CN110632295A - Fe3O4-Au antibody nano magnetic bead and preparation method and application thereof - Google Patents
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Abstract
The invention discloses Fe3O4-Au antibody nano magnetic beads and a preparation method and application thereof. Wherein said Fe3O4The preparation method of the antibody nano magnetic bead is Fe3O4The solution is modified by polydiallyldimethylammonium chloride, and AuNPs is electrostatically adsorbed to the Fe modified by polydiallyldimethylammonium chloride3O4To obtain Fe3O4Au, covalent binding of tubulin light chain 3B antibody to modified Fe3O4-Au, to obtain. The Fe provided by the invention3O4The Au antibody nano magnetic beads fill the vacancy of the existing enrichment and purification secretion type autophagosome, do not need complex treatment on a sample, have excellent anti-interference performance and have high selectivity on the secretion type autophagosome; in addition, Fe3O4The application of the Au antibody nano magnetic beads in enrichment and purification of the secretory autophagosome has the advantages of high enrichment efficiency, high product purity and simple and convenient operation.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a Fe-based material3O4-Au specific enrichment and purification secretory autophagosome nano magnetic bead preparation method and application.
Background
Tumors are diseases with high morbidity and high mortality which seriously threaten human health, and a large amount of research and clinical data prove that early diagnosis and early treatment are the most effective methods for preventing and treating tumors and reducing the mortality of tumor patients. Therefore, the search for tumor markers that contribute to early diagnosis is a focus of constant attention. The novel tumor marker can provide more possibility for early diagnosis of cancer, so that the detection and application of the novel tumor marker have good prospects.
Autophagy refers to the process of phagocytizing and encapsulating autoplasmatic proteins or organelles into vesicles, fusing with lysosomes to form autophagosomes, and degrading the encapsulated contents. Autophagosomes formed in yeast cells and mammalian cells can be released extracellularly, called secretory autophagosomes, the characteristic marker for which is LC3B (tubulin-light chain 3B).
Research proves that tumor cells release autophagosome (TRAP) carrying damage-related molecular pattern (DAMP) molecules, and B cells are induced to differentiate into IL-10 through TLR2-MyD 88-NF-kB signal channel+B cells; neutrophils that phagocytose TRAP by macrophagocytosis can produce Reactive Oxygen Species (ROS) to induce neutrophil apoptosis; TRAP affects macrophage polarization. Various evidences confirm that secreted autophagosome TRAP is closely associated with tumor development.
The secretory autophagosome is contained in hydrothorax and ascites and peripheral blood of a tumor patient, but the content is low, the capture and detection are not easy, and the conventional method for extracting the secretory autophagosome is a differential centrifugation method, but the operation is complicated and the product purity is low. The immunomagnetic beads are commonly used for purification, the operation is simple and convenient, the product purity is higher, and the immunomagnetic beads have wide application prospect in different fields.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing Fe aiming at the defects of the prior art3O4-Au antibody nanobeads.
Further, the invention also provides Fe3O4A preparation method of-Au antibody nano magnetic beads.
Finally, the invention also provides Fe3O4-Au antibody nano magnetic beadThe application in enriching and purifying the secretion type autophagosome.
In order to solve the technical problem, the invention discloses Fe3O4Preparation method of-Au antibody nano magnetic beads, wherein Fe3O4The solution is modified by poly (diallyldimethylammonium chloride) (PDDA), and AuNPs are electrostatically adsorbed to the PDDA modified Fe3O4To obtain Fe3O4Au, covalent binding of the tubulin light chain 3B (LC3B) antibody to modified Fe3O4-Au, to obtain.
Fe as described above3O4The preparation method of the-Au antibody nano magnetic bead specifically comprises the following steps:
1)Fe3O4preparation of Au: mixing Fe3O4Mixing the solution and poly (diallyldimethylammonium chloride), shaking for 10-40 min, preferably 30min, centrifuging to obtain a precipitate, washing the precipitate with water, suspending the precipitate in 0.8-1.5 mL of water, preferably 1mL of water per gram of wet precipitate, adding AuNPs, mixing at room temperature, shaking for 5-10 h, preferably 7 h; centrifuging to obtain a precipitate, washing the precipitate with water, and resuspending the precipitate in 0.8-1.5 mL of water per gram of wet precipitate, preferably 1mL of water, to obtain Fe3O4-an Au solution;
2)Fe3O4-preparation of Au antibody nanobeads: the Fe prepared in the step 1) is added3O4Adding the Au solution into 4-mercaptobutyric acid, and oscillating for 10-24 h at room temperature, preferably overnight; performing magnetic separation, washing with water, preferably washing with water for 3 times, and suspending the wet precipitate per gram in 0.8-1.5 mL of water, preferably 1 mL; adding 1-butanethiol, and oscillating at room temperature for 40-80 min, preferably for 1 h; performing magnetic separation, washing with water, preferably washing with water for 3 times, and suspending the wet precipitate per gram in 0.8-1.5 mL of PBS, preferably 1 mL; adding EDC/NHS solution, oscillating for 100-160 min at room temperature, preferably for 2h, performing magnetic separation, washing with PBS, and suspending the precipitate in 0.8-1.5 mL of PBS, preferably 1mL, per gram of wet precipitate; adding an LC3B antibody, and oscillating for 10-24 h at 2-10 ℃, preferably oscillating overnight at 4 ℃; adding BSA, stirring at 2-10 deg.C for 40-80 min, preferably at 4 deg.C for 1h, magnetically separating, washing with PBS, preferably washing three times, and resuspending the wet precipitate per gram in 0.8-1.5 mL PBS, preferably 1mL to obtainTo obtain Fe3O4-Au antibody nanobead solution.
Wherein, the Fe3O4The preparation method of the solution is as follows: FeCl is added3Dissolving sodium citrate in ethylene glycol, adding sodium acetate, stirring for 10-40 min, pouring into a reaction kettle, and reacting to prepare Fe3O4And (3) solution.
Wherein the FeCl3The mass ratio of the sodium citrate to the ethylene glycol is 2: 1-4: 1, preferably 3.25:1, and the dosage of the ethylene glycol is controlled to ensure that FeCl is added3FeCl at a concentration of 0.01-0.05 g/mL, preferably 0.0325g/mL3The mass ratio of the sodium acetate to the sodium acetate is 1: 1-1: 4, preferably 6.5: 12; the stirring speed is 400-1000r/min, preferably 800r/min, the reaction temperature is 180-220 ℃, the reaction time is 6-12 h, preferably 200 ℃ for 10 h; the reaction kettle is a hydrothermal reaction kettle; centrifuging the material after the reaction to obtain a first precipitate, washing the first precipitate with water, centrifuging to obtain a second precipitate, washing the second precipitate with ethanol to obtain a liquid dissolved in ethanol, namely Fe3O4A solution; said Fe3O4The concentration of the solution is 0.5-2 mg/mL, and the pH value is 8.0-10; wherein, Fe3O4The concentration of the solution is preferably 1mg/mL, and the pH is preferably 9.5.
The preparation method of the AuNPs comprises the following steps: adding HAuCl4Heating the solution to boiling, adding a sodium citrate solution while stirring, continuously heating and boiling for 10-30 min, preferably 15min, and cooling to prepare an AuNPs solution;
wherein, the HAuCl4The solvent of the solution is water, and the mass percent of the solvent is 0.005-0.02%, preferably 0.01%; the solvent of the sodium citrate solution is water, and the mass percent of the solvent is 0.05-2%, preferably 1%; HAuCl4The volume ratio of the solution to the sodium citrate solution is 55: 1-40: 1, preferably 50: 1.
In the step 1), the mass percent of the PDDA is 2-5%, preferably 4%; fe3O4The volume ratio of the solution to the PDDA is 1: 0.6-1: 0.2, preferably 1: 0.4; the pH value of the AuNPs is 6.5-7.8, and the preferred pH value is 7.0; fe3O4The volume ratio of the solution to AuNPs is 1: 6-1: 15, preferably 1: 10; what is needed isFe3O4The concentration of the Au solution is 0.5-2 mg/mL, preferably 1 mg/mL; the water washing is carried out for 2-5 times, preferably 3 times; the rotating speed of the centrifugation is 8000-12000 rpm, and 10000rpm is preferred.
In the step 2), the concentration of the 4-mercaptobutyric acid is 0.8-1.2 mM, preferably 1mM, and Fe3O4The volume ratio of the Au solution to the 4-mercaptobutyric acid is 6000: 1-4000: 1, preferably 5000: 1; the concentration of the 1-butanethiol is 0.8-1.2 mM, preferably 1mM, and Fe3O4The volume ratio of the Au solution to the 1-butanethiol is 6000:1 to 4000:1, preferably 5000: 1; the concentration of EDC in the EDC/NHS solution is 15-30 mg/mL, preferably 20mg/mL, the concentration of NHS is 5-15 mg/mL, preferably 10mg/mL, the solvent is PBS (pH 6.0), Fe3O4The volume ratio of the Au solution to the EDC/NHS solution is 1: 0.8-1: 1.5, preferably 1: 1; the concentration of the LC3B antibody is 0.8-1.2 mg/mL, preferably 1mg/mL, Fe3O4-the volume ratio of the Au solution to the LC3B antibody is 50:1 to 200:1, preferably 100: 1; the mass percent of the BSA is 0.2-2%, preferably 1%; the magnetic separation is that a magnet attracts magnetic nano-particles Fe3O4-Au-LC3B antibody complex.
Fe prepared by the above preparation method3O4-Au antibody nano-magnetic beads are also within the scope of the present invention.
Fe as described above3O4The application of-Au antibody nano magnetic beads in enrichment and purification of secretory autophagosomes is also within the protection scope of the invention.
Wherein, the application is Fe3O4-use of Au antibody nanomagnetic beads for enrichment and purification of secreted autophagosomes in cell culture supernatants, mouse peripheral blood and human peripheral blood, comprising the steps of:
magnetic enrichment and purification of autophagosomes: mixing Fe3O4Dropwise adding an Au antibody nano magnetic bead solution into an autophagosome solution, reacting at 20-50 ℃ for 10-120 min, preferably at 37 ℃ for 100min, then washing with a PBS solution, and suspending in PBS to capture autophagosomes;
and secondly, the autophagosome captured in the step I is used for Western blot, flow cytometry, ELISA detection and the like.
In step (i), the Fe3O4The concentration of the Au antibody nano magnetic bead solution is 10-30 mu g/mL, preferably 10 mu g/mL; the dropping amount is 80-150 mu L, preferably 100 mu L; the volume of the autophagosome solution is 500-1000. mu.L, preferably 500. mu.L; resuspend in 50-100. mu.L PBS, preferably 50. mu.L.
Abbreviations for technical terms in the present invention are as follows:
tumor cells secrete autophagosomes: TRAP; ferroferric oxide: fe3O4(ii) a Gold nanoparticles: AuNPs; poly (diallyldimethylammonium chloride): PDDA; 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride: EDC; nitrogen hydroxysuccinimide: NHS; microtubule-associated protein light chain 3B: LC 3B; bovine serum albumin: BSA.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the nanometer magnetic bead of the specific enrichment and purification secretion type autophagosome fills the vacancy of the enrichment and purification secretion type autophagosome at present, and does not need complex treatment on a sample.
(2) The nano magnetic bead has excellent anti-interference performance and high selectivity on secretory autophagosome.
(3) The invention selects Fe from nanometer magnetic beads3O4As raw materials, the price is low.
(4) The autophagosome obtained by enriching and purifying the nano magnetic bead can be continuously used for subsequent experiments.
(5) The Fe provided by the invention3O4The application of Au antibody nano magnetic beads in enrichment and purification of secretory autophagosomes has the advantages of high enrichment efficiency, high product purity and simple and convenient operation.
Drawings
FIG. 1 Fe in the present invention3O4Scanning electron micrograph (c).
FIG. 2 Fe in the present invention3O4Scanning electron micrograph of Au.
FIG. 3 Fe in the present invention3O4Laser of-Au antibody nano magnetic beadsConfocal images.
FIG. 4 Fe in the present invention3O4Laser confocal mapping of Au antibody nanobeads capture TRAP.
FIG. 5 Fe in the present invention3O4Specificity verification laser confocal mapping of Au antibody nanobeads.
FIG. 6 Fe in the present invention3O4Specific verification ELISA results for Au antibody nano magnetic beads.
FIG. 7 Fe in the present invention3O4Western blot verification of the enrichment efficiency of Au antibody nano magnetic beads.
FIG. 8 Fe in the present invention3O4-flow cytometry verification of the product purity of Au antibody nanobeads.
FIG. 9 Fe in the present invention3O4ELISA verification of the enrichment efficiency of Au antibody nano magnetic beads.
FIG. 10 Fe in the present invention3O4Electron microscopy of TRAP in peripheral blood of tumor patients enriched with Au antibody nanomagnetic beads.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
The LC3B antibody was purchased from NOVUS biotechnology limited, No. 1251A.
Example 1: fe3O4Preparation of-Au antibody nano magnetic beads
(1)Fe3O4Preparation of
0.65g FeCl3Dissolving 0.2g of sodium citrate in 20mL of ethylene glycol, adding 1.2g of sodium acetate, violently stirring for 30min, pouring into a hydrothermal reaction kettle, reacting for 10h at 200 ℃, centrifuging the materials after reaction to obtain a first precipitate, centrifuging the first precipitate after water washing to obtain a second precipitate, washing the second precipitate with ethanol to obtain a liquid dissolved in ethanol to obtain Fe3O4(concentration 1mg/mL, pH 9.5), the scanning electron micrograph of which is shown in FIG. 1.
(2) Preparation of AuNPs
100mL of HAuCl with the mass fraction of 0.01%4Heating the aqueous solution to boiling, adding 2mL of 1% by mass sodium citrate aqueous solution while stirring, continuously heating and boiling for 15min to obtain AuNPs, and cooling for later use.
(3)Fe3O4Preparation of-Au
1mL of Fe prepared in step (1)3O4Mixing with 400 μ L of 4% PDDA, shaking for 30min, centrifuging to obtain precipitate, washing the precipitate with water for 3 times, suspending the wet precipitate per gram in 1mL of water, adding 10mL of AuNPs (pH 7.0), shaking at room temperature for 7h, centrifuging at 10000rpm to obtain precipitate, washing with water for 3 times, and suspending the wet precipitate per gram in 1mL of water to obtain Fe3O4The scanning electron micrograph of the Au solution (concentration of 1mg/mL) is shown in FIG. 2.
(4)Fe3O4Preparation of-Au antibody nano magnetic beads
To 500uL of Fe prepared in step (3)3O40.1uL of 4-mercaptobutanoic acid (dissolved in Fe) was added to the-Au solution3O4Final concentration of Au solution 1mM), shaking overnight at room temperature; performing magnetic separation, washing with water for 3 times, resuspending the wet precipitate per gram with 1mL of water, adding 0.1uL of 1-butanethiol (final concentration of 1mM), and shaking at room temperature for 1 h; magnetic separation, washing 3 times, and resuspending according to 500uLPBS per gram of wet precipitate; 500uL EDC/NHS (20mg/mL, 10mg/mL) was added and shaken at room temperature for 2 h; 5uL of 1mg/mL LC3B antibody was added, and the mixture was shaken overnight at 4 ℃; adding 1% BSA (bovine serum albumin) by mass percent, stirring for 1h at 4 ℃, performing magnetic separation, washing with PBS for three times, and resuspending the precipitate in 1mL PBS according to the wet weight per gram to obtain Fe3O4-Au antibody nanobead solution. The magnetic separation is that a magnet attracts magnetic nano-particles Fe3O4The confocal laser image is shown in fig. 3.
Example 2: fe3O4-Au antibody nano magnetic bead capture TRAP validation
Fe3O4And co-incubating Au antibody nano magnetic beads and TRAP for 2h at room temperature, magnetically separating, washing with PBS for 3 times, and suspending in PBS to find that the nano magnetic beads can capture TRAP. The confocal laser image is shown in fig. 4.
Example 3: fe3O4Specificity verification of-Au antibody nano magnetic beads
One group of Fe3O4Co-incubating Au antibody nano magnetic beads and interference red fluorescent protein for 2h at room temperature, and performing Fe treatment on the other group3O4And (3) incubating Au antibody nano magnetic beads with interference red fluorescent protein and TRAP dyed with green fluorescence for 2h at room temperature, magnetically separating, washing with PBS for 3 times, and suspending in PBS to find that the nano magnetic beads can specifically capture the TRAP without non-specifically adsorbing the interference protein. The confocal laser image is shown in fig. 5.
Example 4: fe3O4Specificity verification of-Au antibody nano magnetic beads
The human hepatitis B core antigen HBcAg is used as an interference protein, the nano magnetic beads and the HBcAg are incubated for 2h at room temperature, magnetic separation is carried out, PBS is washed for 3 times and is resuspended in PBS, and the detection is carried out by using a human hepatitis B core antigen ELISA kit, so that the nano magnetic beads can not be combined with the interference protein HBcAg. The results are shown in FIG. 6.
Example 5: fe3O4Western blot verification of enrichment efficiency of-Au antibody nano magnetic beads
Incubating nano magnetic beads and autophagosome solution for 2 hours at room temperature, carrying out magnetic separation, respectively taking supernatant and precipitate, washing the precipitate for 3 times by using PBS, suspending in the PBS, carrying out Western blot detection on the supernatant and the precipitate, and comparing to find that the precipitate has more LC3B and the supernatant does not have LC3B, which indicates that the nano magnetic beads completely enrich TRAP and have high enrichment efficiency. The results are shown in FIG. 7.
Example 6: fe3O4Product purity verification of-Au antibody nanobead
Respectively enriching TRAP in ascites of tumor patient by differential centrifugation and prepared nanometer magnetic beads, performing purity verification on the enriched product by flow cytometry, and observing LC3B+Proportion of vesicles, 72% of the vesicles enriched by differential centrifugation was found to be LC3B+The 97% of the vesicles enriched by the nano magnetic beads is LC3B+And vesicles indicate that TRAP obtained by enrichment of nano magnetic beads has high purity, and the result is shown in FIG. 8.
Example 7: fe3O4ELISA verification of enrichment efficiency of-Au antibody nano magnetic beads
Incubating nano magnetic beads and autophagosome solution for 2 hours at room temperature, carrying out magnetic separation, respectively taking supernatant and sediment, washing the sediment for 3 times by PBS, suspending in PBS, cracking TRAP obtained by enriching the supernatant and the sediment and TRAP of equal amount of stock solution, and detecting the concentration of LC3B by an ELISA method, comparing to find that the absorbance of the sediment group is the highest and is basically the same as the stock solution, the absorbance of the supernatant group is very low, namely, the sediment LC3B has more amount, and the supernatant does not have LC3B basically, which indicates that the nano magnetic beads completely enrich the TRAP basically and the enrichment efficiency is high. The results are shown in FIG. 9.
Example 8: fe3O4Electron micrograph of TRAP in peripheral blood of-Au antibody nano-magnetic bead enriched tumor patient
Collecting EDTA anticoagulated peripheral whole blood of tumor patient, centrifuging, collecting supernatant, enriching with nanometer magnetic bead, and shooting captured TRAP with transmission electron microscope to find that it has typical double-layer membrane structure of autophagosome, and confirm to be TRAP, wherein the transmission electron microscope image is shown in FIG. 10.
The invention provides Fe3O4The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (10)
1. Fe3O4The preparation method of the-Au antibody nano magnetic bead is characterized in that Fe3O4The solution is modified by polydiallyldimethylammonium chloride, and AuNPs is electrostatically adsorbed to the Fe modified by polydiallyldimethylammonium chloride3O4To obtain Fe3O4Au, covalent binding of tubulin light chain 3B antibody to modified Fe3O4-Au, to obtain.
2. Fe according to claim 13O4The preparation method of the Au antibody nano magnetic bead is characterized by comprising the following steps:
1)Fe3O4preparation of Au: mixing Fe3O4Mixing the solution and poly (diallyldimethylammonium chloride), shaking for 10-40 min, centrifuging to obtain a precipitate, washing the precipitate with water, suspending the precipitate in 0.8-1.5 mL of water per gram of wet precipitate, adding AuNPs, mixing and shaking for 5-10 h at room temperature, centrifuging to obtain a precipitate, washing the precipitate with water, and suspending in water to obtain Fe3O4-an Au solution;
2)Fe3O4-preparation of Au antibody nanobeads: fe prepared in the step 1)3O4Adding the-Au solution into 4-mercaptobutyric acid, and oscillating for 10-24 h at room temperature; performing magnetic separation, washing with water, suspending in water, adding 1-butanethiol, and oscillating at room temperature for 40-80 min; magnetic separation, water washing, and resuspension in PBS; adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide solution, oscillating for 100-160 min at room temperature, performing magnetic separation, washing with PBS (phosphate buffer solution), and suspending the wet precipitate per gram in 0.8-1.5 ml PBS (phosphate buffer solution); adding an LC3B antibody, and oscillating for 10-24 h at 2-10 ℃; adding BSA, stirring at 2-10 ℃ for 40-80 min, performing magnetic separation, washing with PBS, and resuspending in PBS to obtain Fe3O4-Au antibody nanobead solution.
3. Fe according to claim 1 or 23O4-Au antibody nano magnetic bead preparation method, characterized in that Fe3O4The preparation method of the solution is as follows: FeCl is added3Dissolving sodium citrate in ethylene glycol, adding sodium acetate, stirring for 10-40 min, pouring into a reaction kettle, and reacting to prepare Fe3O4And (3) solution.
4. Fe according to claim 33O4The preparation method of the-Au antibody nano magnetic bead is characterized in that FeCl is adopted3The mass ratio of the FeCl to the sodium citrate is 2: 1-4: 1, and the dosage of the ethylene glycol is controlled to ensure that FeCl is added3FeCl with the concentration of 0.01-0.05 g/mL3The mass ratio of the sodium acetate to the sodium acetate is 1: 1-1: 4, the stirring speed is 400-1000r/min, the reaction temperature is 180-220 ℃, and the reaction time is 6-12 hours; centrifuging the material after the reaction to obtain a first precipitate, washing the first precipitate with water, centrifuging to obtain a second precipitate, washing the second precipitate with ethanol to obtain a liquid dissolved in ethanol, namely Fe3O4A solution; said Fe3O4The concentration of the solution is 0.5-2 mg/mL, and the pH value is 8.0-10.0.
5. Fe according to claim 1 or 23O4The preparation method of the-Au antibody nano magnetic beads is characterized in that the preparation method of the AuNPs is as follows: adding HAuCl4Heating the solution to boiling, adding a sodium citrate solution while stirring, continuously heating and boiling for 10-30 min, and cooling to prepare an AuNPs solution;
wherein, the HAuCl4The solvent of the solution is water, and the mass percent is 0.005-0.02%; the solvent of the sodium citrate solution is water, and the mass percent is 0.05-2%; HAuCl4The volume ratio of the solution to the sodium citrate solution is 55: 1-40: 1.
6. Fe according to claim 23O4The preparation method of the-Au antibody nano magnetic beads is characterized in that in the step 1), the mass percentage of the poly (diallyldimethylammonium chloride) is 2% -5%; fe3O4The volume ratio of the solution to the poly (diallyldimethylammonium chloride) is 1: 0.6-1: 0.2; the pH value of the AuNPs is 6.5-7.8; fe3O4The volume ratio of the solution to AuNPs is 1: 6-1: 15; said Fe3O4The concentration of the-Au solution is 0.5-2 mg/mL.
7. Fe according to claim 23O4The preparation method of the-Au antibody nano magnetic bead is characterized in that in the step 2), the concentration of the 4-mercaptobutyric acid is 0.8-1.2 mM, and Fe3O4The volume ratio of the-Au solution to the 4-mercaptobutyric acid is 6000: 1-4000: 1; the concentration of the 1-butanethiol is0.8～1.2mM，Fe3O4The volume ratio of the-Au solution to the 1-butanethiol is 6000: 1-4000: 1; the concentration of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide solution is 15-30 mg/mL, the concentration of N-hydroxysuccinimide is 5-15 mg/mL, and the solvent is PBS (phosphate buffered saline) with the pH value of 6.0 or Fe3O4The volume ratio of the-Au solution to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide solution is 1: 0.8-1: 1.5; the concentration of the LC3B antibody is 0.8-1.2 mg/mL, and the concentration is Fe3O4The volume ratio of the Au solution to the LC3B antibody is 50: 1-200: 1; the mass percent of the BSA is 0.2-2%; the magnetic separation is that a magnet attracts magnetic nano-particles Fe3O4-Au-LC3B antibody complex.
8. Fe produced by the production method according to claim 1 or 23O4-Au antibody nanobeads.
9. Fe as recited in claim 83O4Application of Au antibody nano magnetic beads in enrichment and purification of secretory autophagosomes.
10. Use according to claim 9, characterized in that it comprises the following steps: mixing Fe3O4Dropwise adding an Au antibody nano magnetic bead solution into an autophagosome solution, reacting for 10-120 min at 20-50 ℃, then washing with a PBS solution and suspending in PBS, and magnetically enriching and capturing autophagosomes.
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Cited By (2)
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|---|---|---|---|---|
| CN113804633A (en) * | 2021-09-15 | 2021-12-17 | 中国石油大学(华东) | Based on Au-Fe3O4Preparation method and application of nano-material salmonella identification immune probe |
| CN114602439A (en) * | 2021-07-01 | 2022-06-10 | 中国科学院上海硅酸盐研究所 | ACE2 modified magnetic bead, preparation method and application in SARS-CoV-2 virus detection |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101776691A (en) * | 2010-02-09 | 2010-07-14 | 北京大学 | Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles |
| CN102507714A (en) * | 2011-11-07 | 2012-06-20 | 华中师范大学 | Method for detecting biomarker of organophosphorus pesticide |
| CN102520164A (en) * | 2011-12-21 | 2012-06-27 | 深圳市老年医学研究所 | Method of improving sensitivity and specificity of immunochromatographic detection |
| CN103439495A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Listeria monocytogenes enrichment and rapid detection method |
| CN105738341A (en) * | 2016-02-22 | 2016-07-06 | 中国科学院生态环境研究中心 | Heavy metal mercury ion detection method |
| WO2018081684A1 (en) * | 2016-10-31 | 2018-05-03 | Musc Foundation For Research Development | Polypeptides for improved response to anti-cancer therapy |
| WO2018200829A1 (en) * | 2017-04-26 | 2018-11-01 | University Of South Florida | Nanoparticle-enhanced enzymatic immune biosensor assay for antigen detection |
-
2019
- 2019-09-26 CN CN201910916384.4A patent/CN110632295B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101776691A (en) * | 2010-02-09 | 2010-07-14 | 北京大学 | Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles |
| CN102507714A (en) * | 2011-11-07 | 2012-06-20 | 华中师范大学 | Method for detecting biomarker of organophosphorus pesticide |
| CN102520164A (en) * | 2011-12-21 | 2012-06-27 | 深圳市老年医学研究所 | Method of improving sensitivity and specificity of immunochromatographic detection |
| CN103439495A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Listeria monocytogenes enrichment and rapid detection method |
| CN105738341A (en) * | 2016-02-22 | 2016-07-06 | 中国科学院生态环境研究中心 | Heavy metal mercury ion detection method |
| WO2018081684A1 (en) * | 2016-10-31 | 2018-05-03 | Musc Foundation For Research Development | Polypeptides for improved response to anti-cancer therapy |
| WO2018200829A1 (en) * | 2017-04-26 | 2018-11-01 | University Of South Florida | Nanoparticle-enhanced enzymatic immune biosensor assay for antigen detection |
Non-Patent Citations (3)
| Title |
|---|
| ANTHONY J. DI PASQUA,ET AL: "Preparation of antibody-conjugated gold nanoparticles", 《MATERIALS LETTERS》 * |
| JINGYU YAO,ET AL: "Tumor-released autophagosomes induce IL-10-producing B cells with suppressive activity on T lymphocytes via TLR2-MyD88-NF-κB signal pathway", 《ONCOIMMUNOLOGY》 * |
| ZHI-FA WEN，ET AL: "Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1", 《JOURNAL FOR IMMUNOTHERAPY OF CANCER》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114602439A (en) * | 2021-07-01 | 2022-06-10 | 中国科学院上海硅酸盐研究所 | ACE2 modified magnetic bead, preparation method and application in SARS-CoV-2 virus detection |
| CN114602439B (en) * | 2021-07-01 | 2023-10-13 | 中国科学院上海硅酸盐研究所 | ACE2 modified magnetic bead, preparation method and application thereof in SARS-CoV-2 virus detection |
| CN113804633A (en) * | 2021-09-15 | 2021-12-17 | 中国石油大学(华东) | Based on Au-Fe3O4Preparation method and application of nano-material salmonella identification immune probe |
| CN113804633B (en) * | 2021-09-15 | 2023-07-11 | 中国石油大学(华东) | Based on Au-Fe 3 O 4 Preparation method and application of salmonella recognition immune probe of nano material |
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