CN106000814A - Halloysite coating used for capturing tumour cells and preparation method and application thereof - Google Patents
Halloysite coating used for capturing tumour cells and preparation method and application thereof Download PDFInfo
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- CN106000814A CN106000814A CN201610508849.9A CN201610508849A CN106000814A CN 106000814 A CN106000814 A CN 106000814A CN 201610508849 A CN201610508849 A CN 201610508849A CN 106000814 A CN106000814 A CN 106000814A
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- halloysite
- coating
- tumor cells
- capturing
- cells
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- HPTYUNKZVDYXLP-UHFFFAOYSA-N aluminum;trihydroxy(trihydroxysilyloxy)silane;hydrate Chemical compound O.[Al].[Al].O[Si](O)(O)O[Si](O)(O)O HPTYUNKZVDYXLP-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 229910052621 halloysite Inorganic materials 0.000 title claims abstract description 102
- 238000000576 coating method Methods 0.000 title claims abstract description 78
- 239000011248 coating agent Substances 0.000 title claims abstract description 77
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims abstract description 85
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000000463 material Substances 0.000 claims abstract description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 28
- 201000011510 cancer Diseases 0.000 claims description 25
- 239000006185 dispersion Substances 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000004094 surface-active agent Substances 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 9
- 239000008055 phosphate buffer solution Substances 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 238000005507 spraying Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 5
- 238000010168 coupling process Methods 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 4
- XDLMVUHYZWKMMD-UHFFFAOYSA-N 3-trimethoxysilylpropyl 2-methylprop-2-enoate Chemical compound CO[Si](OC)(OC)CCCOC(=O)C(C)=C XDLMVUHYZWKMMD-UHFFFAOYSA-N 0.000 claims description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- 206010033128 Ovarian cancer Diseases 0.000 claims description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 4
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 4
- 201000007983 brain glioma Diseases 0.000 claims description 4
- 238000013399 early diagnosis Methods 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 238000010907 mechanical stirring Methods 0.000 claims description 4
- 229920001467 poly(styrenesulfonates) Polymers 0.000 claims description 4
- 229910000077 silane Inorganic materials 0.000 claims description 4
- 229910052710 silicon Inorganic materials 0.000 claims description 4
- 239000010703 silicon Substances 0.000 claims description 4
- 229940006186 sodium polystyrene sulfonate Drugs 0.000 claims description 4
- UUEWCQRISZBELL-UHFFFAOYSA-N 3-trimethoxysilylpropane-1-thiol Chemical compound CO[Si](OC)(OC)CCCS UUEWCQRISZBELL-UHFFFAOYSA-N 0.000 claims description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims description 3
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 claims description 3
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 claims description 3
- 235000019982 sodium hexametaphosphate Nutrition 0.000 claims description 3
- 229940048086 sodium pyrophosphate Drugs 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 235000019818 tetrasodium diphosphate Nutrition 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
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- 229940116978 human epidermal growth factor Drugs 0.000 claims description 2
- 210000004072 lung Anatomy 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 claims description 2
- 201000008968 osteosarcoma Diseases 0.000 claims description 2
- 239000002985 plastic film Substances 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 239000010453 quartz Substances 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 abstract description 13
- 239000008280 blood Substances 0.000 abstract description 13
- 239000000758 substrate Substances 0.000 abstract description 9
- 239000012530 fluid Substances 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 230000005859 cell recognition Effects 0.000 abstract 1
- 238000004113 cell culture Methods 0.000 description 18
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 14
- 239000002953 phosphate buffered saline Substances 0.000 description 14
- 239000002086 nanomaterial Substances 0.000 description 8
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 7
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 206010027476 Metastases Diseases 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 239000007850 fluorescent dye Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000007921 spray Substances 0.000 description 6
- 230000008901 benefit Effects 0.000 description 5
- 230000009401 metastasis Effects 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000000089 atomic force micrograph Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002071 nanotube Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000004593 Epoxy Substances 0.000 description 1
- 208000005890 Neuroma Diseases 0.000 description 1
- 206010070308 Refractory cancer Diseases 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- CSDREXVUYHZDNP-UHFFFAOYSA-N alumanylidynesilicon Chemical group [Al].[Si] CSDREXVUYHZDNP-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910001649 dickite Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052622 kaolinite Inorganic materials 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000010445 mica Substances 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 239000002103 nanocoating Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 208000016691 refractory malignant neoplasm Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003376 silicon Chemical class 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- NBXZNTLFQLUFES-UHFFFAOYSA-N triethoxy(propyl)silane Chemical compound CCC[Si](OCC)(OCC)OCC NBXZNTLFQLUFES-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D1/00—Processes for applying liquids or other fluent materials
- B05D1/28—Processes for applying liquids or other fluent materials performed by transfer from the surfaces of elements carrying the liquid or other fluent material, e.g. brushes, pads, rollers
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the field of inorganic nanometer materials and biosensors, and discloses a halloysite coating used for capturing tumour cells and a preparation method and application thereof. Specific halloysite nanometer structures are prepared on different substrates through a coating method, and target cell specific recognition molecules are fixed. Or a rough nanometer structure is directly used, the reinforced three-dimensional topology mutual function between the cell surface structure and the substrate rough nanometer structure is used, and more target cell recognition molecules can be fixed on the nanometer rough structure compared with a smooth surface. The flow speed of fluid in blood can be lowered based on the rough nanometer surface structure, and therefore the chance of interacting with cells is increased, the cell capturing efficiency of the halloysite nanometer structure is improved, and the 3h capturing rate for most tumour cells exceeds 80%. The method is easy to operate, low in energy consumption and high in preparation efficiency. In the whole preparation process of the substrates, no toxic and harmful substances are generated, and no pollution is caused to the environment.
Description
Technical Field
The invention belongs to the field of inorganic nano materials and biosensors, and particularly relates to a halloysite coating for capturing tumor cells, and a preparation method and application thereof.
Background
Cancer is one of the major diseases threatening human health and life in the world at present. The most important cause of refractory cancer and high mortality is the presence of cancer metastasis, and death in more than 90% of cancer patients is directly associated with metastasis of tumor cells. During metastasis, the cancer cells of the primary epithelial tumor are shed and invade the circulating blood system. Such cancer cells present in Circulating blood are called Circulating Tumor Cells (CTCs) as the blood circulation grows into new tumor tissues (metastases) at other tissue and organ sites. The method for detecting the existence and the number change of the CTCs in the circulating blood is a non-destructive, non-invasive and effective cancer metastasis monitoring and cancer prognosis judgment means, and has important significance for in vitro diagnosis, personalized treatment, pathological research and the like of the cancer. In particular, it has been discovered in recent years that CTCs are present in the blood before solid tumors are formed in early stages of cancer, and thus accurate detection of CTCs in the blood can be used as a reliable means for screening for early stage cancer, monitoring cancer progression, and responding to treatment of patients, and thus, more survival opportunities are won for patients. However, the number of CTCs in peripheral blood of tumor patients is small, and it is difficult to separate them from blood by a specific means.
Nanotechnology is rapidly evolving to bring about eosin early diagnosis of tumors. The special antibody coupled to the nano material can be specifically combined with tumor cells, so that the tumor cells can be specifically separated from blood. Because the size and the surface structure of the tumor cells are different from those of blood cells in peripheral blood, the surface of some rough nano surface structures can capture the tumor cells in a nano trap mode even if the surface of some rough nano surface structures is not coupled with antibodies, and then cancer diagnosis can be carried out more simply, conveniently and quickly.
The halloysite nanotube is a one-dimensional nano material formed in nature, has a unique and perfect nano-scale tubular structure, and has a chemical formula of Al2Si2O5(OH)4·H2The halloysite has the advantages that ① halloysite has a size and a structure suitable for preparing a cell capture and separation surface, the diameter is about 50nm, the length-diameter ratio is about 20, the halloysite is good in hydrophilicity and can be stably dispersed and exist in physiological solution and blood, ② halloysite has active groups such as silicon-aluminum hydroxyl groups and can be subjected to various functional modifications, ③ halloysite has good cell affinity and good cell compatibility and cell adhesion, ④ halloysite is cheap and easy to obtain, is convenient to popularize and apply, and can solve the problems of high price, limited raw material source, poor batch stability and the like, and the cost advantage is obvious.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the present invention provides a method for preparing a halloysite coating for capturing tumor cells. The method utilizes a spraying method to prepare the halloysite into the rough surface, can effectively overcome the defect that the forming of the halloysite rough surface prepared by a common direct solution volatilization drying method is difficult, can also overcome the defects of uneven coating and poor adhesion caused by coating a plastic pipe with a solution, can also overcome the defect that the large-area preparation cannot be realized by the traditional nano surface preparation method, and is a novel halloysite nano rough surface preparation method for capturing tumor cells. The spraying method can obtain a large area of halloysite surface, the preparation process is convenient and fast, the adhesion of the coating and the substrate is high, the coating is uniform, and the reproducibility is high.
It is another object of the present invention to provide a halloysite coating prepared by the above method for capturing tumor cells.
Still another object of the present invention is to provide the use of the halloysite coating for capturing tumor cells in the fields of capturing tumor cells and early diagnosis of cancer.
The purpose of the invention is realized by the following scheme:
a method of preparing a halloysite coating for capturing tumor cells, comprising the steps of:
(1) preparation of halloysite dispersion: according to the mass parts, 0.01-40 parts of halloysite, 0-99.9 parts of deionized water, 0-99.9 parts of organic solvent, 0.001-5 parts of silane and 0.001-1 part of surfactant are mixed and mechanically stirred or ultrasonically dispersed to obtain a halloysite dispersion liquid;
(2) spraying the halloysite dispersion liquid prepared in the step (1) on a preheated base material, and then fully drying to obtain a dried coating, namely a halloysite coating for capturing tumor cells;
or,
and (3) repeating the steps (1) and (2), and then coupling the coating dried in the step (2) with a specific recognition antibody of the tumor cells to obtain the halloysite coating modified by the antibody and used for capturing the tumor cells.
The organic solvent in step (1) may be one or more of ethanol, methanol, acetone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, dioxane, dichloromethane or chloroform.
The silane in the step (1) can be at least one of gamma-aminopropyltriethoxysilane, gamma-epoxypropyltriethoxysilane, gamma-mercaptopropyltrimethoxysilane and gamma-methacryloxypropyltrimethoxysilane.
The surfactant in the step (1) is at least one of sodium polystyrene sulfonate, sodium hexametaphosphate, hexadecyl trimethyl ammonium bromide, sodium pyrophosphate and sodium citrate.
The dosage of the deionized water and the organic solvent in the step (1) can not be 0 at the same time.
The ultrasonic dispersion condition in the step (1) is ultrasonic for 5-60 min under the power of 60-1000W.
The mechanical stirring in the step (1) is stirring at a speed of 50-1500 rpm for 3-120 min.
The base material in the step (2) is a glass slide, a quartz plate, a silicon wafer, a polystyrene transparent plastic sheet or a metal sheet, and the preheating temperature is 35-100 ℃.
The drying in the step (2) is drying at 30-100 ℃ for 0.5-600 min.
The specific recognition antibody of the tumor cells can be epithelial cell adhesion molecule antibody anti-EpCAM or human epidermal growth factor anti-HER 2.
The coupling specifically comprises the following steps: and soaking the dried coating in 1-20 mu g/mL antibody PBS solution for 3-30 min, and then taking out and washing the coating for 2-3 times by using phosphate buffer solution.
A halloysite coating prepared by the method for capturing tumor cells.
The halloysite coating for capturing the tumor cells has the characteristics of high tumor cell capturing efficiency, high sensitivity and the like, and can be applied to the fields of tumor cell capturing and early cancer diagnosis.
The tumor cells comprise human breast cancer cells (MCF-7), human prostate cancer cells (PC-3), human liver cancer cells (HePG2), human colon adenocarcinoma cells (SW480), human lung small cell adenocarcinoma cells (A549), human ovarian cancer cells (HO-8910), human brain glioma cells (U87) and human osteosarcoma cells (SW 1353).
The mechanism of the invention is as follows:
the specific halloysite nanostructure is prepared on different substrates, and target cell specific recognition molecules (such as epithelial cell adhesion molecule antibody, anti-EpCAM) are fixed on the nanostructure, or the rough nanostructure is directly utilized, the three-dimensional topological interaction between the cell surface structure and the substrate rough nanostructure is utilized, the nano rough structure can fix more recognition molecules of target cells than a smooth surface, and the structure based on the nano rough surface can reduce the flow speed of fluid in blood so as to increase the interaction chance with cells, so that the nanostructure prepared by the method is the main reason for improving the cell capture efficiency.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention provides a method for rapidly preparing a halloysite nano coating by a spraying method, which has the advantages of low cost, simple operation, low energy consumption, high preparation efficiency, no generation of toxic and harmful substances in the whole preparation process of a substrate and no pollution to the environment, and the prepared coating can be used for enriching and detecting circulating tumor cells.
2. The halloysite nanotube adopted by the invention has the advantages of wide source, low price, no toxicity, harmlessness, environmental friendliness, good compatibility of blood and cells, easiness for dispersing and assembling into an array structure in a polar solvent system, convenience for constructing the micro-nano rough surface, fixation of more recognition molecules of target cells, reduction of the flow speed of fluid in the blood, increase of the interaction chance with the cells and high capture efficiency of the surface on tumor cells.
3. The halloysite coating prepared by the method has good stability and strong adhesive force with a substrate, and active groups on the surface of the halloysite can be conveniently subjected to hydrophobization treatment and coupled with a specific recognition antibody of tumor cells, so that the tumor cells are captured from blood specifically.
4. The prepared coating has the characteristics of high tumor cell capturing efficiency (the capturing rate of 3h exceeds 85 percent), high sensitivity and the like, and can be used for clinical diagnosis and metastasis monitoring of cancer patients. Meanwhile, the activity of the tumor cells captured by the coating is high, the tumor cells can be continuously digested and cultured, real-time monitoring can be carried out, and the coating has important significance for subsequent analysis of the tumor cells.
Drawings
FIG. 1 is a surface atomic force microscope image of an antibody-modified halloysite coating prepared in example 1 of the invention.
FIG. 2 is a graph comparing the capturing effect of the halloysite coating on a silicon wafer prepared in example 2 of the present invention and a general glass substrate on human prostate cancer cell PC-3.
FIG. 3 is a statistical graph showing the capturing effect of the halloysite coating on various cells prepared in example 3 of the present invention;
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
The reagents used in the examples are commercially available without specific reference. Cancer cells and normal cells used in the examples are also commercially available. The parts described in the examples are parts by mass.
Example 1
Preparing halloysite dispersion, wherein the halloysite dispersion comprises 2 parts of halloysite, 20 parts of deionized water, 80 parts of ethanol, 1 part of gamma-aminopropyltriethoxysilane and 0.5 part of surfactant sodium polystyrene sulfonate, uniformly mixing the components through mechanical stirring at room temperature (400r/min, stirring for 30min), and spraying the mixture onto a glass sheet preheated at 100 ℃ by using a spray gun; and (3) fully volatilizing and drying the coating at 60 ℃, soaking the halloysite coating into a5 mu g/mL PBS solution of Anti-EpCAM (purchased from Sigma-Aldrich company) antibody, taking out after 10min, and washing with phosphate buffer solution for 3 times to obtain the halloysite coating modified by the antibody.
FIG. 1 is a surface atomic force microscope image of the antibody-modified halloysite coating prepared in example 1. It can be seen from FIG. 1 that the coating has a uniform surface, uniform thickness and mean square roughness of 52.6nm, and is suitable for cell capture.
The obtained halloysite coating modified by the antibody is captured by human breast cancer cells, the surface of the halloysite modified by the antibody is placed in a cell culture plate, the human breast cancer cells with the measured number are added into a cell culture solution (DMEM medium, 10% fetal bovine serum), then the cell culture solution added with a certain number of cancer cells is added on the surface of the halloysite, and the mixture is kept still and cultured for 3 hours at 37 ℃. After that, the cells were washed at least three times with a PBS solution, stained with DAPI, a fluorescent dye, and the number of the cells was observed by photographing with a fluorescent microscope. The capture efficiency of the 3h halloysite coating on MCF-7 cells was found to be 90%.
Example 2
Preparing halloysite dispersion, wherein 5 parts of halloysite, 10 parts of deionized water, 90 parts of ethanol, 2 parts of gamma-epoxy propyl triethoxysilane and 0.8 part of surfactant sodium hexametaphosphate are uniformly mixed by mechanical stirring (400r/min and stirring for 30min) at room temperature, and are sprayed on a silicon wafer preheated at 80 ℃ by a spray gun; the coating is fully volatilized and dried at 60 ℃, and then the halloysite coating is soaked in 10 mu g/mL of PBS (phosphate buffered saline) solution of anti-HER2 (purchased from Sigma-Aldrich company) antibody for 30min and washed for 3 times by phosphate buffer solution, thus obtaining the halloysite coating modified by the antibody.
The obtained antibody-modified halloysite coating is used for capturing human prostate cancer cells, the surface of the antibody-modified halloysite is placed in a cell culture plate, and a blank glass slide is used as a blank control. A measured number of human prostate cancer cells were added to a cell culture solution (DMEM medium, 10% fetal bovine serum), and then the cell culture solution to which a certain number of cancer cells were added was added to the surface of halloysite, and allowed to stand and culture at 37 ℃ for 2 hours. After that, the cells were washed three times with a PBS solution, stained with DAPI, a fluorescent dye, and the number of the cells was observed by photographing with a fluorescent microscope. The capture efficiency of the 2h halloysite coating on PC-3 cells was found to be 85%.
FIG. 2 is a cytofluorescence image of a halloysite coating and a blank glass slide on an antibody-modified silicon wafer prepared in example 2 at various capture times for human prostate cancer cells (PC 3). As can be seen from fig. 2, the halloysite coating showed higher cell capture efficiency than the blank glass sheet surface at the same capture time.
Example 3
Preparing halloysite dispersion, wherein 10 parts of halloysite, 100 parts of methanol, 0.5 part of gamma-methacryloxypropyltrimethoxysilane and 1.5 parts of sodium pyrophosphate as a surfactant are uniformly mixed under 800W for 30min by ultrasonic treatment, and the mixture is sprayed on an aluminum sheet preheated at 50 ℃ by a spray gun; and (3) fully volatilizing and drying the coating at 80 ℃, soaking the halloysite coating into a PBS (phosphate buffered saline) solution of 30 mu g/mL anti-HER2 antibody for 20min, and washing for 3 times by using a phosphate buffer solution to obtain the halloysite coating modified by the antibody.
The obtained halloysite coating modified by the antibody is subjected to human liver cancer cell capture, the surface of the halloysite modified by the antibody is placed in a cell culture plate, a metered number of human liver cancer cells are added into a cell culture solution (DMEM medium, 10% fetal bovine serum), then the cell culture solution added with a certain number of cancer cells is added onto the surface of the halloysite, and the mixture is subjected to static culture at 37 ℃ for 3 hours. After that, the cells were washed at least three times with a PBS solution, stained with DAPI, a fluorescent dye, and the number of the cells was observed by photographing with a fluorescent microscope. The 3h halloysite coating was found to be 93% efficient at capturing HepG cells.
Example 4
Preparing halloysite dispersion, wherein 8 parts of halloysite, 100 parts of acetone, 1 part of gamma-mercaptopropyl trimethoxy silane and 0.8 part of sodium citrate as a surfactant are uniformly mixed under 500W for 1 hour of ultrasonic sound, and the mixture is sprayed on mica sheets preheated at 90 ℃ by a spray gun; and (3) fully volatilizing and drying the coating at 40 ℃, soaking the halloysite coating into a PBS (phosphate buffer solution) solution of an Anti-EpCAM (Anti-EpCAM) antibody of 2 mu g/mL for 10min, and washing for 3 times by using a phosphate buffer solution to obtain the halloysite coating modified by the antibody.
The obtained halloysite coating modified by the antibody is subjected to human lung cancer tumor cell capture, the surface of the halloysite modified by the antibody is placed in a cell culture plate, the metered number of human lung cancer cells are added into a cell culture solution (DMEM medium, 10% fetal bovine serum), then the cell culture solution added with a certain number of cancer cells is added onto the surface of the halloysite, and the mixture is subjected to static culture at 37 ℃ for 10 min. After that, the cells were washed three times with a PBS solution, stained with DAPI, a fluorescent dye, and the number of the cells was observed by photographing with a fluorescent microscope. The capture efficiency of 10min halloysite coating on a549 cells was found to be 75%.
Example 5
Preparing halloysite dispersion liquid, wherein the halloysite dispersion liquid comprises 0.5 part of halloysite, 100 parts of dichloromethane, 3 parts of gamma-aminopropyltriethoxysilane and 1 part of surfactant sodium polystyrene sulfonate, mechanically stirring the components at the rotating speed of 500rpm for 60 minutes, and spraying the components onto glass sheets preheated at 100 ℃ by a spray gun; and (3) fully volatilizing and drying the coating at 40 ℃, soaking the halloysite coating into a PBS (phosphate buffer solution) solution of an Anti-EpCAM (Anti-EpCAM) antibody of 8 mu g/mL for 10min, and washing for 3 times by using a phosphate buffer solution to obtain the halloysite coating modified by the antibody.
Capturing human ovarian cancer cells on the obtained halloysite coating modified by the antibody, placing the surface of the halloysite modified by the antibody in a cell culture plate, adding the human ovarian cancer cells with a measured number into a cell culture solution (DMEM medium, 10% fetal bovine serum), then adding the cell culture solution added with a certain number of cancer cells to the surface of the halloysite, and standing and culturing at 37 ℃ for 5 min. After that, the cells were washed three times with a PBS solution, stained with DAPI, a fluorescent dye, and the number of the cells was observed by photographing with a fluorescent microscope. The capture efficiency of 5min halloysite coating on HO-8910 cells was found to be 78%.
Example 6
Preparing halloysite dispersion, wherein 20 parts of halloysite, 30 parts of pure water, 60 parts of tetrahydrofuran, 2 parts of gamma-methacryloxypropyltrimethoxysilane and 0.6 part of surfactant cetyl trimethyl ammonium bromide, mechanically stirring the components at the rotating speed of 1000rpm for 30 minutes, uniformly mixing, and spraying the components onto glass sheets preheated at 90 ℃ by using a spray gun; and fully volatilizing and drying the coating at 80 ℃ to obtain the halloysite coating without antibody coupling.
Directly capturing the halloysite coating which is not coupled by the antibody by using human brain glioma cells (U87), placing the halloysite surface in a cell culture plate, adding a metered number of human brain glioma cells into a cell culture solution (DMEM medium, 10% fetal bovine serum), then adding the cell culture solution added with a certain number of cancer cells onto the halloysite surface, and standing and culturing at 37 ℃ for 30 min. After that, the cells were washed three times with a PBS solution, stained with DAPI, a fluorescent dye, and the number of the cells was observed by photographing with a fluorescent microscope. The capture efficiency of U87 cells at 30min was found to be 85%.
Example 7
The antibody modified halloysite coating prepared in example 3 was used for capturing normal cell preosteoblasts (MC3T3-E1), human normal hepatocytes (L02), human hepatoma cells (HEPG-2), human breast cancer cells (MCF-7), mouse brain neuroma cells (Neuro-2a), lung cancer cells (A549) and mouse melanoma cells (B16F10), the specific operation is the same as example 3, the capturing efficiency of various cells is shown in FIG. 3, and it can be seen from FIG. 3 that the capturing rate of the antibody-coupled halloysite surface on normal cells is far lower than that of tumor cells. Thus, the cancer diagnosis can be conveniently carried out.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for preparing a halloysite coating for capturing tumor cells, comprising the steps of:
(1) preparation of halloysite dispersion: according to the mass parts, 0.01-40 parts of halloysite, 0-99.9 parts of deionized water, 0-99.9 parts of organic solvent, 0.001-5 parts of silane and 0.001-1 part of surfactant are mixed and mechanically stirred or ultrasonically dispersed to obtain a halloysite dispersion liquid;
(2) spraying the halloysite dispersion liquid prepared in the step (1) on a preheated base material, and then fully drying to obtain a dried coating, namely a halloysite coating for capturing tumor cells;
or,
and (3) repeating the steps (1) and (2), and then coupling the coating dried in the step (2) with a specific recognition antibody of the tumor cells to obtain the halloysite coating modified by the antibody and used for capturing the tumor cells.
2. The method of preparing a halloysite coating for capturing tumor cells according to claim 1, wherein:
the organic solvent in the step (1) is one or a mixture of more of ethanol, methanol, acetone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, dioxane, dichloromethane or chloroform;
the silane in the step (1) is at least one of gamma-aminopropyltriethoxysilane, gamma-epoxypropyltriethoxysilane, gamma-mercaptopropyltrimethoxysilane and gamma-methacryloxypropyltrimethoxysilane;
the surfactant in the step (1) is at least one of sodium polystyrene sulfonate, sodium hexametaphosphate, hexadecyl trimethyl ammonium bromide, sodium pyrophosphate and sodium citrate.
3. The method of preparing a halloysite coating for capturing tumor cells according to claim 1, wherein:
the dosage of the deionized water and the organic solvent in the step (1) can not be 0 at the same time.
4. The method of preparing a halloysite coating for capturing tumor cells according to claim 1, wherein:
the ultrasonic dispersion condition in the step (1) is ultrasonic for 5-60 min under the power of 60-1000W;
the mechanical stirring in the step (1) is stirring at a speed of 50-1500 rpm for 3-120 min.
5. The method of preparing a halloysite coating for capturing tumor cells according to claim 1, wherein:
the base material in the step (2) is a glass slide, a quartz plate, a silicon wafer, a polystyrene transparent plastic sheet or a metal sheet, and the preheating temperature is 35-100 ℃;
the drying in the step (2) is drying at 30-100 ℃ for 0.5-600 min.
6. The method of preparing a halloysite coating for capturing tumor cells according to claim 1, wherein:
the specific recognition antibody of the tumor cells is epithelial cell adhesion molecule antibody anti-EpCAM or human epidermal growth factor anti-HER 2.
7. The method of preparing a halloysite coating for capturing tumor cells according to claim 1, wherein:
the coupling specifically comprises the following steps: and soaking the dried coating in 1-20 mu g/mL antibody PBS solution for 3-30 min, and then taking out and washing the coating for 2-3 times by using phosphate buffer solution.
8. A halloysite coating for capturing tumor cells prepared according to the method of any one of claims 1-7.
9. Use of a halloysite coating for capturing tumor cells according to claim 8 in the fields of capturing tumor cells and early diagnosis of cancer.
10. Use of a halloysite coating for capturing tumor cells according to claim 9 in the fields of capturing tumor cells and early diagnosis of cancer, characterized in that:
the tumor cells comprise human breast cancer cells, human prostate cancer cells, human liver cancer cells, human colon adenocarcinoma cells, human lung small cell adenocarcinoma cells, human ovarian cancer cells, human brain glioma cells and human osteosarcoma cells.
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| CN108531455A (en) * | 2018-03-09 | 2018-09-14 | 大连理工大学 | Polyphenol coating for circulating tumor cell capture |
| CN110665012A (en) * | 2019-11-20 | 2020-01-10 | 潍坊医学院 | Nano-particles for isolated culture of tumor cells and preparation method thereof |
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| CN104789124A (en) * | 2014-12-30 | 2015-07-22 | 中国科学院兰州化学物理研究所 | A preparing method of a stable superamphiphobic surface |
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| CN104789124A (en) * | 2014-12-30 | 2015-07-22 | 中国科学院兰州化学物理研究所 | A preparing method of a stable superamphiphobic surface |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108531455A (en) * | 2018-03-09 | 2018-09-14 | 大连理工大学 | Polyphenol coating for circulating tumor cell capture |
| CN110665012A (en) * | 2019-11-20 | 2020-01-10 | 潍坊医学院 | Nano-particles for isolated culture of tumor cells and preparation method thereof |
| CN110665012B (en) * | 2019-11-20 | 2022-12-06 | 潍坊医学院 | Nano-particles for isolated culture of tumor cells and preparation method thereof |
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