CN108918854B - Method for determining ultra-trace macromolecular nano-drug carrier based on fluorescence immunoadsorption marked by magnetic fluorescent probe - Google Patents
Method for determining ultra-trace macromolecular nano-drug carrier based on fluorescence immunoadsorption marked by magnetic fluorescent probe Download PDFInfo
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Abstract
The invention provides a method for determining an ultra-trace macromolecular nano-drug carrier based on fluorescence immunoadsorption marked by a magnetic fluorescent probe, which improves the detection sensitivity based on easy separation and excellent fluorescence performance of the magnetic fluorescent probe. I.e. by preparing PSIOAmAntigen-coated, magnetic fluorescent probe-labeled anti-PSIOAmAntibodies, specificity of binding to antigen-antibody reaction vs. oleylamine graft Polysuccinimide (PSI)OAm) The macromolecule nano particles are subjected to ultra trace detection and analysis. Compared with the prior art, the method provided by the invention has the characteristics of simple operation, low background, high sensitivity, strong specificity, high throughput, accurate targeting, nondestructive detection and the like.
Description
Technical Field
The invention relates to functionalization of a magnetic fluorescent probe and quantitative detection of a nano material, in particular to a method for determining an ultra-trace high-molecular nano drug carrier based on fluorescence immunoadsorption marked by the magnetic fluorescent probe.
Background
According to the research and development trend prediction report of 2017 Chinese tumor hospital market published by the Chinese industry research network, at present, the disease rate of tumors in China is increased at a rate of 3% -5% every year, the annual average tumor medical cost is more than 1500 million yuan, the disease rate and death rate of malignant tumors are continuously increased, and the service requirement on tumor treatment is inevitably brought, so that the research of the development and supply and demand conditions of high-efficiency antitumor drugs is more and more necessary.
At present, the treatment of cancer in clinic mainly adopts drugs, chemotherapy and radiotherapy, but the existing chemotherapy drugs have poor water solubility and no targeting property, can kill normal cells while killing cancer cells, thereby causing serious toxic and side effects and hindering the development and application of the chemotherapy drugs.
The research provides an antibody drug carrier system, which is a system for coupling the antitumor drug and the monoclonal antibody through chemical bonds and carries the antitumor drug to a specified position by utilizing the specific reaction of the antigen and the antibody. Although the targeting property and the water solubility of part of easily modified anti-cancer drugs are improved to a certain extent by the antibody drug carrier system, the high toxicity of the anti-cancer drugs seriously influences the specific reaction among antigen and antibodies due to the small drug carrying quantity, and the anti-cancer drugs cannot kill the tumor even reaching the tumor part. Therefore, it has become a great challenge in the biomedical field to improve the drug treatment efficiency, reduce the toxic and side effects of the drug, improve the in vivo distribution of the drug, and the like.
The nano-drug carrier is a novel carrier, is usually made of natural or synthetic polymer materials, and has the main advantages of improving the absorptivity and stability of the drug, improving the property and targeting property of the drug, prolonging the action time of the drug, increasing the curative effect, reducing the toxic and side effects, reducing the toxicity to normal cells and the like, so more and more researchers aim at the nano-drug carrier. At present, the organic metal frame, the inorganic nonmetal frame, the high molecular polymer, the magnetofluid, the liposome and the like are mainly used. The nano carrier has the particle size of about 10-500 nm, can wrap drug molecules in the nano carrier or adsorb the drug molecules on the surface of the nano carrier, and enters cells under the action of cell uptake through the combination of targeting molecules and cell surface specific receptors or magnetic targeting, so that safe and effective targeted drug delivery is realized, and the nano carrier has special value and significance in drug delivery.
The oleylamine grafted polysuccinimide nano material is one of important members of a high molecular carrier material, and plays an important role in the field of biomedicine due to simple and convenient preparation, stable property, multiple types and quantities of drug-loading amount, good biocompatibility and easy surface modification. The targeting polypeptide RGD is modified on the polysuccinimide nano material to be used as a novel targeting drug carrier, so that the nano drug carrier is a more ideal nano drug carrier with intelligent effect, provides a new thought and means for treating cancer, and has a greater breakthrough in the aspects of solving the diagnosis, treatment, prevention and the like of human serious diseases. For example, Wanlerian has developed the oleylamine grafted polysuccinimide polymer nano micelle as a delivery carrier of anticancer drugs such as adriamycin, camptothecin and the like, and the drug loading efficiency and variety are improved on a certain basis.
At present, a great deal of research reports are carried out on polymer micelles as drug carriers, but before the polymer micelles are fully, safely and effectively applied to clinical application, a series of problems such as how to realize real-time dynamic targeting of nano carriers, more accurate targeting substances, more effective therapeutic drugs, more sensitive sensors with more convenient operability, biocompatibility and degradability, encapsulation efficiency and release time, stability and integrity of carried biomacromolecules, dynamic testing and analysis methods of in-vivo carrier action mechanisms and the like are still to be further researched and solved. From the results of the existing research, the application of the nano material in the medical field is generally limited to the aspects of investigating the distribution, degradation, drug release efficiency and the like of the nano material in the body. The nanometer material has high specific surface energy, and can interact with various complex components in blood to form a layer of protein coat during transportation in a human body, and has certain influence on the targeting property of the nanometer drug carrier, and in addition, different dosages can generate different effects or toxic and side effects on the body. Therefore, the dosage detection of the nano-drug carrier is particularly important for investigating whether the material can be applied to clinic. The current method for positioning and characterizing the nano-drug carrier is mainly fluorescence spectroscopy. Fluorescent reagents are generally selected as imaging agents of nano-drug carriers, but due to poor photobleaching resistance of various fluorescent reagents encapsulated in the drug carriers, even some fluorescent imaging agents can react with drugs to cause drug effect change. Therefore, the detection of the single nano-drug carrier labeled with the fluorescent developer cannot achieve real-time dynamic detection for a long time.
However, the quantitative analysis method of nanomaterials commonly used at present is mainly by means of expensive analysis instruments. In addition, the reagent used for testing has certain toxicity and great destructiveness to the sample, the stability and the sensitivity can not meet the requirements, and the detection technology is still not mature enough. Therefore, the methods cannot well realize real-time accurate quantitative detection and analysis of the nano material.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for determining an ultra-trace amount of a polymer nano-drug carrier based on fluorescence immunoadsorption marked by a magnetic fluorescent probe, which improves the detection sensitivity based on easy separation and excellent fluorescence performance of the magnetic fluorescent probe. I.e. by preparing PSIOAmAntigen-coated, magnetic fluorescent probe-labeled anti-PSIOdmAntibodies, specificity of binding to antigen-antibody reaction vs. oleylamine graft Polysuccinimide (PSI)OAm) The macromolecule nano particles are subjected to ultra trace detection and analysis. The method has the characteristics of simple operation, low background, high sensitivity, strong specificity, high throughput, accurate targeting, nondestructive detection and the like.
The specific technical scheme of the invention is as follows:
the invention provides a method for determining an ultra-trace high molecular nano-drug carrier based on fluorescence immunoadsorption marked by a magnetic fluorescent probe, which comprises the following steps:
a. preparation of PSIOAmHydrolyzing the solution;
b. preparation of PSIOAmCoating antigens and immunogens;
c. anti-PSIOAmPreparing an antibody;
d. preparing a magnetic fluorescent probe;
e. anti-PSI labeled by magnetic fluorescent probeOAmPreparing an antibody;
f. general PSIOdmCoating antigen in 96-well plate after being diluted by coating liquid, sealing, adding PSI with different concentrationsOAmStandard substance, labeling anti-PSI with functional magnetic fluorescent probeOAmAntibody, direct competitive fluorescence immunoadsorption assay PSIOAm；
g. In PSIOAmThe logarithm of the concentration of the standard substance is an abscissa, the fluorescence intensity value is an ordinate, and a standard curve is drawn, so that the PSI is quantitatively detectedOAmThe concentration of (c).
Specifically, the step a specifically comprises the following steps: 20-60 mg PSIOAmDissolving the mixture into 1-5 mL of trichloromethane solution, and adding the solution into 10-16 mL of chloroform solution after dissolvingPerforming ultrasonic treatment in 0.001-0.008 mg/mL sodium hydroxide solution for 10-30 min with the power of 300-600W, magnetically stirring for 20-50 min, evaporating the solution at 40-60 ℃ to remove chloroform, centrifuging at 12000-20000 r/min for 10-15 min, washing with PBS (pH 7.4) for 3 times, taking the precipitate, dispersing in 0.5-2 mL PBS (pH 7.4) buffer solution, and obtaining the hydrolyzed PSIOAm-COO-And (3) solution.
The step b specifically comprises the following steps:
b-1, dissolving 1mgOVA in 1mLPBS solution, and then adding PSI hydrolyzed in the step aOAm-COO-1mL of the solution, finally adding 1mLPBS buffer solution, wherein the buffer solution contains 0.1-1 mg of N-hydroxysuccinimide (NHS) and 0.1-1 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, carrying out a mixed spinning reaction for 10-30 min, standing for 30-60 min, carrying out centrifugal separation, washing for 3 times by PBS with pH 7.4, then adding 1-10 mg of bovine serum albumin, incubating for 2-4 h at 25 ℃, then centrifuging for 10-15 min by a centrifuge at 12000-20000 r/min, taking the precipitate to disperse into 1mLPBS (pH 7.4) buffer solution, filling the solution into a dialysis bag, putting into the PBS buffer solution for dialysis for more than 12 h, and obtaining the PSIOAm-an envelope antigen of OVA;
b-2 PSI after hydrolysisOAmAdding hydroxysuccinimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and bovine serum albumin into the solution, incubating for 3-6 h at 20-30 ℃, performing centrifugal separation, taking the precipitate, and dispersing the precipitate in PBS buffer solution with pH of 7.4 to obtain PSIOAmAn immunogen solution;
in the step b-1, the cut-off molecular weight of the dialysis bag is 8000-25000 Da;
the step c specifically comprises the following steps:
c-1, primary immunization: general PSIOAmMixing the immunogen and Freund's complete adjuvant in an equal volume ratio, injecting the mixture into a white rabbit body by adopting a back subcutaneous multipoint injection mode, and injecting 8-10 points, wherein the injection amount is 1-2 mL/rabbit; three weeks after the first immunization, the boosting immunization is carried out;
c-2, boosting immunity: general PSIOAmMixing the immunogen with Freund's incomplete adjuvant at equal volume ratioThen, injecting the mixture into the body of a big white rabbit by the same method, wherein 8-10 points are injected, and the injection amount is 1-2 mL/rabbit; then boosting immunity every two weeks, taking auricular vein blood sampling to measure serum titer in the middle week until titer reaches 1:64000, performing the last boosting immunity, taking blood from carotid artery of animal after one week of immunity, standing to precipitate antiserum, and purifying to obtain anti-PSIOAmAn antibody.
The step d specifically comprises the following steps:
d-1, first 10.8g of FeCl3·6H2Dissolving O and 36.5g of sodium oleate in 80mL of ethanol, sequentially adding 60mL of deionized water and 140mL of cyclohexane, heating at 70 ℃ for 4h, extracting the organic layer with deionized water for 3 times when the organic layer is cooled to room temperature, mixing the obtained solutions, heating at 70 ℃ for 4h to evaporate cyclohexane, and cooling to room temperature to obtain iron oleate for later use;
d-2, adding 9g of iron oleate prepared in the step d-1 into 50g of octadecene, uniformly stirring, adding 1.4g of oleic acid, heating the mixed solution to 100 ℃, keeping the temperature for 30min, vacuumizing, heating the mixture to 320 ℃ in a nitrogen atmosphere, keeping the temperature for 3h, adding 20mL of absolute ethyl alcohol to precipitate the ferroferric oxide nanocrystal when the temperature reaches the room temperature after the reaction, repeatedly washing for 3 times, and drying to obtain the ferroferric oxide nanocrystal;
d-3, dissolving the ferroferric oxide nanocrystal prepared in the step d-2 by using cyclohexane to obtain a ferroferric oxide nanocrystal solution of 100mg/mL, uniformly stirring 14.4mL of cyclohexane, 3.3mL of LTX-100, 0.5mL of CO-520, 3.3mL of hexanol, 1.5mL of deionized water and 0.25mL of ammonia water, adding 1mL of the ferroferric oxide nanocrystal solution of 100mg/mL, adding 50 mu L of tetraethyl silicate, stirring and reacting for 2 hours, centrifuging after the reaction is finished, washing for 3 times by using absolute ethyl alcohol, and freeze-drying to obtain the silicon dioxide functionalized ferroferric oxide nanocrystal for later use;
d-4, weighing 6.7mg of silicon dioxide functionalized ferroferric oxide nanocrystal prepared in the step d-3, dispersing in 50mL of deionized water, adjusting the pH value of the solution to 9.0 by using 2M sodium hydroxide, heating the system to 70 ℃, then sequentially adding 0.5mL of tetraethyl silicate, 0.05mL of rhodamine isothiocyanate labeled 3-aminopropyltriethoxysilane and 3mL of ethyl acetate, stirring for 10min in a dark place, then adding 0.05mL of 3-aminopropyltriethoxysilane, stirring for 3h, cooling to room temperature, washing with ethanol for 3 times, and freeze-drying to obtain the magnetic fluorescent probe for later use.
In the step d, square ferroferric oxide nanocrystals are synthesized according to a high-temperature coprecipitation method, the sizes of the crystals are uniformly distributed, a layer of silicon dioxide is wrapped on the surfaces of the ferroferric oxide nanocrystals by a reverse microemulsion method, and the monodisperse ferroferric oxide nanocrystals are assembled into a spherical structure with high crystallinity and very clear lattice stripes under mild conditions, so that the magnetic fluorescent composite probe is fully shown to be formed by gathering the same ferroferric oxide single crystal nanoparticles; compared with the traditional magnetic fluorescent composite probe, the magnetic fluorescent probe is still assembled into a spherical structure with high crystallinity under the condition of loading the same amount of rhodamine isothiocyanate, so that the application of the magnetic composite material in biological labeling is fully expanded.
The step e specifically comprises the following steps:
e-1, taking the purified anti-PSIOAm0.4mg of antibody is added into 0.07mL of acetic acid buffer solution, then 0.14mL of sodium periodate solution is added, and the mixture is stirred for 2 hours at room temperature in a dark place;
e-2, taking 2mg of the magnetic fluorescent probe prepared in the step d-4, washing the magnetic fluorescent probe for 3 times by using PBS, fully suspending the magnetic fluorescent probe in 0.6mL of PBS, slowly adding the oxidized antibody treated in the step e-1, and stirring the mixture for 20 hours at 4 ℃; then adding 2mg/mL of 0.02mL of sodium borohydride solution, reacting for 2h in a dark place, and washing for 3 times by PBS to obtain the anti-PSI marked by the magnetic fluorescent probeOAmAnd (5) preparing the antibody for later use.
The step f specifically comprises the following steps:
f-1, coating: PSI with coating bufferOAmDiluting the coating antigen to 25 mug/mL, coating a 96-well plate with each well being 100 mug/L, and refrigerating overnight at 4 ℃;
f-2, sealing: PBST solution is washed for 3 times, spin-dried for 3-5 min each time, and unbound PSI is washed offOAmCoating antigen, adding 1wt% of casein, sealing each hole by 200 mu L, and incubating in an oven at 37 ℃ for 1-2 h;
f-3, addSample competition: washing the PBST solution for 3 times, spin-drying for 3-5 min each time, washing off redundant confining liquid, and then labeling the optimized 50 muL of the anti-PSI labeled by the magnetic fluorescent probeOdmAntibodies and 50 μ L of PSI at various concentrationsOAmAdding the standard substance into each hole in a gradient manner to enable the standard substance to generate a competitive reaction, and incubating for 1-2 h in an oven at 37 ℃;
f-4, detection: PBST solution is washed for 3 times and dried for 3-5 min each time, and free PSI is removedOAmAnd (3) measuring the fluorescence intensity value of each hole at the excitation wavelength of 530nm and the emission wavelength of 585nm by using a multifunctional microplate reader.
The linear equation of the standard curve in the step g is F ═ 11477.59-1537.63lgC, wherein F is the fluorescence intensity value, and C is PSIOAmThe concentration of (c). Its coefficient of correlation R2Linear range of 5 × 10 ═ 0.994-5-5×103ng/mL, detection limit of 3X 10-7ng/mL。
The invention provides a method for measuring oleylamine grafted Polysuccinimide (PSI) based on fluorescence immunoadsorption marked by a magnetic fluorescent probeOAm) The method of the macromolecule nano-drug carrier combines the specificity of the antigen and antibody reaction and the multifunctional magnetic fluorescent probe as the marker to realize the immune method to PSIOAmUltra trace detection of (2).
Compared with the prior art, the invention has the following characteristics:
(1) the magnetic fluorescent probe is prepared by a simple and green method and is used as a fluorescent signal molecule to establish high-sensitivity immunoassay quantitative detection PSIOAmA foundation is laid;
(2) the specificity of the reaction of the magnetic fluorescent probe with antigen and antibody is utilized to establish the PSI based on the fluorescence immunoadsorption measurement marked by the magnetic fluorescent probeOAmThe new method provides a simple, rapid and accurate quantitative detection method for the nano materials in the future;
(3) the magnetic fluorescent probe is used for marking the nano-drug carrier antibody, so that the water solution dispersibility of the magnetic fluorescent probe is enhanced, and the targeting detection of the material on the nano-material is improved.
(4) The fluorescent probe has uniform particle size distribution, a large amount of loaded dye, a mesoporous structure which effectively improves the photobleaching resistance of organic dye, and wraps a plurality of ferroferric oxide nanocrystals, so that the magnetic separation effect is further enhanced, and the sensitivity of an experiment is improved;
(6) the method is simple and rapid to operate, high in sensitivity and specificity and capable of realizing high-throughput nondestructive testing.
Drawings
FIG. 1 shows the PSIOAmThe logarithm of the concentration of the standard substance is a standard curve chart established by the abscissa and the fluorescence intensity value is the ordinate.
Detailed Description
Freund's complete adjuvant, Bovine Serum Albumin (BSA) and chicken Ovalbumin (OVA) were purchased from Biotechnology engineering (Shanghai) Inc. Rhodamine isothiocyanate labeled 3-aminopropyltriethoxysilane was purchased from Pobexady, and other reagents were purchased from commercial vendors.
The preparation method of each solution related by the invention comprises the following steps:
PBS solution (0.01mol/L pH 7.4): weighing 8.0g of NaCl, 0.1g of KCl and NaH2PO4·2H2O 0.106g、Na2HPO4·12H2O3.34 g was dissolved in distilled water and made to 1000 mL.
Carbonate buffer CD (0.5mol/L pH 9.6): weighing Na2CO3 1.59g、NaHCO32.94g was dissolved in distilled water and made to 100 mL.
PBST solution (0.01mol/L pH 7.4): add 500. mu.L of Tween-20 to 1000mL of PBS and mix well.
Coating buffer fe (0.05mol/L pH 9.6): weighing Na2CO3 1.59g、NaHCO32.94g was dissolved in distilled water and made up to 1000 mL.
Acetic acid buffer solution: 84.25g of sodium acetate was weighed, dissolved in water, 100ml of acetic acid was added, and the mixture was diluted to 2500ml with water to obtain a (pH 4.2) sodium acetate buffer solution.
1wt% casein solution: as a blocking solution, 0.01g of casein was weighed and dissolved in 1mL of PBS, and mixed well.
Example 1
A method for determining an ultra-trace amount of high molecular nano-drug carriers based on fluorescence immunoadsorption marked by a magnetic fluorescent probe comprises the following steps:
a. preparation of PSIOAmHydrolysis solution:
20-60 mg PSIOAmDissolving the solution into 1-5 mL of trichloromethane solution, adding the dissolved solution into 10-16 mL of sodium hydroxide solution with the concentration of 0.001-0.008 mg/mL, carrying out ultrasonic treatment for 10-30 min at the power of 300-600W, then carrying out magnetic stirring for 20-50 min, evaporating the solution at 40-60 ℃ to remove the trichloromethane, centrifuging the solution for 10-15 min at 12000-20000 r/min, washing the solution for 3 times by PBS (pH 7.4), taking the precipitate, dispersing the precipitate in 0.5-2 mL of PBS (pH 7.4) buffer solution to obtain the hydrolyzed PSI (pressure specific integrated circuit)OAm-COO-And (3) solution.
b. Preparation of PSIOAmCoating antigens and immunogens:
b-1, dissolving 1mgOVA in 1mLPBS solution, and then adding PSI hydrolyzed in the step aOAm-COO-1mL of the solution, finally adding 1mLPBS buffer solution, wherein the buffer solution contains 0.1-1 mg of N-hydroxysuccinimide (NHS) and 0.1-1 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, carrying out a mixed spinning reaction for 10-30 min, standing for 30-60 min, carrying out centrifugal separation, washing for 3 times by PBS with pH 7.4, then adding 1-10 mg of bovine serum albumin, incubating for 2-4 h at 25 ℃, then centrifuging for 10-15 min by a centrifuge at 12000-20000 r/min, taking the precipitate to disperse into 1mLPBS (pH 7.4) buffer solution, filling the solution into a dialysis bag, putting into the PBS buffer solution for dialysis for more than 12 h, and obtaining the PSIOAm-an envelope antigen of OVA;
b-2 PSI after hydrolysisOAmAdding hydroxysuccinimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and bovine serum albumin into the solution, incubating for 3-6 h at 20-30 ℃, performing centrifugal separation, taking the precipitate, and dispersing the precipitate in PBS buffer solution with pH of 7.4 to obtain PSIOAmAn immunogen solution;
the cut-off molecular weight of the dialysis bag in the step b-1 is 8000-25000 Da;
c. anti-PSIOAmPreparation of antibody:
c-1, primary immunization: general PSIOAmMixing the immunogen and Freund's complete adjuvant in an equal volume ratio, injecting the mixture into a white rabbit body by adopting a back subcutaneous multipoint injection mode, and injecting 8-10 points, wherein the injection amount is 1-2 mL/rabbit; three weeks after the first immunization, the boosting immunization is carried out;
c-2, boosting immunity: general PSIOAmMixing the immunogen and Freund's incomplete adjuvant in the same volume ratio, and injecting the mixture into a white rabbit body by the same way, wherein 8-10 points are injected, and the injection amount is 1-2 mL/rabbit; then boosting immunity every two weeks, taking auricular vein blood sampling to measure serum titer in the middle week until titer reaches 1:64000, performing the last boosting immunity, taking blood from carotid artery of animal after one week of immunity, standing to precipitate antiserum, and purifying to obtain anti-PSIOAmAn antibody.
d. Preparation of magnetic fluorescent probe:
d-1, first 10.8g of FeCl3·6H2O and 36.5g of sodium oleate were dissolved in 80mL of ethanol, 60mL of deionized water and 140mL of cyclohexane were added in this order, and the mixture was heated at 70 ℃ for 4 hours. When the solution is cooled to room temperature, the organic layer is extracted for 3 times by deionized water, the extracted solution is mixed, heated for 4 hours at 70 ℃ to evaporate cyclohexane, and a reddish brown sticky substance appears when the solution is cooled to room temperature, namely the successfully synthesized iron oleate;
d-2, adding 9g of the iron oleate prepared in the step d-1 into 50g of octadecene, uniformly stirring, adding 1.4g of oleic acid, heating the mixed solution to 100 ℃, keeping the temperature for 30min, vacuumizing, heating the mixture to 320 ℃ in a nitrogen atmosphere, keeping the temperature for 3h, adding 20mL of absolute ethyl alcohol to precipitate iron nanocrystals when the temperature reaches the room temperature after reaction, repeatedly washing for 3 times, and drying to obtain the ferroferric oxide nanocrystals for later use;
d-3, dissolving the ferroferric oxide nanocrystal prepared in the step d-2 by using cyclohexane to obtain a ferroferric oxide nanocrystal solution of 100mg/mL, respectively taking 14.4mL of cyclohexane, 3.3mL of TX-100, 0.5mL of CO-520, 3.3mL of hexanol, 1.5mL of deionized water and 0.25mL of ammonia water to be uniformly stirred in a 100mL beaker, then adding 1mL of the ferroferric oxide nanocrystal solution of 100mg/mL, adding 50 mu L of tetraethyl silicate to be stirred for 2 hours, centrifuging after the reaction is finished, washing 3 times by using absolute ethyl alcohol, and freeze-drying to obtain the silicon dioxide functionalized ferroferric oxide nanocrystal for later use;
d-4, accurately weighing 6.7mg of a sample prepared in d-3, dispersing the sample in 50mL of deionized water, adjusting the pH of the solution to 9.0 by using 2M sodium hydroxide, heating the system to 70 ℃, sequentially adding 0.5mL of tetraethyl silicate, 0.05mL of rhodamine isothiocyanate labeled 3-aminopropyltriethoxysilane and 3mL of ethyl acetate, stirring for 10min in a dark place, adding 0.05mL of 3-aminopropyltriethoxysilane, stirring for 3h, cooling to room temperature, washing with ethanol for 3 times, and freeze-drying to obtain the magnetic fluorescent probe for later use.
e. anti-PSI labeled by magnetic fluorescent probeOAmPreparation of antibody:
e-1, taking the purified anti-PSIOAm0.4mg of the antibody was added to 0.07mL of acetic acid buffer (0.05mol/L, pH 4.2), and then 0.14mL of sodium periodate solution (1.5mg/mL, pH 4.2) was added and stirred for 2 hours with exclusion of light;
e-2, washing the 2mg multifunctional magnetic fluorescent probe with PBS for 3 times, fully suspending the multifunctional magnetic fluorescent probe in 0.6mL PBS, slowly adding the oxidized antibody, and stirring for 20 hours at 4 ℃; 0.02mL of 2mg/mL sodium borohydride solution is added, the mixture is reacted for 2 hours in a dark place, and the reaction product is washed for 3 times by PBS for standby.
f. General PSIOdmCoating antigen in 96-well plate after being diluted by coating liquid, sealing, adding PSI with different concentrationsOAmStandard substance, labeling anti-PSI with functional magnetic fluorescent probeOAmAntibody, direct competitive fluorescence immunoadsorption assay PSIOAm:
f-1, coating: PSI was diluted with 0.05M carbonate buffer pH 9.6OAmDiluting the coating antigen solution to 25 mu g/mL, coating in a 96-well plate with each well being 100 mu L, and keeping the temperature in a refrigerator at 4 ℃ overnight;
f-2, sealing: taking out 96-well plate, washing with PBST for 3 times (3 min each time), and washing away unbound PSIOAmCoating antigen, adding 1wt% casein for sealing, sealing at 37 deg.C for 1.5 hr, and sealing at 200 μ L per well;
f-3, sample addition competition: PBST was washed 3 times, spun-dried for 3min each time, and 50. mu.L of the solution was added at a concentration of 5X 10-5ng/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、103PSI of ng/mLOAmThe standard solution is sequentially added into each row of a 96-well plate, namely each concentration gradient is repeated three times, and then 50 mu L of anti-PSI marked by the functionalized magnetic fluorescent probe is added into each holeOAmAntibody, competition 1.5h at 37 ℃;
f-4, washing with a washing solution for 3 times, each time for 3 minutes, and spin-drying; PSI of different concentrationsOAmThe preparation method of the standard solution comprises the following steps: PSI is treated with 0.01mol/L PBS buffer solution with pH 7.4OAmDiluting to a specified standard concentration; the fluorescence intensity of each well at an excitation wavelength of 530nm and an emission wavelength of 585nm was measured on a microplate reader.
General PSIOAmCoating antigen in 96-well plate after being diluted by coating liquid, sealing, adding PSI with different concentrationsOAmStandard substance, PSI labeled with functionalized magnetic fluorescent probeOAmThe antibody is used as a primary antibody to establish direct competition fluoroimmunoassay quantitative detection PSIOAm. PSI labeled with functionalized magnetic fluorescent probesOAmAntibodies and PSIOdmThe coating antigen is subjected to direct competition fluoroimmunoassay which utilizes anti-PSIOAmAntibodies and PSIOAmThe coating antigen is specifically combined, the aim of quantitatively detecting the antigen is achieved by detecting the fluorescent signal of the complex of the antigen and the antibody, and compared with the method for detecting the antigen by directly competing the optical signal of the enzyme-linked immunosorbent assay, the method has lower detection limit and higher sensitivity.
g. In PSIOAmThe logarithm of the standard solution concentration is an abscissa, the fluorescence intensity value is an ordinate, a standard curve is established, the prepared standard curve is shown in figure 1, and the linear equation of the standard curve is as follows: 11477.59-1537.63lgC, wherein F is fluorescence intensity value, and F is PSIOAmOf the concentration of (A), its correlation coefficient R2Linear range of 5 × 10 ═ 0.994-5-1×103ng/mL, detection limit of 3X 10-7ng/mL。
Repeating the above steps except for the PSI with different concentrations in step f-3OAmPSI with unknown concentration replaced by standard solutionOAmMeasuring the fluorescence intensity of each hole with excitation wavelength of 530nm and emission wavelength of 585nm on a microplate reader to obtain average fluorescence intensity value, and calculating PSI according to the standard curveOAmThe concentration of the solution to be tested.
The method is the optimal experimental method after multiple experimental verifications, and the standard curve obtained by the method has the best linear relation and the widest linear range.
The above reference example quantitatively detects the oleylamine grafted Polysuccinimide (PSI) by the fluorescence immunoadsorption method based on the labeling of the functionalized magnetic fluorescent probeOAm) The detailed description of the method of the polymer nano-drug carrier is illustrative and not restrictive, and several examples can be enumerated according to the limited scope, so that changes and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.
Claims (1)
1. A method for determining an ultra-trace amount of high molecular nano-drug carriers based on fluorescence immunoadsorption marked by a magnetic fluorescent probe is characterized by comprising the following steps:
a. preparation of PSIOAmHydrolyzing the solution;
b. preparation of PSIOAmCoating antigens and immunogens;
c. anti-PSIOAmPreparing an antibody;
d. preparation of magnetic fluorescent probe:
d-1, first 10.8g of FeCl3·6H2Dissolving O and 36.5g of sodium oleate in 80mL of ethanol, sequentially adding 60mL of deionized water and 140mL of cyclohexane, heating at 70 ℃ for 4h, extracting the organic layer with deionized water for 3 times when the temperature is cooled to room temperature, mixing the obtained solutions, heating at 70 ℃ for 4h to evaporate cyclohexane,cooling to room temperature to obtain iron oleate for later use;
d-2, adding 9g of iron oleate prepared in the step d-1 into 50g of octadecene, uniformly stirring, adding 1.4g of oleic acid, heating the mixed solution to 100 ℃, keeping the temperature for 30min, vacuumizing, heating the mixture to 320 ℃ in a nitrogen atmosphere, keeping the temperature for 3h, adding 20mL of absolute ethyl alcohol to precipitate the ferroferric oxide nanocrystal when the temperature reaches the room temperature after the reaction, repeatedly washing for 3 times, and drying to obtain the ferroferric oxide nanocrystal;
d-3, dissolving the ferroferric oxide nanocrystal prepared in the step d-2 by using cyclohexane to obtain a ferroferric oxide nanocrystal solution of 100mg/mL, uniformly stirring 14.4mL of cyclohexane, 3.3mL of LTX-100, 0.5mL of CO-520, 3.3mL of hexanol, 1.5mL of deionized water and 0.25mL of ammonia water, adding 1mL of the ferroferric oxide nanocrystal solution of 100mg/mL, adding 50 mu L of tetraethyl silicate, stirring and reacting for 2 hours, centrifuging after the reaction is finished, washing for 3 times by using absolute ethyl alcohol, and freeze-drying to obtain the silicon dioxide functionalized ferroferric oxide nanocrystal for later use;
d-4, weighing 6.7mg of silicon dioxide functionalized ferroferric oxide nanocrystal prepared in the step d-3, dispersing in 50mL of deionized water, adjusting the pH of the solution to be =9.0 by using 2M sodium hydroxide, heating the system to 70 ℃, then sequentially adding 0.5mL of tetraethyl silicate, 0.05mL of 3-aminopropyltriethoxysilane marked by rhodamine isothiocyanate and 3mL of ethyl acetate, stirring for 10min in a dark place, then adding 0.05mL of 3-aminopropyltriethoxysilane, stirring for 3h, cooling to room temperature, washing for 3 times by using ethanol, and freeze-drying to obtain the magnetic fluorescent probe for later use;
e. anti-PSI labeled by magnetic fluorescent probeOAmPreparation of antibody:
e-1, taking the purified anti-PSIOAm0.4mg of antibody is added into 0.07mL of acetic acid buffer solution, then 0.14mL of sodium periodate solution is added, and the mixture is stirred for 2 hours at room temperature in a dark place;
e-2, taking 2mg of the magnetic fluorescent probe prepared in the step d-4, washing the magnetic fluorescent probe for 3 times by using PBS, fully suspending the magnetic fluorescent probe in 0.6mL of PBS, slowly adding the oxidized antibody treated in the step e-1, and stirring the mixture for 20 hours at 4 ℃; adding 2mg/mL of 0.02mL sodium borohydride solution, reacting for 2h in a dark place, washing 3 times with PBSThen obtaining the anti-PSI marked by the magnetic fluorescent probeOAmAntibody, for use;
f. general PSIOdmCoating antigen in 96-well plate after being diluted by coating liquid, sealing, adding PSI with different concentrationsOAmStandard substance, labeling anti-PSI with functional magnetic fluorescent probeOAmAntibody, direct competitive fluorescence immunoadsorption assay PSIOAmThe method specifically comprises the following steps:
f-1, coating: PSI with coating bufferOAmDiluting the coating antigen to 25 mug/mL, coating a 96-pore plate with each pore being 100 muL, and standing overnight in a refrigerator at 4 ℃;
f-2, sealing: PBST solution is washed for 3 times, spin-dried for 3-5 min each time, and unbound PSI is washed offOAmCoating antigen, adding 1wt% of casein, sealing each hole by 200 mu L, and incubating in an oven at 37 ℃ for 1-2 h;
f-3, sample addition competition: washing the PBST solution for 3 times, spin-drying for 3-5 min each time, washing off redundant confining liquid, and then labeling the optimized 50 muL magnetic fluorescent probe with the PSI resistanceOdmAntibody and PSI of different concentrations of 50 muLOAmAdding the standard substance into each hole in a gradient manner to enable the standard substance to generate a competitive reaction, and incubating for 1-2 h in an oven at 37 ℃;
f-4, detection: PBST solution is washed for 3 times and dried for 3-5 min each time, and free PSI is removedOAmMeasuring the fluorescence intensity value of each hole at the position where the excitation wavelength is 530nm and the emission wavelength is 585nm by using a multifunctional enzyme label instrument;
g. in PSIOAmThe logarithm of the concentration of the standard substance is an abscissa, the fluorescence intensity value is an ordinate, and a standard curve is drawn, so that the PSI is quantitatively detectedOAmThe concentration of (c); the linear equation of the standard curve is F =11477.59-1537.63lgC, wherein F is the fluorescence intensity value, and C is PSIOAmOf the concentration of (A), its correlation coefficient R2=0.994, linear range 5 × 10-5-5×103ng/mL, detection limit of 3X 10-7ng/mL。
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