CN112129939A - Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor - Google Patents
Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor Download PDFInfo
- Publication number
- CN112129939A CN112129939A CN202010776818.8A CN202010776818A CN112129939A CN 112129939 A CN112129939 A CN 112129939A CN 202010776818 A CN202010776818 A CN 202010776818A CN 112129939 A CN112129939 A CN 112129939A
- Authority
- CN
- China
- Prior art keywords
- sio
- exosomes
- tio
- prostate cancer
- psma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 title claims abstract description 137
- 210000001808 exosome Anatomy 0.000 title claims abstract description 132
- 229910052681 coesite Inorganic materials 0.000 title claims abstract description 75
- 229910052906 cristobalite Inorganic materials 0.000 title claims abstract description 75
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 229910052682 stishovite Inorganic materials 0.000 title claims abstract description 75
- 229910052905 tridymite Inorganic materials 0.000 title claims abstract description 75
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 title claims abstract description 45
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 title claims abstract description 45
- 206010060862 Prostate cancer Diseases 0.000 title claims abstract description 38
- 208000000236 Prostatic Neoplasms Diseases 0.000 title claims abstract description 38
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 31
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 title claims abstract 9
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 65
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 38
- 238000000926 separation method Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 14
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 13
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 230000005284 excitation Effects 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 7
- 238000007885 magnetic separation Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- -1 and after sonication Chemical compound 0.000 claims description 5
- YHWCPXVTRSHPNY-UHFFFAOYSA-N butan-1-olate;titanium(4+) Chemical compound [Ti+4].CCCC[O-].CCCC[O-].CCCC[O-].CCCC[O-] YHWCPXVTRSHPNY-UHFFFAOYSA-N 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 238000000527 sonication Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 210000005267 prostate cell Anatomy 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 abstract description 18
- 238000002965 ELISA Methods 0.000 abstract description 8
- 108091023037 Aptamer Proteins 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000000956 alloy Substances 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 12
- 238000005199 ultracentrifugation Methods 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102000007066 Prostate-Specific Antigen Human genes 0.000 description 4
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000003917 TEM image Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 description 2
- APQHKWPGGHMYKJ-UHFFFAOYSA-N Tributyltin oxide Chemical compound CCCC[Sn](CCCC)(CCCC)O[Sn](CCCC)(CCCC)CCCC APQHKWPGGHMYKJ-UHFFFAOYSA-N 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000499489 Castor canadensis Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 101000736929 Homo sapiens Proteasome subunit alpha type-1 Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 101500021084 Locusta migratoria 5 kDa peptide Proteins 0.000 description 1
- 235000011779 Menyanthes trifoliata Nutrition 0.000 description 1
- 108010001441 Phosphopeptides Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 102100036042 Proteasome subunit alpha type-1 Human genes 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention discloses a Fe-based alloy3O4@SiO2@TiO2Methods for nanoparticle enrichment and PSMA sensors for detecting prostate cancer exosomes. It is made of Fe3O4@SiO2@TiO2The nanoparticles enrich exosomes, and then hairpin-shaped PSMA aptamer sensors are constructed to quantify exosomes by measuring changes in fluorescence intensity. The detection method can successfully distinguish the prostate cancer cell exosomes from the normal prostate cancer cell exosomes, and the detection limit is 9 multiplied by 103mu.L/L. The method is used for clinical human serum samples of prostate cancer patients and normal human serum samples, can quickly and accurately detect the prostate cancer patients, and is verified by the traditional ELISA method.The method for enriching the exosomes is simple and rapid, has high enrichment efficiency, can complete the exosome enrichment within 8 minutes, and has the enrichment efficiency of 91.5 percent. High sensitivity, strong specificity and detection limit of 9X 103The dose is mu L, and clinical prostate cancer patients and healthy people can be remarkably distinguished.
Description
Technical Field
The invention is applied to exosome marker detection and analysisThe technical field relates to a method based on TiO2And rapid and accurate assay of prostate cancer exosomes by PSMA aptamers.
Background
Prostate cancer (PCa) is the most common solid malignancy in men worldwide. It has a high cure rate in the early stages, and therefore, the treatment of prostate cancer depends on early diagnosis. Prostate Specific Antigen (PSA) is a serum biomarker for the diagnosis of prostate cancer. However, while PSA has a high sensitivity, its specificity is poor, especially in men with serum PSA levels of 2-10 ng/mL, and therefore, urology requires other specific PCa biomarkers. Exosomes are biological vesicles secreted by normal and tumor cells, encapsulated within lipid bilayers, ranging in size from 30 to 200 nanometers. Exosomes play different roles in cell-cell communication and are found in a wide range of body fluids, including blood, urine, saliva, etc. Tumor exosomes contain tumor-specific proteins, RNAs, and are involved in tumor development, pathogen spread, and the like. Many studies have shown that tumor exosomes are ideal markers for diagnosing cancer, but at present, the methods for their isolation and detection remain challenging. Ultracentrifugation is the most commonly used method for exosome separation, but the steps are complicated and the separation efficiency is low. The commercial ELISA kit is based on an immunoaffinity method and takes CD-63, CD-9 (exosome surface antibody) and the like as targets to separate and detect exosomes, and has high sensitivity, but the process is complex and the cost is high.
TiO2Due to the reversible binding effect with phosphate group, the method is widely applied to separation and enrichment of phosphopeptide fragments and proteins, and the surface of an exosome is rich in phosphate group, so that TiO can be adopted2And separating and enriching exosomes. There are reports of TiO2The enrichment of exosomes can be successfully achieved, but a large amount of exosomes may be lost by subsequent solid-liquid separation using centrifugation.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for detecting prostate cancer exosomes quickly, sensitively and specifically.
The technical scheme of the invention is specifically introduced as follows.
The invention provides a Fe-based alloy3O4@SiO2@TiO2A method for detecting prostate cancer exosomes with a nanoparticle enrichment and PSMA sensor, comprising the steps of:
1) synthesis of Fe3O4@SiO2@TiO2The nano particles and a sample containing the prostate cell exosomes are incubated together and magnetically separated, so that the exosomes are separated;
2) and co-incubating the separated exosome and a Prostate Specific Membrane Antigen (PSMA) sensor, obtaining a fluorescence intensity signal, and quantifying the exosome by measuring the change of the fluorescence intensity.
Further, in step 1), Fe3O4@SiO2@TiO2The preparation method of the nano particles comprises the following steps:
mixing Fe3O4Dissolving in the mixture of ethanol, deionized water and ammonia water, performing ultrasonic treatment, adding TEOS, and continuously stirring at room temperature for reaction to obtain Fe3O4@SiO2；
Mixing Fe3O4@SiO2Dissolved in a mixture of ethanol and aqueous ammonia, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. Continuously stirring for reaction, washing the prepared product with ultrapure water and ethanol, and drying to obtain Fe3O4@SiO2@TiO2Nanoparticles.
Further, the preferred conditions for exosome isolation in the present invention are: 1.2mg Fe3O4@SiO2@TiO2And incubating for 8 minutes at normal temperature.
Further, in the step 1), after magnetic separation, alkali liquor is eluted.
Further, the elution step uses 10% ammonia water and magnetically separated Fe3O4@SiO2@TiO2The/exosome complex was shake co-incubated.
Preferably, the elution step uses 10% ammonia water to magnetically separate the Fe obtained3O4@SiO2@TiO2The/exosome complex was incubated at 4 ℃ with shaking for 10 min to reach full elution.
Further, in step 2), a hairpin-shaped PSMA sensor is constructed as follows: PSMA antibody sequences were designed to form hairpin-like structures with carboxytetramethylrhodamine fluorophore (TAMRA) and azobenzoic acid (Dabcyl) quenching groups at the respective 3 'and 5' end modifications.
Further, in the step 2), the nucleotide sequence of the PSMA antibody is shown as SEQ No. 3.
Further, the PSMA sensor concentration was 400nM, and incubated at 25 degrees for 30 minutes.
Further, in step 2), the conditions for obtaining the fluorescence intensity: excitation 557nm and emission 580 nm.
The invention also provides Fe3O4@SiO2@TiO2The preparation method of the nano-particles comprises the following steps:
mixing Fe3O4Dissolving in the mixture of ethanol, deionized water and ammonia water, performing ultrasonic treatment, adding TEOS, and continuously stirring at room temperature for reaction to obtain Fe3O4@SiO2；
Mixing Fe3O4@SiO2Dissolved in a mixture of ethanol and aqueous ammonia, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. Continuously stirring for reaction, washing the prepared product with ultrapure water and ethanol, and drying to obtain Fe3O4@SiO2@TiO2Nanoparticles.
The invention also provides Fe3O4@SiO2@TiO2Use of nanoparticles in prostate cancer exosome detection, using Fe3O4@SiO2@TiO2The nano particles and the exosomes are incubated together and magnetically separated, so that the exosomes are separated and enriched.
The invention provides a prostate cancer exosome detection device, which adopts Fe3O4@SiO2@TiO2The nano particles separate and enrich exosomes and are separated and enriched with PSMA sensors were co-incubated.
In particular, in one embodiment of the present invention,
the invention synthesizes Fe3O4@SiO2@TiO2Nanoparticles are used for exosome enrichment, based mainly on reversible binding of TiO2 to phosphate groups. Prostate Specific Membrane Antigen (PSMA) sensors were then designed.
PSMA is a specific protein on the surface of prostate cancer exosome, is highly expressed on the surface of prostate cancer exosome and is lowly expressed on normal exosome, and the specificity detection of the prostate cancer exosome is realized by detecting the PSMA. The PSMA sensor is of a hairpin-shaped structure consisting of a PSMA protein aptamer, a TAMRA fluorescent group and a Dabcyl quenching group, after construction is completed, the fluorescence of the TAMRA is quenched due to a fluorescence resonance energy transfer effect of the PSMA sensor, when an exosome is encountered, the PSMA aptamer is specifically combined with the PSMA protein on the surface of the exosome, the fluorescence resonance energy transfer effect disappears, the TAMRA emits fluorescence again, and detection of the exosome is achieved through change of the fluorescence.
Compared with the prior art, the invention has the following beneficial effects:
the invention synthesizes Fe3O4@SiO2@ TiO2 with Fe3O4As core, SiO2The method is favorable for the connection of two materials, and can realize rapid magnetic separation after enrichment is completed, thereby reducing the loss of exosomes. Binding of Fe according to the invention3O4Advantages of fast magnetic separation and SiO2Extensive compatibility, synthesize Fe3O4@SiO2@TiO2The nanoparticles separate exosomes with rapid and efficient efficiency. Meanwhile, a prostate cancer specific protein PSMA sensor is designed, and prostate cancer exosomes are specifically identified. The invention integrates enrichment and detection into a whole, and realizes rapid and accurate analysis and identification of the prostate cancer exosome
Specifically, the exosome enrichment method is based on Fe3O4@ SiO2@ TiO2 nanoparticles. The enrichment of exosomes is based on TiO2Specific binding to phosphate groups, TiO2The size is about 600 nanometers, and compared with the size of an exosome, the specific surface area is large, and the size can be measuredTo bind more exosomes. Therefore, the enrichment efficiency is high and is 91.5%, and the Fe3O4@ SiO2@ TiO2 nano-particles synthesized by the invention can also enrich other polypeptides, proteins and the like containing phosphoric acid groups. With Fe3O4As the kernel, the magnetic separation can be rapidly carried out after enrichment, the enrichment can be completed within 8 minutes, and the method is simple and rapid. The method for detecting the exosome has high sensitivity, strong specificity and detection limit of 9 multiplied by 103mu.L/uL, can significantly distinguish clinical prostate cancer patients from healthy people (P)<0.01) and verified by a conventional ELISA method. The invention comprises two parts of enrichment and exosome detection, the separation method is simple and quick, the efficiency is high, the detection method has high sensitivity and good specificity, and the method is suitable for early diagnosis of clinical prostate cancer.
Drawings
FIG. 1 is Fe3O4@SiO2@TiO2And (4) characterization of (1). (a) Fe3O4Transmission Electron Microscope (TEM) images of (a). Scale, 100 nm. (b) Fe3O4@SiO2The TEM of (4). Scale 0.2 μm. (c) Fe3O4@SiO2@TiO2The TEM of (4). The scale is 500 nm. (d) Fe3O4@SiO2@TiO2And (5) magnifying the image by using a transmission electron microscope. The scale is 200 nm. (e) Fe3O4@SiO2@TiO2Scanning Electron Microscope (SEM) images of (a). The scale is 200 nm. (f) Fe3O4@SiO2@TiO2Particle size distribution of (d).
FIG. 2 shows the optimal conditions for fluorescence imaging and exosome separation. (a-c) Bright field and fluorescence image of exosomes, Fe3O4@SiO2@TiO2Exosome, magnetic separation of washed laundry after repeated washing. Scale 50 μm. (d) Optimizing Fe3O4@SiO2@TiO2Amount used for exosome isolation. (e) The incubation time for exosome isolation was optimized. (f) Fe in serum and PBS3O4@SiO2@TiO2The efficiency of nanoparticle and ultracentrifugation for exosome separation was compared. All experiments were performed at least three times independently. Error bars represent the standard deviation of triplicate measurements for each sample.
FIG. 3 shows the optimal detection conditions for exosomes. (a) Fluorescence spectra of single detection sensor 1, sensor 2 and sensor 3. (b) Scatter plots of the same exosome content in the presence of different concentration sensors. (c) Fluorescence intensity time and temperature profiles. (d) Fluorescence spectrum. At different concentrations of exosomes (a-h: 0.09, 0.69, 6.93, 2.08, 3.12, 6.25, 8.3, 10, 12.5 × 10, respectively5μ L/μ L). (e) Linear relationship of exosomes of different concentrations to the sensor. (f) A specificity histogram. Error bars represent standard error based on three replicate experiments.
FIG. 4 exosome quantification of clinical serum samples. (a) The detection result of the PSMA sensor. (b) Dot plots for PSMA sensor clinical sample analysis. (c) And (5) detecting results by ELISA. (d) Dot-plot of ELISA clinical sample analysis.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the method of the present invention are not limited thereto.
The reagents and instrumentation used in the examples were as follows:
1. reagent
Fe3O4Purchased from beaver, Suzhou
Tetraethoxysilane (TEOS) and tetrabutyl titanate (TBOT) are both produced by Shanghai Hu test Co., Ltd
PBS buffer (pH 7.4), ammonia, 0.22 μm ultrafiltration membrane, PKH26 were purchased from sigma, USA
ELISA was purchased from SBI of America
PSMA aptamers were purchased from Shanghai Biotech
2. Instrument for measuring the position of a moving object
Transmission Electron microscope 200KV (model: JEM 2011, manufacturer: JEOL Ltd., Japan), F7000F fluorescence spectrometer (Stand alone)
Japan), heliodynamic S-4800 field emission scanning electron microscope, ZS90 nanometers (morvin, uk).
Example 1
Synthesis of Fe3O4@SiO2@TiO2Nanoparticles
1. 0.15g of Fe3O4Dissolved in a mixture of ethanol (280mL), deionized water (70mL) and aqueous ammonia (5mL,28 wt%) and sonicated for 15 min. Then 4mL TEOS is added, and the mixture is continuously stirred and reacted for 10h at room temperature to obtain Fe3O4@SiO2. FIG. 1a is Fe3O4TEM image of (1 b) Fe3O4@SiO2A TEM image of (a). The two figures illustrate SiO2Successfully wrapped in Fe3O4And (5) outside.
2. 0.05g of Fe3O4@SiO2Dissolved in a mixture of ethanol (100mL) and aqueous ammonia (0.3mL,28 wt%), sonicated for 15min, and 0.75mL of ethanol-dissolved TBOT was added dropwise with continuous stirring. Continuously stirring and reacting for 24h at 45 ℃, washing the prepared product with ultrapure water and ethanol for three times, and drying at 60 ℃ overnight. FIG. 1c is Fe3O4@SiO2@TiO2TEM image of (1 d) Fe3O4@SiO2@TiO2Enlarged partial views of (A), illustrating Fe3O4@SiO2@TiO2The synthesis is successful, and the particle size is uniform. FIG. 1e is Fe3O4@SiO2@TiO2The SEM image of (B) shows successful synthesis and uniform particle size. FIG. 1f is Fe3O4@SiO2@TiO2The particle size distribution diagram of (a) shows that the particle size is about 600 nm.
Example 2
Exosome isolation.
1. Two parallel samples were prepared: LNCaP exosome solution (10)7μ L/μ L). One sample of exosomes was stained with PKH26 dye. Another sample was stained with 1.2mg Fe after exosome staining3O4@SiO2@TiO2After incubation at room temperature for 8 minutes, magnetic separation was rapidly performed using a magnetic frame, and washing was repeated 3 times with PBS. FIG. 2a is a graph of the bright field and fluorescence after exosome staining, and FIG. 2b is Fe3O4@SiO2@TiO2Bright field and fluorescence of exosomes, fig. 2c is a bright field and fluorescence of washes after repeated washes. These three sheets illustrate Fe3O4@SiO2@TiO2Can successfully separate the secretionBody, and the number of exosomes enriched is large.
2. Preparation of exosome concentration of 107Mu.l, stained with PKH26 and fluorescence intensity recorded as F0. Preparing exosomes of the same concentration with 0.2, 0.4, 0.6, 0.8, 1.2, 1.4, 1.6mg Fe3O4@SiO2@TiO2The nanoparticles were incubated for 8 minutes, magnetically separated, washed repeatedly 3 times with 150. mu.L PBS, and the fluorescence intensity of the supernatant was measured as F1Each set of experiments was performed in triplicate and F was calculated0-F1/F0The separation efficiency is obtained. FIG. 2d shows different masses of Fe3O4@SiO2@TiO2Influence on exosome separation efficiency. With Fe3O4@SiO2@TiO2The increase in the amount (0.2-1.2mg) resulted in a gradual increase in exosome-separating efficiency followed by Fe3O4@SiO2@TiO2The increase in the amount (1.2-1.6mg) and the slow decrease in exosome separation efficiency, therefore 1.2mg Fe was chosen3O4@SiO2@TiO2As the optimum condition.
3. The fluorescent intensity of exosome staining was recorded as F0(107μ L), same concentration of exosomes and optimal Fe obtained in step 23O4@SiO2@TiO2Incubated for 1-10 min each by mass, magnetically separated rapidly, and washed repeatedly 3 times with PBS, with fluorescence intensity recorded as F1Each set of experiments was performed in triplicate and F was calculated0-F1/F0The separation efficiency is obtained. Figure 2e is the effect of different incubation times on exosome separation efficiency. The separation efficiency gradually increased with increasing incubation time (1-10 min) and then hardly changed, so that an incubation time of 8 min was chosen.
4. Preparation of model exosomes (exosomes secreted by standard cells) dissolved in PBS (10)7μ L), stained and recorded fluorescence intensity as F0The fluorescence intensity of the supernatant obtained by the conventional ultracentrifugation method after staining exosomes of the same concentration was recorded as F1Same concentration of exosomes stained with 1.2mg Fe3O4@SiO2@TiO2Incubated for 8 min, fast magneticThe separation was repeated three times with PBS, and the fluorescence intensity of the supernatant was recorded as F1Each set of experiments was performed in triplicate. By calculating F0-F1/F0The separation efficiency is obtained. Preparation of serum exosomes (10)7μ L), stained and recorded fluorescence intensity as F0The fluorescence intensity of the supernatant obtained by the conventional ultracentrifugation method after staining exosomes of the same concentration was recorded as F1Same concentration of exosomes stained with 1.2mg Fe3O4@SiO2@TiO2Incubated for 8 min, magnetically separated rapidly, washed three times with PBS and the fluorescence intensity of the supernatant noted F1Each set of experiments was performed in triplicate. By calculating F0-F1/F0The separation efficiency is obtained. FIG. 2f shows the conventional ultracentrifugation method and Fe3O4@SiO2@TiO2Exosome separation method the contrast map of model exosomes and serum exosomes was separated, Fe3O4@SiO2@TiO2The separation efficiency of the separated exosomes, whether the separated exosomes are model exosomes or serum exosomes, is far higher than that of the traditional ultracentrifugation method.
Example 3 optimal condition exploration for exosome detection, comprising the following steps:
optimization of PSMA sensor. Three PSMA sensors were first designed and constructed, with the PSMA1 sequence shown in the following table:
after synthesis, the three sensors were subjected to high temperature 100 ℃ chain unwinding, then to an annealing experiment, and then diluted to a concentration of 200nM and their fluorescence intensities measured (excitation: 557nM, emission 580 nM). Figure 3a is a plot of the fluorescence intensity of three sensors of the same concentration, with sensor 3 having the lowest fluorescence intensity and the lowest background, and therefore sensor 3 was experimentally selected for exosome detection.
2. An exosome concentration of 106/. mu.L was prepared, incubated with sensor 3 at different concentrations (0, 20, 50, 100, 200, 300, 400, 500, 600nM) for 30 minutes at ambient temperature, and the fluorescence intensity (excitation: 557nM, emission 580nM) was measured, with each set of experiments performed in triplicate. FIG. 3b shows the fluorescence intensity obtained by incubating the same concentration of exosomes with different concentrations of sensor 3, and the fluorescence intensity is almost constant after increasing with the increase of the concentration of sensor 3, so 400nM was chosen as the detection concentration.
3. Preparation of exosome concentration of 106mu.L, incubated with 200nM sensor 3 at different temperatures (4 deg., 25 deg., 37 deg.) for different times and measured for fluorescence intensity (excitation: 557nM, emission 580nM), with each set of experiments being performed in triplicate. FIG. 3c is a graph showing the change of fluorescence intensity with time at different temperatures. The fluorescence intensity increases with time, the fluorescence increases more rapidly at 25 degrees and is stronger, so 25 degrees, 30 minutes is chosen as the detection temperature and time.
PSMA sensor concentration 400nM, respectively at different concentrations (0.09, 0.69, 0.93, 2.08, 3.12, 6.25, 8.3, 10, 12.5 × 105mu.L/uL of LNCaP exosomes were incubated at 25 ℃ for 30 min and the fluorescence intensity was measured (excitation: 557nm, emission 580 nm). FIG. 3d is a graph of the fluorescent response signal of the PSMA sensor to exosomes of different concentrations. The fluorescence intensity gradually increased with increasing exosome concentration.
5. Step 4, each set of experiments is performed in parallel for three times, the obtained average fluorescence intensity and the exosome concentration are subjected to curve fitting, as shown in fig. 3e, the fluorescence intensity and the exosome concentration form a good linear relation y of 39.62X +131.14, and R20.9942, indicating the feasibility of the PSMA sensor to detect exosomes, and the practical detection limit was 9 × 103mu.L/L.
Specificity of PSMA sensor. Preparation of LNCaP exosome concentration at 106One/μ L of PrEC exosomes and breast cancer MB-231 exosomes with the same concentration were incubated with 400nM PSMA sensors at 25 ℃ for 30 minutes, and fluorescence intensity (excitation: 557nM, emission 580nM) was measured, with each set of experiments being performed in triplicate. FIG. 3f is the fluorescent response signal of the same concentration of different types of exosomes to the PSMA sensor. The fluorescence response signal of the LNCaP exosome is maximum, the MB-231 exosome is second, and the response of the PrEC exosome is minimum, which indicates that the PSMA sensor has specificity to the LNCaP exosome and can successfully distinguish prostate cancer from normal people.
Example 4
Detection of serum exosomes
1. The serum samples of patients with prostate cancer and normal human are filtered through a 0.22 mu m filter membrane, and the macromolecules are removed for later use. Serum passes through the Fe3O4@SiO2@TiO2After separation and detection by PSMA sensor, fluorescence response signals (excitation: 557nm, emission 580nm) are obtained and converted into exosome concentration according to linear relation. The same samples were experimentally verified by ELISA to obtain the corresponding exosome concentrations. Each set of experiments was performed in triplicate. FIG. 4a shows the results of the PSMA sensor on serum from prostate cancer patients and normal human serum. The serum of prostate cancer patients contains a higher amount of exosomes than that of normal persons. FIG. 4b is a dot-plot representation of the data of FIG. 4a, showing significant differences between prostate cancer serum and normal, P<0.01. FIG. 4c shows the results of the commercial ELISA on the same samples. Compared with the method, the prostate cancer serum exosomes are far larger than normal human serum exosomes, and the feasibility of the method for detecting the exosomes is shown. FIG. 4d is a dot plot of ELISA4c data showing significant differences between prostate cancer sera and normal persons, P<0.01. ELISA is a detection method for all exosomes, including normal exosomes. Our approach is based on the specificity of PSMA, which is highly specific for detecting prostate cancer exosomes. Meanwhile, compared with the traditional method, the detection method is simple and convenient to operate, and can complete detection within 30 minutes.
SEQUENCE LISTING
<110> Ningbo university
<120> method for detecting prostate cancer exosomes based on Fe3O4@ SiO2@ TiO2 nanoparticle enrichment and PSMA sensor
Method of
<130> 2020
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 60
<212> DNA
<213> Artificial sequence
<400> 1
accccagcgt tttcgctttt gcgttttggg tcatctgctt acgatagcaa tgcttggggt 60
<210> 2
<211> 60
<212> DNA
<213> Artificial sequence
<400> 2
accccagcgt tttcgctttt gcgttttggg tcatctgctt acgatagcaa tgcttggggt 60
<210> 3
<211> 40
<212> DNA
<213> Artificial sequence
<400> 3
cggacgcttt tcgcttttgc gttttgggtc atctgctccg 40
Claims (10)
1. Based on Fe3O4@SiO2@TiO2A method for detecting prostate cancer exosomes with a nanoparticle enrichment and PSMA sensor, comprising the steps of:
1) synthesis of Fe3O4@SiO2@TiO2The nano particles and a sample containing the prostate cell exosomes are incubated together and magnetically separated, so that the exosomes are separated;
2) and co-incubating the exosome obtained by separation with a PSMA sensor, obtaining a fluorescence intensity signal, and quantifying the exosome by measuring the change of the fluorescence intensity.
2. The method according to claim 1, wherein in step 1), Fe3O4@SiO2@TiO2The preparation method of the nano particles comprises the following steps:
1) mixing Fe3O4Dissolving in the mixture of ethanol, deionized water and ammonia water, performing ultrasonic treatment, adding TEOS, and continuously stirring at room temperature for reaction to obtain Fe3O4@SiO2；
2) Mixing Fe3O4@SiO2Dissolved in a mixture of ethanol and aqueous ammonia, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. Continuously stirring for reaction, washing the prepared product with ultrapure water and ethanol, and drying to obtain Fe3O4@SiO2@TiO2Nanoparticles.
3. The method of claim 1, wherein in step 1), after the magnetic separation, a lye is eluted.
4. The method of claim 3, wherein the elution step uses 10% ammonia water to elute Fe that is magnetically separated from Fe3O4@SiO2@TiO2The/exosome complex was shake co-incubated.
5. The method of claim 1, wherein in step 2), the hairpin PSMA sensor is constructed as follows: through designing a PSMA antibody sequence, a hairpin structure is formed, and carboxyl tetramethyl rhodamine fluorescent group and azobenzoic acid quenching group are modified at the corresponding 3 'end and 5'.
6. The method according to claim 6, wherein the nucleotide sequence of the PSMA antibody in step 2) is shown as SEQ No. 3.
7. The method according to claim 1, wherein in step 2), the conditions for obtaining the fluorescence intensity are: excitation 557nm and emission 580 nm.
8.Fe3O4@SiO2@TiO2Nanoparticles, characterized in that the preparation method of the nanoparticles is as follows:
1) mixing Fe3O4Dissolving in the mixture of ethanol, deionized water and ammonia water, performing ultrasonic treatment, adding TEOS, and continuously stirring at room temperature for reaction to obtain Fe3O4@SiO2；
2) Mixing Fe3O4@SiO2Dissolved in a mixture of ethanol and aqueous ammonia, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. Continuously stirring for reaction, washing the prepared product with ultrapure water and ethanol, and drying to obtain Fe3O4@SiO2@TiO2Nanoparticles.
9.Fe3O4@SiO2@TiO2Use of nanoparticles in the detection of prostate cancer exosomes, characterized in that Fe3O4@SiO2@TiO2The nano particles and the exosomes are incubated together and magnetically separated, so that the exosomes are separated and enriched.
10. The prostate cancer exosome detection device is characterized in that Fe is adopted in the detection device3O4@SiO2@TiO2The nanoparticles were isolated and enriched for exosomes and co-incubated with PSMA sensors.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010776818.8A CN112129939A (en) | 2020-08-05 | 2020-08-05 | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor |
| PCT/CN2021/082043 WO2022027976A1 (en) | 2020-08-05 | 2021-03-22 | Method for detecting prostate cancer exosome on basis of fe3o4@sio2@tio2 nanoparticle enrichment and psma sensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010776818.8A CN112129939A (en) | 2020-08-05 | 2020-08-05 | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112129939A true CN112129939A (en) | 2020-12-25 |
Family
ID=73850644
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010776818.8A Pending CN112129939A (en) | 2020-08-05 | 2020-08-05 | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112129939A (en) |
| WO (1) | WO2022027976A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402052A (en) * | 2018-10-15 | 2019-03-01 | 宁波美晶医疗技术有限公司 | It is a kind of for capturing the preparation method and applications of the magnetic nanoparticle of excretion body in blood |
| CN112858680A (en) * | 2021-01-14 | 2021-05-28 | 四川大学 | Fluorescence resonance energy transfer magnetic sensor for exosome detection and preparation method thereof |
| WO2022027976A1 (en) * | 2020-08-05 | 2022-02-10 | 宁波大学 | Method for detecting prostate cancer exosome on basis of fe3o4@sio2@tio2 nanoparticle enrichment and psma sensor |
| CN114459877A (en) * | 2021-12-17 | 2022-05-10 | 中国计量科学研究院 | DNA tetrahedral composite magnetic nano material for enriching exosome and preparation |
| CN115057472A (en) * | 2022-06-21 | 2022-09-16 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
| CN115992092A (en) * | 2023-02-18 | 2023-04-21 | 浙江洛兮医学检验实验室有限公司 | Method for extracting exosomes based on transition metal oxyhydroxide |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116593693B (en) * | 2023-07-11 | 2023-09-19 | 四川大学华西医院 | Magnetic aptamer probe, preparation method and application thereof, and migration body enrichment method |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1447117A (en) * | 2002-03-21 | 2003-10-08 | 生命扫描有限公司 | Direct immunity sensor and analyzing method |
| CN101812528A (en) * | 2010-04-26 | 2010-08-25 | 湖南大学 | Switch mode aptamer probe and application thereof in tumor living cell and vital detection |
| US20110236495A1 (en) * | 2010-03-26 | 2011-09-29 | Board Of Trustees Of The University Of Arkansas | Anti-cancer nanoparticle compositions and methods of use |
| CN102921435A (en) * | 2012-10-31 | 2013-02-13 | 湖北大学 | Magnetic Fe3O4/SiO2/TiO2/quantum dot compounded nanometer photocatalyst and preparation method and application thereof |
| US20170014521A1 (en) * | 2013-09-27 | 2017-01-19 | Wake Forest University Health Sciences | Pharmaceutical compositions for high-capacity targeted delivery |
| CN108387746A (en) * | 2018-03-02 | 2018-08-10 | 浙江大学 | A kind of superparamagnetic nanoparticle and preparation method thereof of capture excretion body and specific excretion body electrochemiluminescent immunoassay immue quantitative detection reagent box |
| CN109187473A (en) * | 2018-09-15 | 2019-01-11 | 福建医科大学 | Fluorescence resonance energy transfer based on up-conversion nano material and tetramethylrhodamine is used for the detection of excretion body |
| CN110082531A (en) * | 2019-04-11 | 2019-08-02 | 南方医科大学南方医院 | A kind of tumour excretion body nano fluorescent detection kit and its application |
| CN110118765A (en) * | 2018-02-05 | 2019-08-13 | 武汉大学 | A kind of preparation of SERS substrate and its application in terms of cancer detection |
| CN110343664A (en) * | 2018-04-03 | 2019-10-18 | 中国人民解放军军事科学院军事医学研究院 | The method for extracting excretion body and excretion body protein |
| CN110376260A (en) * | 2019-07-18 | 2019-10-25 | 济南大学 | Detect the preparation method of the optical electro-chemistry aptamer sensor of prostate-specific antigen |
| CN110531061A (en) * | 2019-07-11 | 2019-12-03 | 宁波大学 | A kind of preparation method and its recyclable immune detection application of ferroso-ferric oxide/titanium dioxide/silver core-shell nano material |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101596456B (en) * | 2009-07-06 | 2011-06-29 | 中北大学 | TiO2/SiO2/Fe3O4 nano particle with nuclear shell structure and manufacturing method thereof |
| CN101776683A (en) * | 2010-01-06 | 2010-07-14 | 东华大学 | Luminescent and magnetic nano material-based method for detecting carcinoembryonic antigen |
| US20210389314A1 (en) * | 2017-08-22 | 2021-12-16 | Hiroshima University | Method for isolating exosome and exosome isolation kit |
| KR102308738B1 (en) * | 2018-12-18 | 2021-10-05 | 한국과학기술연구원 | A novel method for isolating extracellular visicles |
| CN110297030B (en) * | 2019-08-02 | 2021-11-16 | 盐城工学院 | Construction method of high-selectivity progesterone photoelectrochemical biosensor |
| CN110596060B (en) * | 2019-09-12 | 2020-08-21 | 江南大学 | Construction method and application of fluorescence sensor in spectral analysis for detecting prostate specific antigen |
| CN112129939A (en) * | 2020-08-05 | 2020-12-25 | 宁波大学 | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor |
-
2020
- 2020-08-05 CN CN202010776818.8A patent/CN112129939A/en active Pending
-
2021
- 2021-03-22 WO PCT/CN2021/082043 patent/WO2022027976A1/en active Application Filing
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1447117A (en) * | 2002-03-21 | 2003-10-08 | 生命扫描有限公司 | Direct immunity sensor and analyzing method |
| US20110236495A1 (en) * | 2010-03-26 | 2011-09-29 | Board Of Trustees Of The University Of Arkansas | Anti-cancer nanoparticle compositions and methods of use |
| CN101812528A (en) * | 2010-04-26 | 2010-08-25 | 湖南大学 | Switch mode aptamer probe and application thereof in tumor living cell and vital detection |
| CN102921435A (en) * | 2012-10-31 | 2013-02-13 | 湖北大学 | Magnetic Fe3O4/SiO2/TiO2/quantum dot compounded nanometer photocatalyst and preparation method and application thereof |
| US20170014521A1 (en) * | 2013-09-27 | 2017-01-19 | Wake Forest University Health Sciences | Pharmaceutical compositions for high-capacity targeted delivery |
| CN110118765A (en) * | 2018-02-05 | 2019-08-13 | 武汉大学 | A kind of preparation of SERS substrate and its application in terms of cancer detection |
| CN108387746A (en) * | 2018-03-02 | 2018-08-10 | 浙江大学 | A kind of superparamagnetic nanoparticle and preparation method thereof of capture excretion body and specific excretion body electrochemiluminescent immunoassay immue quantitative detection reagent box |
| CN110343664A (en) * | 2018-04-03 | 2019-10-18 | 中国人民解放军军事科学院军事医学研究院 | The method for extracting excretion body and excretion body protein |
| CN109187473A (en) * | 2018-09-15 | 2019-01-11 | 福建医科大学 | Fluorescence resonance energy transfer based on up-conversion nano material and tetramethylrhodamine is used for the detection of excretion body |
| CN110082531A (en) * | 2019-04-11 | 2019-08-02 | 南方医科大学南方医院 | A kind of tumour excretion body nano fluorescent detection kit and its application |
| CN110531061A (en) * | 2019-07-11 | 2019-12-03 | 宁波大学 | A kind of preparation method and its recyclable immune detection application of ferroso-ferric oxide/titanium dioxide/silver core-shell nano material |
| CN110376260A (en) * | 2019-07-18 | 2019-10-25 | 济南大学 | Detect the preparation method of the optical electro-chemistry aptamer sensor of prostate-specific antigen |
Non-Patent Citations (3)
| Title |
|---|
| QIUXIA ZHANG ET AL.: "Universal Ti3C2 MXenes Based Self-Standard Ratiometric Fluorescence Resonance Energy Transfer Platform for Highly Sensitive Detection of Exosomes", 《ANALYTICAL CHEMISTRY》 * |
| 张楠等: "高磁性微球的制备及在冶金废水处理中的应用", 《武钢技术》 * |
| 王子帅等: "磁性纳米Fe3O4@TiO2可见光下光催化还原Cr（Ⅵ）", 《化工学报》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402052A (en) * | 2018-10-15 | 2019-03-01 | 宁波美晶医疗技术有限公司 | It is a kind of for capturing the preparation method and applications of the magnetic nanoparticle of excretion body in blood |
| CN109402052B (en) * | 2018-10-15 | 2022-02-22 | 宁波美晶医疗技术有限公司 | Preparation method and application of magnetic nanoparticles for capturing exosomes in blood |
| WO2022027976A1 (en) * | 2020-08-05 | 2022-02-10 | 宁波大学 | Method for detecting prostate cancer exosome on basis of fe3o4@sio2@tio2 nanoparticle enrichment and psma sensor |
| CN112858680A (en) * | 2021-01-14 | 2021-05-28 | 四川大学 | Fluorescence resonance energy transfer magnetic sensor for exosome detection and preparation method thereof |
| CN114459877A (en) * | 2021-12-17 | 2022-05-10 | 中国计量科学研究院 | DNA tetrahedral composite magnetic nano material for enriching exosome and preparation |
| CN115057472A (en) * | 2022-06-21 | 2022-09-16 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
| CN115057472B (en) * | 2022-06-21 | 2023-10-27 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
| CN115992092A (en) * | 2023-02-18 | 2023-04-21 | 浙江洛兮医学检验实验室有限公司 | Method for extracting exosomes based on transition metal oxyhydroxide |
| CN115992092B (en) * | 2023-02-18 | 2023-11-17 | 湖州科元生物科技有限公司 | Method for extracting exosomes based on transition metal oxyhydroxide |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2022027976A1 (en) | 2022-02-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112129939A (en) | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor | |
| Huang et al. | A dual-signal amplification platform for sensitive fluorescence biosensing of leukemia-derived exosomes | |
| Wang et al. | DNase I enzyme-aided fluorescence signal amplification based on graphene oxide-DNA aptamer interactions for colorectal cancer exosome detection | |
| Tian et al. | Highly sensitive detection of exosomes by SERS using gold nanostar@ Raman reporter@ nanoshell structures modified with a bivalent cholesterol-labeled DNA anchor | |
| Wu et al. | Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin a using upconversion nanoparticles as labels | |
| Liang et al. | Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles | |
| CN109187473B (en) | Energy resonance transfer technology based on up-conversion fluorescence for detecting exosome | |
| CN110095608A (en) | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly | |
| CN106525814A (en) | PSA detection method based on magnetic core-gold satellite assembly body | |
| Yao et al. | Fast detection of E. coli with a novel fluorescent biosensor based on a FRET system between UCNPs and GO@ Fe 3 O 4 in urine specimens | |
| CN106366196B (en) | EpCAM antibody immunomagnetic beads and preparation method thereof | |
| CN108469445B (en) | Kit for detecting content of cancer antigen in blood without background based on lanthanide fluorescent nanoparticles excited by X-rays and preparation method thereof | |
| CN106366197B (en) | HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic beads and preparation method thereof | |
| Li et al. | Rapid and specific detection nanoplatform of serum exosomes for prostate cancer diagnosis | |
| Song et al. | The chemical redox modulated switch-on fluorescence of carbon dots for probing alkaline phosphatase and its application in an immunoassay | |
| CN108802360B (en) | Kit capable of exchanging copper and ceruloplasmin in serum and used for one-step simultaneous detection, preparation method and application | |
| CN112501173A (en) | GPC1DNA aptamer and application thereof | |
| Hou et al. | A sandwich-type surface-enhanced Raman scattering sensor using dual aptamers and gold nanoparticles for the detection of tumor extracellular vesicles | |
| Li et al. | Graphene quantum dots-based magnetic relaxation switch involving magnetic separation for enhanced performances of endoglin detection using ultra-low-field nuclear magnetic resonance relaxometry | |
| Li et al. | A magnetic nanoparticle-based aptasensor for selective and sensitive determination of lysozyme with strongly scattering silver nanoparticles | |
| Kong et al. | Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation | |
| CN105259118B (en) | A kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal | |
| Zhu et al. | Competitive adsorption on gold nanoparticles for human papillomavirus 16 L1 protein detection by LDI-MS | |
| CN110806484A (en) | Sarcosine detection method based on single-walled carbon nanotube and aptamer | |
| CN113504374B (en) | Utilize Fe 3 O 4 Method for realizing accurate detection of beta-amyloid by adopting nano-composite with @ MXene core-shell structure |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201225 |