CN110568184B - Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier - Google Patents
Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier Download PDFInfo
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention discloses a nano-drug carrier fluorescence immunoassay quantitative detection method for loading anticancer drug camptothecin, which is implemented by using CPT @ PSI OAm Coating antigen in the ELISA plate, sealing, adding CPT @ PSI with different concentrations OAm Standard and FITC-labeled anti-PSI OAm Antibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI OAm (ii) a By detecting CPT @ PSI OAm CPT @ PSI (quantitative detection of fluorescent signal of coating antigen and FITC (fitful binding protein) labeled antibody complex) achieving quantitative detection OAm The detection method is based on antigen-antibody specificity, is combined with a fluorescent marker, further improves immunoreaction signals, can perform high-throughput measurement, has a linear range of 0.12-1638.7ng/mL and a detection limit of 0.04ng/mL, and provides a more sensitive detection scheme for the nano-drug carrier loaded with the anti-cancer drug.
Description
Technical Field
The invention relates to a quantitative detection method of a drug-loaded nano-drug carrier, in particular to a fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin.
Background
Now, cancer has become one of the biggest killers of human health and is a major challenge that is difficult to solve in world medicine, and over the past decade, the global incidence of cancer has risen by 33%, and in 2018 alone, 1810 thousands of people have been diagnosed with cancer and 980 thousands of people die as a result. The anticancer drugs approved to be on the market at present are only hundreds, however, the development of cancer treatment is influenced by low water solubility, poor biocompatibility, low blood clearance rate, poor tumor targeting, serious toxic and side effects on normal tissues and the like of many anticancer drugs, the nano-drug carrier can greatly improve the water solubility and stability of the drugs, prolong the blood circulation time, enhance the permeability and retention (EPR) effect of passive accumulation of tumors, and the macromolecular nano-drug carrier is oleamide grafted polysuccinimide PSI OAm The carrier is an emerging drug carrier in recent years, can load anti-cancer drugs, has a wide application prospect in the aspect of cancer treatment, and provides a more feasible experimental scheme for the research on the kinetic processes of quantitative detection, delivery, tracing, drug release and the like of the anti-cancer drugs.
Although many liposome drug carriers are in clinical use, the high molecular drug carrier is used as a nano material, and the biological effect generated by the complex chemical structure of the high molecular drug carrier and the quantitative analysis method of drug carrier loaded drugs have no unified evaluation standard and evaluation method internationally. At present, researchers have provided many challenges faced by nano-drug carriers, and meanwhile, the real-time dynamic monitoring and quantitative analysis research of processes of the nano-drug carriers loaded with drugs, such as circulation in blood after entering a human body, transportation to the surface of cancer cells for enrichment, permeation into the cancer cells, drug release of the drug carriers and the like, are few, so that the establishment of high-sensitivity quantitative detection of the nano-drug carriers loaded with anticancer drugs camptothecin is urgent.
At present, the detection of the anticancer drugs mainly takes HPLC and ultraviolet detection as main parts, and if an immunoassay method is established, the quantitative detection of the anticancer drug loaded nano-drug carrier has more advantages of more sensitively quantifying the anticancer drugs.
Disclosure of Invention
The invention provides a fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin, which is used for quantitatively detecting the drug loaded on the nano-drug carrier by adopting a direct competition fluorescence immunoassay method, is simple and quick, does not need a complex sample pretreatment process, and is combined with high specificity and high sensitivity of an antigen and an antibody.
The technical scheme adopted by the invention is as follows:
a fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin comprises the following steps:
(1) Preparation of CPT @ PSI OAm (camptothecin-loaded oleylamine-grafted polysuccinimide) coating antigen;
(2) Preparation of FITC-labeled anti-PSI OAm An antibody;
(3) Will CPT @ PSI OAm Coating the coating antigen in an enzyme label plate after being diluted by a coating solution, sealing, and adding CPT @ PSI with different concentrations OAm Standard and FITC-labeled anti-PSI OAm Antibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI OAm ；
(4) With CPT @ PSI OAm The logarithm of the standard concentration is the abscissa and the fluorescence intensity value is the ordinateDrawing a standard curve by coordinates to obtain a linear equation, thereby quantitatively detecting CPT @ PSI OAm The concentration of (c).
Further, the step (1) specifically includes the steps of:
(1-1) mixing PSI OAm Dissolving (oleylamine graft polysuccinimide), camptothecin (CPT) and polyethylene-b-polyethylene glycol in chloroform, adding into sodium hydroxide solution, performing ultrasonic reaction, evaporating to remove chloroform, centrifuging, and dispersing the precipitate in PBS buffer solution to obtain hydrolyzed CPT @ PSI OAm -COO - A hapten solution;
(1-2) to CPT @ PSI OAm -COO - Adding PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride into hapten solution for reaction for 10-20 min, then adding egg white albumin, incubating, centrifuging, dispersing obtained precipitate into PBS buffer solution, and obtaining the CPT @ PSI OAm The envelope antigen of (1).
In step (1-1), the PSI OAm The dosage ratio of the camptothecin to the polyethylene-b-polyethylene glycol (PE-b-PEG) to the trichloromethane to the sodium hydroxide is (10-60) mg: (0.5-2) mg: (0.5-2) mL: (2-16) mL; the ultrasonic reaction condition is 200-400W ultrasonic for 5-10 min. The PSI OAm Has an average particle diameter of 200nm.
The step (1-2) specifically comprises the following steps: to 1mL CPT @ PSI OAm -COO - Adding 1mL PBS buffer solution containing 0.1-1 mg of N-hydroxysuccinimide and 0.1-1 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the hapten solution, reacting for 10-20 min, adding 1-10 mg of chicken egg albumin, incubating for 2-4 h at 25 ℃, centrifuging for 10-15 min, taking the precipitate, dispersing into 3mL neutral PBS buffer solution, and obtaining the CPT @ PSI OAm The coating antigen of (1).
The step (2) specifically comprises the following steps: general PSI OAm Mixing the antibody solution with fluorescein isothiocyanate solution, stirring in the dark for reaction for 3-6 h, and purifying by dialysis and gel filtration after the reaction to obtain FITC labeled anti-PSI OAm An antibody.
The PSI OAm The antibody solution is PSI OAm The antibody is dissolved in PBS buffer solution with 0.01M pH7.1 to obtain, and the protein concentration of the antibody is 10-20 mg/mL; the fluorescein isothiocyanate solution is obtained by dissolving fluorescein isothiocyanate in a carbonate buffer solution with pH =9.6, and the concentration of the fluorescein isothiocyanate solution is 2mg/mL.
The PSI OAm The volume ratio of the antibody solution to the fluorescein isothiocyanate solution is 10; the eluent after gel filtration and purification is 0.01M PBS buffer solution with pH value of 7.1, and the flow rate is 0.1-0.5 mL/min.
The step (3) specifically comprises the following steps:
(3-1) coating: CPT @ PSI with coating buffer OAm Diluting the coating antigen by 80 times, coating a 96-hole enzyme label plate with 100 mu L of each hole, and keeping the temperature in a refrigerator at 4 ℃ overnight;
(3-2) sealing: washing the 96-well enzyme label plate for 3 times by using PBST solution, wherein each time is 3-5 min, then adding 1wt% casein for sealing, keeping the volume of each well at 200 mu L, and incubating for 1-2 h at 37 ℃; the purpose of the PBST solution wash is to wash away unbound CPT @ PSI OAm Coating antigen;
(3-3) sample addition Competition: PBST solution washes 96-hole enzyme label plate 3 times, each time 3-5 min, then 50 μ L CPT @ PSI with different concentration OAm Standard solution and 50. Mu.L of FITC-labeled PSI OAm Mixing the antibody in advance, adding the mixture into each hole in gradient, incubating for 1-2 h at 37 ℃, washing for 2-4 times by using a washing solution, each time for 3-5 minutes, and spin-drying;
and (3-4) measuring the fluorescence intensity of each hole on a microplate reader when the excitation wavelength is 485nm and the emission wavelength is 525 nm.
In the step (4), the linear equation of the standard curve is F =8116.6-311.7lgC, F is fluorescence intensity, C is CPT @ PSI OAm The concentration of (d); coefficient of correlation R 2 =0.991, linear range 0.12-1638.7ng/mL, detection limit 0.04ng/mL.
In the preparation method provided by the invention, PSI with the grain diameter of 200nm is used OAm High-titer PSI obtained by immunizing white rabbits with nanoparticles as immunogen OAm An antibody. By combining CPT and PSI Oam The polymer and polyethylene-b-polyethylene glycol are dissolved in chloroformThen adding the mixture into sodium hydroxide solution for ultrasonic reaction to realize drug loading and PSI OAm Hydrolysis is completed in one step to obtain CPT @ PSI OAm -COO - Hapten, and then CPT @ PSI is prepared OAm The coating antigen of (1). The polyethylene-b-polyethylene glycol as an amphiphilic block polymer can play a role in modification and protection, can prolong the circulation time of the nano-drug carrier in vivo and promote accumulation of target tissues. In addition, the polyethanol block part can provide good hydrophilicity, and the hydrophobic polyethylene block part can promote the encapsulation of hydrophobic drugs, so that a uniform and stable nano-drug carrier is formed.
The oleylamine grafted polysuccinimide is a pH response nano-drug carrier, the inner core of the carrier has hydrophobicity, and the anticancer drug Camptothecin (CPT) can be mixed with PSI OAm The hydrophobic part of the nano-drug carrier forms hydrogen bonds, and the hydrophobic drug is successfully wrapped in the hydrophobic cavity of the nano-drug carrier; meanwhile, the shell has hydrophilicity, can provide good biocompatibility and can convey the anti-cancer drug to a target position. Camptothecin (CPT) and PSI due to acidity of environmental pH of cancer cells OAm The hydrogen bonds are easy to break, so that the medicine is released to achieve the purpose of treating cancer.
General PSI OAm After loading the anticancer drug camptothecin, CPT @ PSI OAm The nanoparticles have a specific PSI OAm The smaller size of the nanoparticles (-100 nm) indicates that the loading of the drug gives the nanoparticles a more compact appearance. Meanwhile, experimental results prove that the nano-drug carrier PSI OAm Not only with anti-PSI OAm The antibody has high specificity combination, and the nano-drug carrier and the anti-PSI after the antibody loads the anti-cancer drug camptothecin OAm Antibodies also bind with high specificity. Based on the method, a nano-drug carrier (CPT @ PSI) for loading the anticancer drug camptothecin is established OAm ) Detection, which provides a better quantitative detection scheme and wider practical application value for the nano-drug carrier loaded with the anticancer drug.
The invention establishes a nano-drug carrier (CPT @ PSI) for loading an anticancer drug camptothecin OAm ) Compared with the prior art, the fluorescence immunoassay quantitative detection method has the following advantages:
(1) The invention realizes the quantitative detection of the nano-drug carrier loaded with the anticancer drug camptothecin by using a direct competitive fluorescence immunoassay method for the first time, and provides a more effective detection means for the future anticancer drug detection.
(2) The invention uses a nano-drug carrier (CPT @ PSI) loaded with an anticancer drug camptothecin OAm ) As coating antigen, and whether the coated medicine can be combined with anti-PSI OAm Antibody specific binding and further validation of PSI using fluorescence immunoassay OAm Wrapping with PSI OAm The antibody still has specificity and meets the requirement of quantitative detection of CPT @ PSI OAm The purpose is.
(3) At present, the detection of the anticancer drugs mainly takes HPLC and ultraviolet detection as main materials, and the quantitative detection of the nano-drug carrier loaded with the anticancer drug camptothecin is more advantageous for the high-sensitivity quantification of the anticancer drugs by establishing an immunoassay method, so that a more sensitive detection scheme is provided for the detection of the nano-drug carrier and the drugs.
(4) The method establishes direct competitive fluorescence immunoassay for quantitatively detecting CPT @ PSI OAm By detecting CPT @ PSI OAm Envelope antigen, FITC-labeled PSI OAm Quantitative detection CPT @ PSI achieved by antibody complex fluorescent signal OAm The object of (1) is to further improve an immunoreaction signal by binding to a fluorescent label based on the antigen-antibody specificity, and to enable high-throughput measurement.
(5) Nano medicine carrier PSI OAm The antibody can be further applied to wrapping other anti-cancer drugs, can specifically identify the nano-drug carrier loaded by other anti-cancer drugs by utilizing one antibody, has wide application range and has practical application value.
Drawings
In FIG. 1, (a) is PSI OAm TEM image of hapten, (b) CPT @ PSI OAm A TEM image of the hapten;
CPT @ PSI in FIG. 2 OAm -COO - The hapten is (a) an infrared characteristic pattern and (b) an XRD characteristic pattern;
FIG. 3 shows the CPT @ PSI OAm Logarithm of standard concentration is abscissa, fluorescence intensity at 525nmThe values establish a standard curve plot for the ordinate.
Detailed Description
The present invention will be described in detail with reference to examples.
The raw materials used in the invention are as follows:
polysuccinimide (Mw = 6000) was purchased from shijiazhuang dessai chemical ltd;
camptothecin was purchased from Shanghai Mecline Biotechnology, inc.;
FITC was purchased from Aladdin;
polyethylene-b-polyethylene glycol (PE-b-PEG, mn = 1400) was purchased from ALDRICH;
the chicken egg white albumin was purchased from bio-engineering (Shanghai) Co., ltd;
other reagents are commercially available from commercial vendors.
High molecular polymer PSI OAm The preparation method comprises the following steps: heating 32mL of N, N-dimethylformamide to 90 deg.C, adding 1.6g of polysuccinimide and 2.17mL of oleylamine, heating at 100 deg.C for 5 hr, adding 360mL of methanol to precipitate, centrifuging, collecting precipitate, and weighing to obtain high molecular polymer PSI OAm 。
anti-PSI OAm The preparation method of the antibody refers to the method disclosed in CN201810459487.8, and the anti-PSI OAm The titer of the antibody was 1.
The preparation method of each solution used in the invention comprises the following steps:
PBS buffer: weighing 8.0g of NaCl, 0.1g of KCl and NaH 2 PO 4 ·2H 2 O 0.29g、Na 2 HPO 4 ·12H 2 Dissolving O2.96 g in distilled water, and diluting to 1000mL to obtain PBS buffer solution with the concentration of 0.01mol/LpH = 7.4;
PBST solution: adding 500 mu L of Tween-20 into 1000mL of PBS, and uniformly mixing;
coating buffer CB: weighing Na 2 CO 3 1.59g、NaHCO 3 2.94g of the extract is dissolved in distilled water and the volume is up to 1000mL; obtaining coating buffer solution with 0.05mol/L and pH = 9.6;
1wt% casein: 0.3g of casein was weighed out and dissolved in 30mL of 0.01mol/L PBS buffer solution with pH =7.4, and stirred well.
Example 1
A research on a nano-drug carrier fluorescence immunoassay quantitative detection method for loading an anticancer drug camptothecin comprises the following steps:
(1) Preparation of CPT @ PSI OAm The coating antigen of (camptothecin-loaded oleylamine grafted polysuccinimide) specifically comprises the following steps:
(1-1) 60mg of high molecular weight Polymer PSI OAm Dissolving 1mg Camptothecin (CPT) and 2mg polyethylene-b-polyethylene glycol (PE-b-PEG) into 1mL chloroform solution, mixing, adding the above solution into 10mL sodium hydroxide solution with concentration of 0.5mmol/L, performing ultrasound for 5min with power of 200W, evaporating the solution at 60 deg.C to remove chloroform, centrifuging at 12000r/min for 10min, dispersing the precipitate in 10mL PBS buffer solution with pH =7.4 to obtain hydrolyzed CPT @ PSI OAm -COO - A hapten solution, the TEM image of which is shown in fig. 1 (b); compared with PSI OAm -COO - TEM image of hapten solution, CPT @ PSI loaded with camptothecin OAm -COO - The hapten solution is in a core-shell structure, the particle size is about 100nm, and the wrapping of the camptothecin in PSI is proved OAm Of (c).
PSI OAm -COO - TEM image of hapten solution, shown in FIG. 1 (a), PSI OAm -COO - The preparation method of the hapten solution comprises the following steps: 60mg of high molecular polymer PSI OAm Dissolving into 1mL of trichloromethane solution, mixing uniformly, adding the solution into 2-16 mL of sodium hydroxide solution with the concentration of 0.1-1mmol/L, carrying out ultrasonic treatment for 5min at the power of 200W, evaporating the solution at 65 ℃ to remove the trichloromethane, centrifuging for 10min, taking the precipitate, dispersing in 10mL of PBS buffer solution with the pH value of 7.4, and obtaining hydrolyzed PSI (pressure-sensitive polyimide) OAm -COO - A hapten.
CPT@PSI OAm -COO - Haptens were characterized by Fourier Transform Infrared (FTIR) spectroscopy. FIG. 2 (a) shows CPT and PSI OAm CPT and PSI OAm Physical hybrid, CPT @ PSI OAm -COO - Infrared spectrum of hapten, as shown, for PSI OAm Methylene (-CH) in oleylamine was also observed in FTIR 2 ) About 2920 and 2850cm in C-H tensile vibration -1 。1724cm -1 Is attributed to PSI OAm the-NH-COO-group of the support. CPT @ PSI OAm -COO - Hapten is 1651, 1136, 1076, 827, 532cm -1 The absorption peaks reflect the characteristic peaks of CPT. 1651cm -1 Is due to the stretching vibration of the C = C bond of CPT. Obviously, at CPT @ PSI OAm -COO - The hapten is loaded with CPT at 1136, 1076, 827 and 532cm -1 The intensity of the absorption peak at (A) is significantly increased. FTIR confirmed that CPT was successfully wrapped.
Analysis of PSI by XRD OAm The physical state of CPT. FIG. 2 (b) shows CPT and PSI OAm CPT and PSI OAm Physical hybrid, CPT @ PSI OAm -COO - The XRD spectrum of the hapten can be seen, and pure CPT shows a plurality of sharp diffraction peaks, which indicates that the drug has larger crystallinity; in pure PSI OAm One broad peak and very weak crystallization peak appear in the solution, indicating PSI OAm Is in an amorphous fluffy state. CPT @ PSI OAm -COO - The diffraction peak of CPT medicine can be detected in the infrared image of hapten, and is simultaneously matched with pure PSI OAm By contrast, CPT @ PSI OAm -COO - The hapten has a plurality of diffraction peaks (26, 31, 46, 56, 67, 76) in which the diffraction signals 31 and 46 are significantly enhanced, but no pure drug signal is detected, which can be interpreted as the fact that the drug intercalates through hydrogen bonds and hydrophobic interactions, and no new functional groups are present, thus leading to PSI OAm The hapten of the loaded CPT exhibits a highly crystalline state.
(1-2) taking 1mL of hydrolyzed CPT @ PSI OAm -COO - Adding 1mL PBS buffer solution containing NHS 0.1mg and EDAC 0.7mg into the solution, reacting for 20min, adding egg white albumin 2mg, incubating at 25 deg.C for 4h, centrifuging for 10min, and dispersing the precipitate into 3mL neutral PBS buffer solution to obtain CPT @ PSI OAm An envelope antigen.
(2) Preparation of FITC-labeled anti-PSI OAm The antibody specifically comprises the following steps:
(2-1) weighing the purified 15mg PSI OAm The antibody was dissolved in PBS buffer solution (0.01M, pH = 7.1) to give a PSI concentration of 15mg/mL OAm An antibody solution;
(2-2) dissolving 2mg of Fluorescein Isothiocyanate (FITC) in 1mL of carbonate buffer solution with pH =9.6 to prepare a FITC solution with a concentration of 2 mg/mL;
(2-3) mixing the solutions obtained in the step d-1 and the step d-2 according to the volume ratio of 10;
(2-4) filling the mixed solution obtained in the step d-3 into a dialysis bag, dialyzing in a PBS solution for 2 hours, and purifying by adopting a gel filtration method to obtain PSI marked by FITC OAm A fluorescent antibody; the purification method by the gel filtration method comprises the following steps:
gel filtration chromatography column: 2X 30cm glass chromatographic column filled with fully swollen and air-exhausted dextran gel G-50; eluent: 0.01m, ph=7.1 PBS buffer solution; flow rate: 0.1-0.5 mL/min; sample adding amount: loading 10-15% of the gel column volume.
(3) Will CPT @ PSI OAm Coating the coating antigen in an enzyme label plate after being diluted by a coating solution, sealing, and adding CPT @ PSI with different concentrations OAm Standard and FITC-labeled anti-PSI OAm Antibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI OAm (ii) a The method specifically comprises the following steps:
(3-1) coating: CPT @ PSI with coating buffer OAm Diluting the coating antigen by 80 times, coating the antigen in 24 holes in a 96-hole enzyme label plate, namely taking 3 holes in each row for experiment, wherein each hole is 100 mu L, and keeping the temperature in a refrigerator at 4 ℃ overnight;
(3-2) sealing: taking out the enzyme label plate, washing for 3 times by PBST (basic-bound-thereto) for 3min each time, adding 1wt% casein for sealing, sealing at 37 ℃ for 1h, wherein each hole is 200 mu L;
(3-3) sample addition Competition: PBST was washed 3 times for 3min, and 50. Mu.L of the solution was added to 10 4 ng/mL、10 3 ng/mL、10 2 ng/mL、5×10 2 ng/mL、10 1 ng/mL、5ng/mL、10 -1 CPT @ PSI of ng/mL OAm Standard solutions and50 μ L anti-PSI OAm The antibody is sequentially added into each row of the ELISA plate, namely, each concentration gradient is repeated for three times, and competition is carried out for 2 hours at 37 ℃;
(3-4) measuring the fluorescence intensity values of the holes at the excitation wavelength of 485nm and the emission wavelength of 525nm on an enzyme-labeling instrument, and averaging the same concentration gradient;
(4) With CPT @ PSI OAm The logarithm of the concentration of the standard substance is an abscissa, the fluorescence intensity value is an ordinate, a standard curve is established, the prepared standard curve is shown in figure 2, and the linear equation of the standard curve is as follows: f =8116.6-311.7lgC, F is fluorescence intensity, C is CPT @ PSI OAm The unit of (2) is ng/mL; coefficient of correlation R 2 =0.991, linear range 0.12-1638.7ng/mL, detection limit 0.04ng/mL.
Repeating the above steps except for different concentrations of CPT @ PSI in step (3-3) OAm Standard solution is replaced by CPT @ PSI with unknown concentration OAm Measuring the fluorescence intensity of each hole at 485nm excitation wavelength and 525nm emission wavelength on enzyme-labeling instrument, and calculating the average value to obtain the final product OAm The concentration of the solution to be tested.
The method is an optimal experimental method after multiple experimental verifications, the linear relation of the standard curve obtained by the method is best, and the detection limit is lowest.
The detailed description of the method for the quantitative fluorescence immunoassay of a nano-drug carrier loaded with an anticancer drug camptothecin with reference to the above embodiments is illustrative and not restrictive, and several embodiments can be enumerated according to the limited scope, so that variations and modifications thereof without departing from the general concept of the present invention shall fall within the protection scope of the present invention.
Claims (5)
1. A fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin is characterized by comprising the following steps:
(1) Preparation of camptothecin-loaded oleylamine-grafted polysuccinimide CPT @ PSI OAm The coating antigen of (1);
(2) Preparation of FITC-labeled anti-PSI OAm An antibody;
(3) Will CPT @ PSI OAm Coating the coating antigen in an enzyme label plate after being diluted by a coating solution, sealing, and adding CPT @ PSI with different concentrations OAm Standard and FITC-labeled anti-PSI OAm Antibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI OAm ；
(4) With CPT @ PSI OAm The logarithm of the concentration of the standard substance is an abscissa, the fluorescence intensity value is an ordinate, and a standard curve is drawn to obtain a linear equation, so that CPT @ PSI is quantitatively detected OAm The concentration of (d);
the step (1) specifically comprises the following steps:
(1-1) grafting oleylamine to polysuccinimide PSI OAm Dissolving CPT and polyethylene-b-polyethylene glycol in chloroform, adding into sodium hydroxide solution, performing ultrasonic reaction, evaporating to remove chloroform, centrifuging, and dispersing the precipitate in PBS buffer solution to obtain hydrolyzed CPT @ PSI OAm -COO - A hapten solution;
(1-2) to CPT @ PSI OAm -COO - Adding PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into hapten solution for reaction for 10-20min, then adding chicken egg white albumin, incubating, centrifuging, dispersing obtained precipitate into PBS buffer solution, and obtaining the CPT @ PSI OAm The coating antigen of (1);
in step (1-1), the PSI OAm The dosage ratio of the camptothecin to the polyethylene-b-polyethylene glycol to the chloroform to the sodium hydroxide is (10 to 60) mg: (0.5 to 2) mg: (0.5 to 2) mL: (2-16) mL; the ultrasonic reaction condition is 200-400W ultrasonic for 5-10min;
the step (1-2) specifically comprises the following steps: to 1mL CPT @ PSI OAm -COO - Adding 1mL of PBS buffer solution containing 0.1-1 mg of N-hydroxysuccinimide and 0.1-1 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the hapten solution, reacting for 10-20min, adding 1-10mg of chicken egg white albumin, incubating for 2-4 h at 25 ℃, centrifuging for 10-10 15min, and taking out a precipitateDispersing into 3mL neutral PBS buffer solution to obtain CPT @ PSI OAm The coating antigen of (1);
the step (2) specifically comprises the following steps: general PSI OAm Mixing the antibody solution with fluorescein isothiocyanate solution, stirring in the dark for reaction for 3-6 h, and purifying by dialysis and gel filtration after the reaction is finished to obtain the FITC-labeled anti-PSI OAm An antibody.
2. The detection method according to claim 1, wherein the PSI OAm The antibody solution is PSI OAm Dissolving the antibody in 0.01M PBS buffer solution with pH7.1, wherein the protein concentration is 10-20mg/mL; the fluorescein isothiocyanate solution is obtained by dissolving fluorescein isothiocyanate in a carbonate buffer solution with pH =9.6, and the concentration of the fluorescein isothiocyanate solution is 2mg/mL.
3. Detection method according to claim 1 or 2, characterized in that said PSI OAm The volume ratio of the antibody solution to the fluorescein isothiocyanate solution is 10; the eluent after gel filtration and purification is 0.01M PBS buffer solution with pH7.1, and the flow rate is 0.1 to 0.5mL/min.
4. The detection method according to claim 1, wherein the step (3) comprises the following steps:
(3-1) coating: CPT @ PSI with coating buffer OAm Diluting the coated antigen by 80 times, coating a 96-hole enzyme label plate with 100 mu L of each hole, and standing overnight in a refrigerator at 4 ℃;
(3-2) sealing: washing a 96-hole enzyme label plate for 3 times by using a PBST solution, wherein each time lasts for 3 to 5min, then adding 1wt% of casein for sealing, and incubating for 1 to 2h at 37 ℃ with 200 muL per hole; the purpose of the PBST solution wash was to wash away unbound CPT @ PSI OAm A coating antigen;
(3-3) sample addition Competition: washing a 96-hole enzyme label plate for 3 times by using a PBST solution, 3 to 5min each time, and then washing CPT @ PSI with different concentrations of 50 mu L OAm Standard solution and 50 mu L FITC mark PSI OAm Mixing the antibody in advance, adding the mixture into each hole in gradient, incubating for 1 to 2h at 37 ℃, and washing with washing solution for 2~4 times, 3 timesDrying for 5 minutes;
and (3-4) measuring the fluorescence intensity of each hole on a microplate reader when the excitation wavelength is 485nm and the emission wavelength is 525 nm.
5. The detection method according to claim 1, wherein in the step (4), the linear equation of the standard curve is F =8116.6-311.7lgC, F is fluorescence intensity, C is CPT @ PSI OAm The concentration of (c); coefficient of correlation R 2 =0.991, linear range 0.12-1638.7ng/mL, detection limit 0.04ng/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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