CN109142712B - The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ - Google Patents
The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ Download PDFInfo
- Publication number
- CN109142712B CN109142712B CN201810580389.XA CN201810580389A CN109142712B CN 109142712 B CN109142712 B CN 109142712B CN 201810580389 A CN201810580389 A CN 201810580389A CN 109142712 B CN109142712 B CN 109142712B
- Authority
- CN
- China
- Prior art keywords
- tube array
- nano
- dendritic
- cell
- substrate film
- 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.)
- Active
Links
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
- G01N33/543—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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer 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
- G01N33/57484—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
- G01N33/57488—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 involving compounds identifable in body fluids
Abstract
The invention belongs to the detection technique field of medical cancer cell, in particular to a kind of preparation method of nano-tube array, the microfluidic devices that cancer cell is used to capture and regulated and controled in situ with dendritic nano-tube array come the method for specific recognition circulating tumor cell and by nano-tube array.The microfluidic devices, including PDMS module pond, PDMS module pond include cell culture insert and microchannel, and the substrate film of the dendritic nano-tube array is used to separate the cell culture insert on upper layer and the microchannel of bottom；Two ends of the microchannel are equipped with the I/O port for conveying solution.The present invention high efficiency and can specifically capture cancer cell by force, by modifying specific recognition antibody molecule on dendritic nanotube, cancer cell is effectively captured and isolated from blood, while dendritic nanotube has excellent biological safety to cell, does not influence the normal function of cancer cell.
Description
Technical field
The invention belongs to the detection technique field of medical cancer cell, in particular to the preparation method of dendritic nano-tube array,
It is captured with dendritic nano-tube array come the method for specific recognition circulating tumor cell and by nano-tube array and in situ
Regulate and control the microfluidic devices of cancer cell.
Background technique
In the prior art, cancer is to threaten one of human health and the major disease of life in the world at present.It is biomedical
Research has been devoted to disclose cell carcinogenesis mechanism, exploitation cancer diagnosis technology and proposes prevention and control of cancer scheme, and develops novel
Cancer cell detection technique, and realize the behavior of drug regulation cancer cell, precision to Development of Novel and personalized swollen
Tumor treatment method has very important value.
Traditional diagnosing tumor means mainly have based on X-ray, CT scan (abbreviation CT), positive electron hair
The iconography of the technologies such as emitting computerized tomograph (abbreviation PET) and magnetic resonance (abbreviation MRI) is based on tumor-marker analyte detection
Hematology and pathology method based on tissue biopsy.However, these conventional means are faced with testing result lag, nothing
The problems such as method completes real-time monitoring, and specificity is insufficient and materials are difficult.
In recent years, biologist's discovery, during cancer metastasis, tumour cell, which can occur to be detached from, to be entered blood and follows
Loop system and be transferred to other positions of body, by directly taking blood and being screened, whether can be diagnosed to be with cancer cell, with
And whether metastases occur.It is this to be detected based on circulating tumor cell (circulating tumor cells, abbreviation CTCs)
Diagnostic means, it is noninvasive and real-time due to having many advantages, such as, external early diagnosis and individualized treatment can be effectively applied to
Detection including clinical sieve medicine, drug resistance, monitoring and the exploitation of tumour novel drugs of tumor recurrence etc. are truly realized the full course of disease
The treatment clinical course of tracking of knub, it is considered to be most innovative and Transformation Potential tumor diagnosis method at present.
CTCs detection technique common at present mainly has immunomagnetic beads method, micro-pore-film filtration method and micro-fluidic chip etc., main
If containing specific recognition molecules or physical property such as size using the special chemical property of tumour such as cell membrane surface, can
Morphotropism, density and dielectric constant etc. are different, and tumour cell is separated from blood sample, reaches concentration effect.Wherein,
Microfluidic chip technology, due to having many advantages, such as that required sample size is small, at low cost, small to cellular damage and be easy to be miniaturized,
Increasingly by everybody concern.Novel micro-fluidic device is constructed by advanced micro-nano technology technology, is expected to develop into
Fast and convenient, universality and multifunctional unit Clinical CT Cs chip.The Tonor seminar of Harvard University passes through Soft lithograph skill
Art designs the cluster chip (Cluster-Chip) that multiple rows of micro- triangle pillar is constituted, these pillars are with every two pillar handle
The mode that cell drains into the top of third root pillar arranges.Based on the micro-fluidic chip of this particular array structure, using thin
The physical property of born of the same parents group, can efficiently sub-argument goes out CTC groups from the whole blood without any pre-treatment.In addition, by micro-fluidic
Chip and other CTCs detection techniques are combined, and can greatly improve the detection performance of device.For example, Tonor seminar is again by CTC core
Piece and immuno magnetic cell separation technology tandem are combined, and realize up to 97% CTCs separative efficiency.Peripheral blood sample is logical first
It crosses silicon nano column array and completes first order separation, then by the leucocyte of marked by magnetic bead and unlabelled CTC under magnetic fields
Cell is separated.This CTC chip has many advantages, such as that separating rate is fast, high specificity and high-efficient, solves using single
Immuno magnetic cell separation technology when the amount of samples that encounters is more, testing cost is high and the problems such as detection time is long.
In recent years, with the development of advanced micro-nano technology technology and emerging in large numbers for nano material abundant, it is various to receive
Rice structure be used to construct bionic interface, be widely used for the research of CTC separation and detection.Researcher has found nano junction
The characteristics such as small-size effect, high-specific surface area that structure is shown are capable of increasing the contact probability of tumour cell and substrate, so that
Minimal amount of tumour cell is captured from blood to be possibly realized.The Chinese Academy of Sciences it is physical and chemical Wang Shutao seminar etc. deposited by the Nature
Nanostructure inspiration, construct it is various based on the micro-nano bionical interface such as silicon nanowires, nanometer acanthosphere, it is thin for tumour
The detection of born of the same parents.For example, they grow three-dimensional dendritic ITO nanowire structure using chemical vapour deposition technique, then substrate is repaired
The antibody of specific recognition tumour cell such as epithelial cell adhesion molecule antibody (anti-EpCAM), is caught using immunization on decorations
Breast cancer cell line is obtained, separative efficiency can reach 90%.By showing to work as nanometer to nanostructure-cell membrane Interface Study
Structure is contacted with coarse cancer cell membrane surface, generates obviously enhancement effect to cell capture.By by nanostructure
It is combined with micro-fluidic device, CTCs detection sensitivity and recall rate can be significantly improved.University of California in Los Angeles
Silicon nano column array is integrated into micro-fluidic chip by Tseng seminar, is used for reference flow morphology and is improved connecing for cell and substrate
Frequency is touched, it can be by the simple separative efficiency (45~65%) for using silicon nano column array, promotion up to 95%.In addition, logical
It crosses and nanometer-bioelectric interface is carried out different surface-functionalized, various multi-functional micro-fluidic chips can be constructed, realized thin to cancer
Born of the same parents such as separate, capture and discharge at the operation.Heat is modified on graphene oxide by the Nagrath seminar of University of Michigan
Quick property macromolecular chain and Specific antibody molecules construct the CTCs chip of temperature-responsive.When room temperature is tested, specific antibody
Molecule can be incorporated on macromolecular chain, and cancer cell is effectively captured from blood, then by reducing temperature damage high score
The combination of subchain and antibody, realizes and controllably discharges cell.
To sum up, by the way that nanostructure substrate and microfluidic device are integrated, cancer cell can be completed capture, separation and
The operation such as release, is expected to develop into very popular CTCs detection technique.
Most of CTCs micro-fluidic chip is all more confined to capture, separation and release cancer cell, few originals at present
Position carries out more complicated and more accurate controllable operation, such as release drug and the row for regulating and controlling cancer cell to the cancer cell captured
For.The limitation that these cancer cells regulate and control and test and analyze in situ limits the cognition to cancer cell active mechanism.It is examined in CTCs
In the research and development of survey technology, cancer cell capture and separation and situ drug regulation cancer cell behavior can be collected by also lacking one kind
Method.
Summary of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, specifically disclose the preparation method of dendritic nano-tube array, use
Dendritic nano-tube array carrys out the method for specific recognition circulating tumor cell and by dendritic nano-tube array for capture and original
The microfluidic devices of position regulation cancer cell.The present invention high efficiency and can specifically capture cancer cell by force, by by specificity knowledge
Other antibody molecule modification effectively captures from blood and isolates cancer cell, while dendritic nanotube on dendritic nanotube
There is excellent biological safety to cell, do not influence the normal function of cancer cell.
In order to reach above-mentioned technical purpose, the present invention is realized by following technical scheme:
A kind of preparation method of dendritic nano-tube array of the present invention: it is comprised the concrete steps that:
(1) atomic layer deposition (abbreviation ALD) technology is used, the diethyl zinc of gas phase is used to mix as presoma with vapor
It is passed through reaction chamber, upper ZnO film is all deposited on the surface of substrate film and hole inner wall；
(2) continue the trimethyl aluminium and vapor that are passed through a period of time gas phase, in ZnO film surface depositing Al2O3Nanometer
Layer；
(3) in BCl3And Cl2Under mixed gas, plasma etching falls the Al of substrate film surface2O3/ ZnO layer, it is then sharp
Use O2Plasma etching falls the polycarbonate membrane of part, exposes nano-tube array structure；
(4) ZnO/Al is being obtained2O3After nano-tube array, using zinc nitrate and hexa as raw material, pass through hydro-thermal
Method is reacted in outer tube wall growing ZnO nano-wire, and dendritic nano-tube array is prepared；
(5) the dendritic nanotube surface also sputters one layer of Al2O3Protective film layer.
As the further improvement of above-mentioned technology, in above-mentioned steps (1), the thickness value of the ZnO film is 30~40nm.
As the further improvement of above-mentioned technology, in above-mentioned steps (2), the Al2O3The thickness value of nanometer layer be 10~
20nm。
As the further improvement of above-mentioned technology, in above-mentioned steps (5), the thickness range of the Al2O3 protective film layer
It is 10~20nm.
The invention also discloses the methods that above-mentioned dendritic nano-tube array carrys out specific recognition circulating tumor cell, specific
Step is:
(1) mercapto groups are modified on dendritic nanotube；
(2) processing of substrate film；
(3) it will be added dropwise with phosphate buffer solution (abbreviation PBS) prepared Streptavidin (streptavidin) solution
It is reacted in substrate film surface；
(4) PBS of the EpCAM antibody molecule of end biotin labeling (biotinylated anti-EpCAM) is molten
Drop is added on substrate film, and the antibody molecule of the specific recognition is fixed on substrate surface after reaction.
In above-mentioned steps (1), the detailed process that mercapto groups are modified on the dendritic nanotube is: substrate film is impregnated
12h is reacted in the ethanol solution containing 4% 3- mercaptopropyl trimethoxysilane (abbreviation MPTMS), then with a large amount of ethyl alcohol
Rinse surface, N2Air-flow drying.
In above-mentioned steps (2), the processing detailed process of substrate film is: will contain amino-mercapto crosslinking agent γ-Malaysia acyl
The aqueous solution of imido grpup butyric acid-N- hydroxy thiosuccinimide sour (Sulfo-GMBS) handles substrate film, places 30min, so
After be rinsed with water surface, N2Air-flow drying.
In above-mentioned steps (3), the reaction in substrate film surface is added dropwise in Streptavidin (streptavidin) solution
Journey is specifically: will be added dropwise with phosphate buffer solution (abbreviation PBS) prepared Streptavidin (streptavidin) solution
Substrate film surface, react 45min after with PBS rinse surface, N2Air-flow drying.
In above-mentioned steps (4), the antibody molecule of the specific recognition is fixed on the specific mistake of substrate surface after reaction
Cheng Shi: the PBS solution of the EpCAM antibody molecule (biotinylated anti-EpCAM) of end biotin labeling is added dropwise
On substrate film, after reacting 30min, PBS rinses surface, and biotin can be directly in conjunction with Streptavidin, to make specificity
The antibody molecule of identification is fixed on substrate surface.
In addition, regulating and controlling the micro- of cancer cell with original position for capturing by dendritic nano-tube array the invention also discloses a kind of
Device is flowed, including PDMS module pond, PDMS module pond include:
Cell culture insert is placed in the upper part in PDMS module pond for containing culture solution, is internally provided with dendritic nanotube
Array, the substrate film of the dendritic nano-tube array bottom are the bottom surface of cell culture insert；
Microchannel is placed in the lower part in PDMS module pond for conveying drug solution and collecting intracellular molecules；
The substrate film of the dendritic nano-tube array is used to separate the cell culture insert on upper layer and the microchannel of bottom；
Two ends of the microchannel are equipped with the I/O port for conveying solution.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention high efficiency and can specifically capture cancer cell by force, by repairing specific recognition antibody molecule
Decorations effectively capture from blood and isolate cancer cell on dendritic nanotube, at the same dendritic nanotube have to cell it is excellent
Good biological safety does not influence the normal function of cancer cell.
(2) microfluidic devices of the present invention can be realized accurately drug regulation, and dendritic nano-tube array and miniflow are logical
There is sufficient substance diffusion transport between road, drug can be delivered to upper layer training by nanometer lumen in conjunction with microflow control technique
Pond is supported, the biobehavioral of captured cancer cell is precisely controllably regulated and controled.
Detailed description of the invention
The present invention is described in detail in the following with reference to the drawings and specific embodiments:
Fig. 1 is microfluidic devices structural schematic diagram of the present invention；
Fig. 2 is dendritic nano tube structure schematic diagram in the present invention.
Specific embodiment
A kind of preparation method of dendritic nano-tube array of the present invention: it is comprised the concrete steps that:
(1) atomic layer deposition (abbreviation ALD) technology is used, the diethyl zinc of gas phase is used to mix as presoma with vapor
It is passed through reaction chamber, upper ZnO film is all deposited on the surface of substrate film and hole inner wall, the thickness value of the ZnO film is 30
~40nm；
(2) continue the trimethyl aluminium and vapor that are passed through a period of time gas phase, in ZnO film surface depositing Al2O3Nanometer
Layer, the Al2O3The thickness value of nanometer layer is 10~20nm；
(3) in BCl3And Cl2Under mixed gas, plasma etching falls the Al of substrate film surface2O3/ ZnO layer, it is then sharp
Use O2Plasma etching falls the polycarbonate membrane of part, exposes nano-tube array structure, at this point, can be by changing O2Plasma
The length of time of body etching adjusts the length of nanotube；
(4) ZnO/Al is being obtained2O3After nano-tube array, using zinc nitrate and hexa as raw material, pass through hydro-thermal
Method is reacted in outer tube wall growing ZnO nano-wire, and dendritic nano-tube array is prepared；
(5) the dendritic nanotube surface also sputters one layer of Al2O3Protective film layer, the Al2O3The thickness model of protective film layer
Enclosing is 10~20nm, Al2O3Protective film layer can be effectively reduced the toxic effect that ZnO generates cell.
The invention also discloses the methods that above-mentioned dendritic nano-tube array carrys out specific recognition circulating tumor cell, specific
Step is:
(1) mercapto groups are modified on dendritic nanotube: substrate film is immersed in the 3- mercapto propyl trimethoxy containing 4%
12h is reacted in the ethanol solution of silane (abbreviation MPTMS), then rinses surface, N with a large amount of ethyl alcohol2Air-flow drying.
(2) amino-mercapto crosslinking agent γ-maleimidobutyric acid-N- hydroxy amber the processing of substrate film: will be contained
The aqueous solution of amber acid imide sour (Sulfo-GMBS) handles substrate film, places 30min, is then rinsed with water surface, N2Air-flow is blown
It is dry.
(3) it will be added dropwise with phosphate buffer solution (abbreviation PBS) prepared Streptavidin (streptavidin) solution
Reacted in substrate film surface: the reaction process tool in substrate film surface is added dropwise in Streptavidin (streptavidin) solution
Body is: will be added dropwise with phosphate buffer solution (abbreviation PBS) prepared Streptavidin (streptavidin) solution in substrate
Film surface, react 45min after with PBS rinse surface, N2Air-flow drying.
(4) PBS of the EpCAM antibody molecule of end biotin labeling (biotinylated anti-EpCAM) is molten
Drop is added on substrate film, and after reacting 30min, PBS rinses surface, and biotin can be directly in conjunction with Streptavidin, to make
The antibody molecule of specific recognition is fixed on substrate surface.
As shown in Figure 1, the invention also discloses a kind of using nano-tube array for capturing and regulation cancer cell in situ
Microfluidic devices, including PDMS module pond 10, PDMS module pond 10 include:
Cell culture insert 1 is placed in the upper part in PDMS module pond 10 for containing culture solution, is internally provided with dendritic nanometer
Pipe array 2, the substrate film of the dendritic nano-tube array bottom are the bottom surface of cell culture insert；
Microchannel 3 is placed in the lower part in PDMS module pond 10 for conveying 4 solution of drug and collecting intracellular molecules；
The miniflow of cell culture insert 1 and bottom that the substrate film 21 of the dendritic nano-tube array 2 is used to separate upper layer leads to
Road 3；
Two ends of the microchannel 3 are equipped with the I/O port for conveying solution.
When being captured using microfluidic devices of the present invention to cancer cell, cancer cell can be divided from human blood sample
It separates out and, achieve the effect that separation and detection.It is described through the invention after tumour cell is captured on nano-tube array
Microfluidic devices carry out drug regulation in situ to cancer cell, and detailed process is:
Firstly, captured cancer cell will continue culture for 24 hours, guarantee cell adherent growth and drawout；
Secondly, being injected into small-molecule drug 4 (such as propidium iodide) by plastic injection pipe 5 into the microchannel 3 of bottom
Deng, at this point, the drug 4 in microchannel 3 can regulate and control upper layer culture pond inner cell by nanometer lumen, research drug work
With the physiological behavior of lower cancer cell；
Finally, realizing precisely controllable drug release to cancer cell by adjusting drug concentration and action time.
As shown in Fig. 2, the periphery of dendritic nanotube 22 is equipped with ZnO nano-wire 221, the ZnO nano-wire 221 passes through hydro-thermal
Method reaction is grown in outer tube wall.
It is specifically described below by way of studying and evaluating different performance:
Embodiment 1:
Model system of the human breast cancer cell (MCF7) as cell capture is selected, 50 μ L are contained 106Cells/mL's
MCF7 cell suspending liquid is injected into the upper cell culture pond 1 of microfluidic devices, is put into culture 60 minutes in cell incubator, then
After being rinsed with a large amount of cell culture fluid, in fluorescence microscope, it is possible to find a large amount of cells are captured, and capture rate is reachable
90% or more.
Embodiment 2
Study the specific detection ability of dendritic nanotube:
With human breast cancer cell (MCF7) and human prostate cancer cell line (PC-3) for EpCAM positive cell line controls,
Human cervical cancer cell lines (Hela) and human T lymphocyte (Jurkat T) compare as EpCAM negative cells system, and 50 μ L are contained
106The cell suspending liquid of cells/mL is injected into the upper cell culture pond 1 of microfluidic devices, is put into culture in cell incubator
90 minutes, then with a large amount of cell culture fluid rinse after, in fluorescence microscope, it is possible to find in EpCAM positive cell line group
In the middle, a large amount of cells are captured, and capture rate is up to 90% or more；In EpCAM negative cells system group, a small amount of cell is caught
It obtains, capture rate is lower than 40%.
Embodiment 3
Evaluate the biological safety of dendritic nano-tube array:
After the completion of cell capture, continuation is cultivated 48 hours in the upper cell culture pond 1 of microfluidic devices, yellowish green using calcium
Plain (calcein AM) and the double staining reagents of propidium iodide (abbreviation PI) mark living cells and dead cell respectively, observe after dyeing
The proliferative conditions of cell, it is possible to find cell still has good activity, cell normal proliferative after 48 hours.
Embodiment 4
Evaluate the effect of the drug regulation release cell of dendritic nano-tube array:
After the completion of cell capture, the Trypsin EDTA for being 2.5% to 3 implantation concentration of microchannel of microfluidic devices is molten
Liquid is transported solution is injected to pipeline by nanometer lumen.Trypsin EDTA is that one kind can be used for destroying specific resist
The reagent of body and the compound of antigen binding is commonly used for the subsequent cell release of cell capture.After injection solution 15 minutes, use
A large amount of PBS solution is rinsed, in fluorescence microscopy microscopic observation, it can be seen that the cell in channel is considerably less than outside channel
Cell.
Embodiment 5
Study the Apoptosis behavior of drug regulation cancer cell:
Based on microfluidic devices of the present invention, staurosporin (Staurosporine, letter are added in microchannel 3
Claim STS) solution regulation cancer cell apoptosis behavior, assess STS using 3/7 competent cell apoptosis detection kit of Caspase
Influence to Apoptosis.
It is all that the present invention is not departed to various changes or modifications of the invention the invention is not limited to above embodiment
Spirit and scope, if these modification and variations belong within the scope of claim and equivalent technologies of the invention, then this hair
It is bright to also imply that comprising these modification and variations.
Claims (10)
1. a kind of preparation method of dendritic nano-tube array: it is comprised the concrete steps that:
(1) technique for atomic layer deposition is used, uses the diethyl zinc of gas phase to mix as presoma with vapor and is passed through reaction chamber,
Upper ZnO film is all deposited on the surface of substrate film and hole inner wall；
(2) continue the trimethyl aluminium and vapor that are passed through a period of time gas phase, in ZnO film surface depositing Al2O3Nanometer layer；
(3) in BCl3And Cl2Under mixed gas, plasma etching falls the Al of substrate film surface2O3/ ZnO layer, followed by O2Deng
Plasma etching falls the polycarbonate membrane of part, exposes nano-tube array structure；
(4) ZnO/Al is being obtained2O3It is anti-by hydro-thermal method using zinc nitrate and hexa as raw material after nano-tube array
Dendritic nano-tube array should be prepared in outer tube wall growing ZnO nano-wire；
(5) the dendritic nanotube surface also sputters one layer of Al2O3Protective film layer.
2. the preparation method of dendritic nano-tube array according to claim 1, it is characterised in that:
In above-mentioned steps (1), the thickness value of the ZnO film is 30~40nm.
3. the preparation method of dendritic nano-tube array according to claim 1, it is characterised in that:
In above-mentioned steps (2), the Al2O3The thickness value of nanometer layer is 10~20nm.
4. the preparation method of dendritic nano-tube array according to claim 1, it is characterised in that: in above-mentioned steps (5), institute
State Al2O3The thickness range of protective film layer is 10~20nm.
5. a kind of carry out specific recognition circulating tumor using the described in any item dendritic nano-tube arrays of the claims 1 to 4
The method of cell, comprises the concrete steps that:
(1) mercapto groups are modified on dendritic nanotube；
(2) processing of substrate film；
(3) will with the prepared Streptavidin of phosphate buffer solution (streptavidin) solution be added dropwise substrate film surface into
Row reaction；
(4) phosphoric acid buffer of the EpCAM antibody molecule of end biotin labeling (biotinylated anti-EpCAM) is molten
Drop is added on substrate film, and the antibody molecule of the specific recognition is fixed on substrate surface after reaction.
6. the method that dendritic nano-tube array according to claim 5 carrys out specific recognition circulating tumor cell, feature
It is:
The detailed process that mercapto groups are modified on above-mentioned steps (1) the dendritic nanotube is: substrate film is immersed in containing 4%
3- mercaptopropyl trimethoxysilane ethanol solution in react 12h, then with a large amount of ethyl alcohol rinse surface, N2Air-flow drying.
7. the method that dendritic nano-tube array according to claim 5 carrys out specific recognition circulating tumor cell, feature
It is:
The processing detailed process of above-mentioned steps (2) described substrate film is: will contain amino-mercapto crosslinking agent γ-maleimide
The aqueous solution of base butyric acid-N- hydroxy thiosuccinimide sour (Sulfo-GMBS) handles substrate film, places 30min, then uses
Water rinses surface, N2Air-flow drying.
8. the method that dendritic nano-tube array according to claim 5 carrys out specific recognition circulating tumor cell, feature
Be: the reaction process in substrate film surface is added dropwise in Streptavidin (streptavidin) solution described in above-mentioned steps (3)
Specifically: it will be added dropwise with the prepared Streptavidin of phosphate buffer solution (streptavidin) solution in substrate film surface,
Surface, N are rinsed with phosphate buffer solution after reaction 45min2Air-flow drying.
9. the method that dendritic nano-tube array according to claim 5 carrys out specific recognition circulating tumor cell, feature
Be: the antibody molecule of the specific recognition is fixed on the detailed process of substrate surface after reaction described in above-mentioned steps (4)
It is: the phosphate buffer solution of the EpCAM antibody molecule (biotinylated anti-EpCAM) of end biotin labeling is dripped
Be added on substrate film, react 30min after, phosphate buffer solution rinse surface, biotin can directly in conjunction with Streptavidin, from
And the antibody molecule of specific recognition is made to be fixed on substrate surface.
10. a kind of microfluidic devices for capturing with regulation cancer cell in situ using nano-tube array described in claim 1,
It is characterized by:
Including PDMS module pond, PDMS module pond includes:
Cell culture insert, the upper part for being placed in PDMS module pond are used to contain culture solution, are internally provided with dendritic nano-tube array,
The substrate film of the dendritic nano-tube array bottom is the bottom surface of cell culture insert；
Microchannel is placed in the lower part in PDMS module pond for conveying drug solution and collecting intracellular molecules；
The substrate film of the dendritic nano-tube array is used to separate the cell culture insert on upper layer and the microchannel of bottom；
Two ends of the microchannel are equipped with the I/O port for conveying solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810580389.XA CN109142712B (en) | 2018-06-07 | 2018-06-07 | The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810580389.XA CN109142712B (en) | 2018-06-07 | 2018-06-07 | The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109142712A CN109142712A (en) | 2019-01-04 |
| CN109142712B true CN109142712B (en) | 2019-11-29 |
Family
ID=64801977
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810580389.XA Active CN109142712B (en) | 2018-06-07 | 2018-06-07 | The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109142712B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11781100B2 (en) | 2020-12-03 | 2023-10-10 | Applied Materials, Inc. | All-in-one bioreactor for therapeutic cells manufacturing |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907416A (en) * | 2019-11-05 | 2020-03-24 | 中山大学 | Circulating tumor cell detection device based on hollow nano needle tube electroporation system and detection method thereof |
| CN112869259A (en) * | 2020-08-18 | 2021-06-01 | 天津大学 | Respiratory detects gauze mask for virus |
| CN114317201B (en) * | 2021-12-15 | 2023-10-20 | 深圳先进技术研究院 | Microfluidic chip for capturing circulating tumor cells and manufacturing method thereof |
| CN114875378A (en) * | 2022-05-08 | 2022-08-09 | 中国科学院重庆绿色智能技术研究院 | Preparation method of cell screening device with vertical structure tungsten diselenide array thin film structure |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN202279815U (en) * | 2011-07-19 | 2012-06-20 | 西门子公司 | Circulating tumor cell capturing device |
| CN102951591A (en) * | 2012-11-20 | 2013-03-06 | 华中科技大学 | Micro-channel structure for catching circulating tumor cells and preparation method thereof |
| CN107988586A (en) * | 2017-11-29 | 2018-05-04 | 华南理工大学 | The method that atomic layer deposition prepares zinc oxide nano mitron |
-
2018
- 2018-06-07 CN CN201810580389.XA patent/CN109142712B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN202279815U (en) * | 2011-07-19 | 2012-06-20 | 西门子公司 | Circulating tumor cell capturing device |
| CN102951591A (en) * | 2012-11-20 | 2013-03-06 | 华中科技大学 | Micro-channel structure for catching circulating tumor cells and preparation method thereof |
| CN107988586A (en) * | 2017-11-29 | 2018-05-04 | 华南理工大学 | The method that atomic layer deposition prepares zinc oxide nano mitron |
Non-Patent Citations (2)
| Title |
|---|
| Atomic Layer Deposition of Al-Doped ZnO/Al2O3 Double Layers on Vertically Aligned Carbon Nanofiber Arrays;Gary A. Malek et al;《Appl. Mater. Interfaces》;20140401;第6卷(第9期);第6865–6871页 * |
| ZnO/Al2O3 nanolaminates fabricated by atomic layer deposition growth and surface roughness measurements;J.W. Elam et al;《Thin Solid Films Thin Solid Films Thin Solid Films Thin Solid Films Thin Solid Films Thin Solid Films Thin Solid Films》;20020701;第414卷(第1期);第43–55页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11781100B2 (en) | 2020-12-03 | 2023-10-10 | Applied Materials, Inc. | All-in-one bioreactor for therapeutic cells manufacturing |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109142712A (en) | 2019-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109142712B (en) | The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ | |
| CN102732415B (en) | High-efficiency rare-cell-capturing integrated chip, manufacturing method thereof, and application thereof | |
| Cui et al. | ZnO nanowire-integrated bio-microchips for specific capture and non-destructive release of circulating tumor cells | |
| CN106148315B (en) | A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof | |
| CN102435655A (en) | Field effect transistor-based tumor diagnosis apparatus and assay method thereof | |
| CN101221182A (en) | Novel method for blood serum tumor series diagnosis by fluorescent protein chip | |
| CN206244793U (en) | A kind of micro-fluidic chip | |
| CN108351348A (en) | A kind of drug screening method and its device | |
| Khosravi et al. | Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube–CTC chip | |
| CN111796104A (en) | Exosome detection and typing microfluidic chip and exosome detection and typing method | |
| CN104164360B (en) | Integrated microfluidic chip and for three-dimensional nodule location, build, recovery method | |
| CN202279815U (en) | Circulating tumor cell capturing device | |
| Chang et al. | High-throughput immunomagnetic cell detection using a microaperture chip system | |
| Li et al. | Highly efficient isolation and release of circulating tumor cells based on size-dependent filtration and degradable ZnO nanorods substrate in a wedge-shaped microfluidic chip | |
| Chao et al. | Applications of field effect transistor biosensors integrated in microfluidic chips | |
| CN110907416A (en) | Circulating tumor cell detection device based on hollow nano needle tube electroporation system and detection method thereof | |
| CN105349421B (en) | Particulate release chip, preparation method and the method for releasing of nanometic zinc oxide rod array | |
| CN105755135B (en) | A kind of No. 17 chromosome centromere probe reagent boxes of people and its preparation method and application | |
| CN111893023A (en) | Tumor cell capturing device and preparation and application thereof | |
| Zhou et al. | Degradable porous nanoflower substrate-embedded microfluidic device for capture, release and in situ manipulation of cancer cells | |
| CN107523472A (en) | The cell capture device and its round-robin method of antibacterial and reusable edible | |
| US11193888B2 (en) | Method and system for separating biomolecules from a mixture containing same | |
| CN106442459A (en) | Gold-based extraction material functionalized by different affinity ligands, and application thereof in surface Plasmon optical affinity sandwich analysis | |
| CN110317804A (en) | The method and its kit of RNA are extracted from paraffin-embedded tissue | |
| CN1477397A (en) | Microbial body sorting and detection method, its special-purpose equipment and its kit |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |