in middle Zhou Jinhang ear veins blood sampling, until potency reaches 1:64000, then last time booster immunization is carried out, and
From the blood sampling of animal arteria carotis after immune one week, stand and separate out antiserum and purified, obtain anti-SiO2- FO antibody, its is dense
Spend for 15~20mg/mL.
8. according to the method described in claim 1, it is characterised in that the step c specifically includes following steps：
C-1, take anti-SiO after purification2- FO antibody, every milliliter of inner protein is diluted to carbonate buffer solution
8~15mg, is made antibody-solutions；
C-2, fluorescein isothiocynate (FITC) is dissolved in carbonate buffer solution, the FITC that concentration is 2mg/mL is made molten
Liquid；
C-3, at 25~28 DEG C FITC solution is slowly added in the antibody-solutions that step c-1 is obtained, 4~6h of magnetic agitation；
4~6h of magnetic agitation；
C-4, the solution for obtaining step c-3 carry out Purification by filtration with sephadex, obtain the anti-SiO of FITC marks2- FO resists
Body.
9. the method according to claim 7 or 8, it is characterised in that in the step c-3, FITC solution and antibody-solutions
The ratio between volume be 1:10.
10. according to the method described in claim 1, it is characterised in that the step d specifically includes following steps：
D-1, coating：With coating buffer solution by SiO2- FO envelope antigens dilute 80 times, are coated with 96 hole elisa Plates, per the μ L of hole 100,4
DEG C refrigerator overnight；
D-2, closing：Dry, PBST solution is washed 3 times, and 3~5min, washes away the SiO not being combined every time2- FO envelope antigens,
1wt% caseins are added, are closed per the μ L of hole 200,37 DEG C of baking ovens incubate 1~2h；
D-3, sample-adding competition：Dry, PBST solution is washed 3 times, and 3~5min, washes away unnecessary confining liquid every time, then by 50 μ L
The SiO of various concentrations2The anti-SiO of-FO standard items and 50 μ L FITC marks2- FO antibody point gradients are added in each hole, are allowed to occur
Competitive reaction, 37 DEG C of baking ovens incubate 2~4h；
D-4, detection：Dry, PBST solution is washed 3 times, and 3~5min, removes the SiO of free state every time2- FO standard items or antibody
Conjugate, it is 485nm that each hole is determined with multi-function microplate reader in excitation wavelength, and launch wavelength is the fluorescence intensity level at 528nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710489337.7A CN107238710B (en) | 2017-06-24 | 2017-06-24 | A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710489337.7A CN107238710B (en) | 2017-06-24 | 2017-06-24 | A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107238710A true CN107238710A (en) | 2017-10-10 |
| CN107238710B CN107238710B (en) | 2019-07-19 |
Family
ID=59987152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710489337.7A Active CN107238710B (en) | 2017-06-24 | 2017-06-24 | A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107238710B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108918854A (en) * | 2018-05-15 | 2018-11-30 | 安徽师范大学 | A method of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104817639A (en) * | 2015-05-08 | 2015-08-05 | 河北大学 | Preparation method and application of hexamethylenetetramine complete antigen |
| 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 |
| CN106841595A (en) * | 2017-01-20 | 2017-06-13 | 安徽师范大学 | A kind of quantitative detecting method of the coated ferroferric oxide magnetic nano-material of oxidized dextran |
-
2017
- 2017-06-24 CN CN201710489337.7A patent/CN107238710B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104817639A (en) * | 2015-05-08 | 2015-08-05 | 河北大学 | Preparation method and application of hexamethylenetetramine complete antigen |
| 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 |
| CN106841595A (en) * | 2017-01-20 | 2017-06-13 | 安徽师范大学 | A kind of quantitative detecting method of the coated ferroferric oxide magnetic nano-material of oxidized dextran |
Non-Patent Citations (1)
| Title |
|---|
| 余宇燕等: "直接竞争荧光免疫法测定中药材中的黄曲霉毒素 B1", 《化学通报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107782893B (en) * | 2017-11-18 | 2019-06-04 | 安徽师范大学 | A method of detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier |
| CN108918854A (en) * | 2018-05-15 | 2018-11-30 | 安徽师范大学 | A method of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107238710B (en) | 2019-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5313249B2 (en) | Fluorescence resonance energy transfer detection using nanoparticles | |
| CN103157118B (en) | Composite nano-grade novel material based on cancer early-stage integrated detection, diagnoses, and treatment, and preparation method thereof | |
| CN107238710B (en) | A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier | |
| CN105193831A (en) | Preparation method and application of indocyanine green loaded self-assembled multifunctional nano targeting system | |
| CN107091826B (en) | A method of based on trace fluorescence immunoassay quantitative detection PSI-OAm-NAPI amphiphilic polymer/nanometer pharmaceutical carrier | |
| CN106706894A (en) | Method for quantitatively detecting oleylamine grafted polysuccinimide macromolecule nanometer drug carrier based on indirect competitive enzyme-linked immunosorbent assay | |
| CN102174474A (en) | Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil | |
| CN108245688A (en) | Diagnosis of hepatoma reagent and preparation method thereof | |
| CN105181958B (en) | A kind of method that 80nmAuNPs is quantitatively detected based on Indirect cELISA | |
| CN106075469A (en) | Gd3+induction gold nano cluster is self-assembled into method and the application of gold nano grain | |
| CN107782893B (en) | A method of detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier | |
| CN103864921A (en) | Folic acid-adriamycin immunological preparation for treating cancer through dual targets and preparation method thereof | |
| Di Paola et al. | Human hepatocarcinoma cell targeting by glypican-3 ligand peptide functionalized silica nanoparticles: Implications for ultrasound molecular imaging | |
| CN107957492A (en) | A kind of method of the fluorescence immunoassay determining adsorption ultra trace oleyl amine scion grafting polysuccinimide based on carbon dots mark | |
| CN102336831A (en) | Anti-sildenafil specific antibody | |
| CN105999262A (en) | Nanometer drug carrier (bevacizumab medicated-SiO2@LDH (sodium dioxide @ double hydroxide)) with active tumor targeting function, preparation method and application | |
| CN107228938B (en) | A kind of quantitative detecting method of folic acid functionalized SiO 2 targeted nano pharmaceutical carrier | |
| CN107727844A (en) | A kind of method that fluorescence immunoassay quantitatively detects MFAP nano-medicament carriers | |
| CN108918854A (en) | A method of the fluorescence immunoassay determining adsorption ultra trace polymeric nano medicine carrier based on magnetic fluorescence probe label | |
| CN106267198A (en) | Targeting photo-thermal therapy combined immunization treatment antitumor compound formulation and preparation method and application | |
| CN113941006B (en) | Fluorescent probe containing oxatinib, preparation method and application thereof | |
| CN1921891B (en) | Growth factor-binding compounds connecting with Calixarene | |
| CN110101876A (en) | Purposes of the novel optoacoustic probe in preparation medicine targeting photoacoustic imaging reagent or drug | |
| CN110568184B (en) | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier | |
| CN113289014A (en) | Zwitterionic polypeptide/gallic acid/iron coordination nanoparticle with ultra-small particle size as well as preparation method and application thereof |
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 | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221115 Address after: Room 1322, building 1, No. 33, Science Park Road, Changping District, Beijing 102200 Patentee after: UNITED-POWER PHARMA TECH CO.,LTD. Address before: 241000 Wuhu Road, Yijiang District, Anhui, Patentee before: ANHUI NORMAL University |