CN105255719B - A kind of three-dimensional porous micro-fluidic chip and preparation method and application - Google Patents
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Abstract
The invention discloses a kind of three-dimensional porous micro-fluidic chip and preparation method and application.Its structural integrity is replicated, then removes metal form obtaining three-dimensional porous material by the present invention using foam metal as template with micro-fluidic；Again by its silylation modification, temperature sensing material is filled in hole, after being integrated with micro-fluidic chip, temperature sensing material is removed and obtains three-dimensional porous micro-fluidic chip.After the functional group activation on three-dimensional porous material surface three-dimensional porous micro-fluidic detection chip will be obtained with biomolecule coupling.The three-dimensional porous micro-fluidic chip of the present invention can be used in bio-medical analysis, such as be used for the separation of circulating tumor cell.The present invention three-dimensional porous micro-fluidic chip there is porous, high transparency, obdurability, good biocompatibility, its three-dimensional porous structure can fix substantial amounts of biomolecule, can high efficiency, determinand is separated with high throughput.The three-dimensional porous micro-fluidic chip of the present invention to prepare simple and easy to do, cost low, reproducible.
Description
Technical field
The invention belongs to biological, chemistry and materials science field, and in particular to a kind of three-dimensional porous micro-fluidic chip and its
Preparation method and application.
Background technology
Circulating tumor cell is the tumour cell that peripheral blood is shedded into by cancer focus, the detection pair of circulating tumor cell
Assessment, drug evaluation and the operation prognosis of early diagnosis, personalized treatment in tumour etc. are significant.Because circulation is swollen
Number is few in blood for oncocyte, and it is separated and causes great challenge.Currently urgently develop simple, efficient, quick
Separate the new method of circulating tumor cell.
Micro-fluidic chip is the technology quickly grown in recent years, with easy of integration, analyze speed fast, high flux, safety etc.
Advantage, in fields such as tumor-marker analyte detection, the sorting of tumour cell, genetic analysis, pharmaceutical synthesis screening, biological reagent detections
It is widely used.But at present micro-fluidic chip preparation synthesis steps are cumbersome, technical sophistication, expensive etc., therefore limit
Its application in bio-medical analysis field is made.
The content of the invention
The primary and foremost purpose of the present invention is to overcome the shortcoming and deficiency of prior art, and there is provided the three-dimensional porous micro-fluidic core of one kind
The preparation method of piece and the three-dimensional porous micro-fluidic chip obtained by this method.
Another object of the present invention is to provide a kind of preparation method of three-dimensional porous micro-fluidic detection chip and by this
The three-dimensional porous micro-fluidic detection chip that method is obtained.
It is still another object of the present invention to provide the application of the three-dimensional porous micro-fluidic chip or detection chip.
The purpose of the present invention is achieved through the following technical solutions：
A kind of preparation method of three-dimensional porous micro-fluidic chip, comprises the following steps：
（1）Foam metal is successively put into organic solvent, water and is cleaned by ultrasonic respectively 1-60 minutes.
（2）Foam metal after cleaning is immersed in liquid micro-fluidic, in 1000-8000rpm rotating speed
Lower centrifugation makes micro-fluidic fully infiltrate foam metal.
（3）The foam metal for having infiltrated micro-fluidic is centrifuged with except de-soak under 1000-6000rpm rotating speed
Micro-fluidic in foam metal hole, afterwards makes the foam metal heating for being coated with micro-fluidic micro-fluidic
Chip material solidifies.
（4）By step（3）The obtained coated foam metal of micro-fluidic is immersed in the acid molten of 5-14mol/L
In liquid 1-12 hours to remove foam metal, obtain three-dimensional porous material.
（5）The silylating reagent solution that concentration is 1-50% is immersed in after three-dimensional porous material Plasma is handled 1-5 minutes
It is middle 1-10 hours, obtain the three-dimensional porous material of silylating reagent processing.
（6）The three-dimensional porous material of silanization treatment is immersed in the temperature sensing material solution that concentration is 1-50%, made temperature sensitive
The abundant impregnating porous structure of material, transformation temperature solidifies temperature sensing material, obtains the three-dimensional porous material of temperature sensing material closure.
（7）By step（6）The three-dimensional porous material of obtained temperature sensing material closure is placed on the silicon chip of cleaning, pours into liquid
Micro-fluidic makes it be totally submerged three-dimensional porous material, and placement makes micro-fluidic fully solidify in 1-24 hours.
（8）The part that the three-dimensional porous material of solidification micro-fluidic will be covered is cut, and card punch is respectively at two ends
Inlet and liquid outlet are beaten, Plasma is bonded after handling 1-5 minutes with slide, obtains the micro-fluidic core of temperature sensing material closing
Piece.
（9）Change step（8）The temperature of obtained micro-fluidic chip dissolves temperature sensing material, is passed through 1-1000 points of ultra-pure water
Clock, which rinses temperature sensing material well, obtains three-dimensional porous micro-fluidic chip.
Step（1）Described in foam metal include nickel foam, foam copper, foamed aluminium, foamed alloy etc..
Step（1）Described in organic solvent include acetone, toluene, ethanol etc..
Step（2）Described in micro-fluidic include dimethyl silicone polymer（PDMS）, methyl methacrylate
（PMMA）, makrolon（PC）, cycloolefin copolymer resins（COC）Deng.
Step（4）Described in acid solution include nitric acid, hydrochloric acid, sulfuric acid solution etc..
Step（5）Described in silylating reagent include the sodium alkoxide of carboxyethylsilane three, gamma-aminopropyl-triethoxy silicon
Alkane etc. can derive silylating reagents of functional group such as carboxyl, amino etc. in chip surface.
Step（6）Described in temperature sensing material include gelatin, temperature sensitive chitosan etc..
A kind of three-dimensional porous micro-fluidic chip, is prepared by the above method.Described three-dimensional porous micro-fluidic chip
Size adjustable, aperture is suitable with the aperture of foam metal material.
A kind of preparation method of three-dimensional porous micro-fluidic detection chip, comprises the following steps：By above-mentioned three-dimensional porous miniflow
After control chip is activated with chemical cross-linking agent, the biomolecule for being passed through 1~500mg/mL is incubated 0.5~10 hour, then abundant through PBS
The three-dimensional porous micro-fluidic chip of biomolecule modification, i.e., three-dimensional porous micro-fluidic detection chip are obtained after washing.
Described biomolecule is preferably used for the albumen or nucleic acid of specific recognition and capture biomolecule or cell, such as
Antibody, Streptavidin, agglutinin, growth factor, aptamer etc..
A kind of three-dimensional porous micro-fluidic detection chip, is prepared by the above method.
Described three-dimensional porous micro-fluidic chip or three-dimensional porous micro-fluidic detection chip answering in bio-medical analysis
With such as the separation of circulating tumor cell specific cells in blood, the separating of cell excretion body, free RNA separation, albumen
Separation etc..
A kind of method based on circulating tumor cell in above-mentioned three-dimensional porous micro-fluidic chip separation blood, including following step
Suddenly：
（1）The solution of bovine serum albumin(BSA) containing 1-10% and 0.1-10% surfactants is passed through specific recognition and capture
The biomolecule of tumour cell（Such as antibody or aptamer）In the three-dimensional porous micro-fluidic chip of modification, closing is incubated
1-1000min, afterwards PBS flushings.
（2）The new blood 1-10mL of cancer patient is taken, step is passed through with 1-200 μ L/min flow velocity（1）Obtained life
In the three-dimensional porous micro-fluidic chip of thing molecular modification, so that the circulating tumor cell in blood is captured by the biomolecule.
Step（1）Described in surfactant include tween, skimmed milk power etc..
The present invention copies three-dimensional porous material by the use of foam metal as template is sacrificed, and recycles temperature sensing material conduct
Three-dimensional porous material is incorporated into micro-fluidic chip by secondary sacrifice template, so as to obtain three-dimensional porous micro-fluidic chip.Biological target
It is passed through to molecule in the three-dimensional porous micro-fluidic chip of crosslinking agent activation and is fixed on porous material surface, obtains biological targeting molecule
The three-dimensional porous micro-fluidic detection chip of functionalization, and it is used successfully to the separation of circulating tumor cell.
The invention has the advantages that and effect：
Three-dimensional porous micro-fluidic chip prepared by the present invention has porous, high transparency, obdurability, good biocompatibility
The advantages of.The three-dimensional porous micro-fluidic detection chip of biomolecule is modified, its three-dimensional porous structure can fix substantial amounts of life
Thing molecule, realizes high efficiency to determinand, high-throughput isolation.Easy to operation, cost of the invention is low, reproducible,
General chemistry and Biochemistry Experiment room can be completed.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of nickel foam.
Fig. 2 is the scanning electron microscope (SEM) photograph that obtained three-dimensional porous PDMS material is replicated by nickel foam.
Fig. 3 is that the secondary antibody of FITC marks characterizes the three-dimensional porous micro-fluidic detection of anti-Epithelial Cell Adhesion factor antibody modification
The fluorescence microscopy figure of chip.
Fig. 4 is that the three-dimensional porous micro-fluidic detection chip capture human breast carcinoma of anti-Epithelial Cell Adhesion factor antibody modification is thin
The fluorescence microscopy figure of born of the same parents.
Fig. 5 is the structure chart that blood is passed into three-dimensional porous micro-fluidic detection chip, and the PDMS of 1- solidifications, 2- is three-dimensional more
Porous materials, 3- inlets, 4- liquid outlets, 5- slides, 6- feed tubes, 7- drain pipes.
Fig. 6 is the three-dimensional porous micro-fluidic detection chip of anti-Epithelial Cell Adhesion factor antibody modification from cancer patient blood sample
In the circulating tumor cell that captures and leucocyte result figure；Wherein, A, B, C, D are the cancer cell captured, and E, F, G, H are
Leucocyte, A, E be DAPI dyeing, B, F be CK-FITC dye, C, G be CD45-PE dye, D, H be foregoing three kinds dye fold
Plus figure.
Embodiment
Further detailed description is done to the present invention with reference to embodiment and accompanying drawing, but embodiments of the present invention are not limited
In this.
The concentration for being related to solution if not specified, in this specification is mass fraction.
Embodiment 1
（1）By nickel foam（0.5mm×4mm×20mm）Priority acetone, ethanol, ultra-pure water are cleaned by ultrasonic 30 points respectively
Clock, afterwards oven drying.Fig. 1 is the stereoscan photograph of nickel foam, shows its three-dimensional porous structure.
（2）Dried nickel foam is immersed in and fills uncrosslinked PDMS（Dimethyl silicone polymer）The 2mL of liquid from
In heart pipe, 8000rpm centrifugations make PDMS fully infiltrate nickel foam in 5 minutes.
（3）By step（2）The nickel foam of obtained PDMS infiltrations is transferred in the 2mL centrifuge tubes of sky, 3000rpm centrifugations 3
Minute, 80 DEG C of baking ovens heating afterwards solidified PDMS in 3 hours to remove the PDMS in nickel foam hole.
（4）By step（3）The nickel foam of obtained PDMS claddings is immersed in 7mol/L salpeter solution, is etched 1 hour
To remove nickel template, then with ultra-pure water the salpeter solution of residual is washed, obtain three-dimensional porous material.Fig. 2 is three-dimensional porous material
The stereoscan photograph of material, shows its complete three-dimensional porous structure for having copied nickel foam.
（5）It is immersed at once containing 5% carboxyethylsilane three after obtained three-dimensional porous material Plasma is handled 3 minutes
The phosphate buffer of sodium alkoxide（PBS）In, it is incubated 4 hours at room temperature, then clean with ultra-pure water unnecessary carboxyethylsilane
The phosphate buffer of three sodium alkoxides, obtains the three-dimensional porous material of carboxylated.
（6）The three-dimensional porous material of carboxylated, which is immersed in 15% gelatin solution, makes it be totally submerged filling pore, takes
Go out three-dimensional porous material be placed in less than 35 DEG C treat gelatin solidify, obtain gelatin closure three-dimensional porous material.
（7）By step（6）The three-dimensional porous material of obtained gelatin closure is transferred to the silicon chip surface of cleaning, and addition is not handed over
The PDMS liquid of connection makes it flood covering three-dimensional porous material completely, and place makes the full cross-linked solidifications of PDMS for 12 hours at room temperature.
（8）The part of three-dimensional porous material that solidification PDMS will be covered is cut, card punch beaten respectively at two ends inlet and
Liquid outlet, Plasma is bonded after handling 3 minutes with slide, obtains the micro-fluidic chip of gelatin closing.
（9）The micro-fluidic chip that gelatin is closed is heated to 50 DEG C, and warm water is passed through with 100 μ L/min flow velocity（38 DEG C of left sides
It is right）Fully rinse out remaining gelatin within 40 minutes, obtain three-dimensional porous micro-fluidic chip.
（10）10mmol/L 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides will be contained（EDC·HCl）
With 10mmol/L n-hydroxysuccinimides（NHS）Mixed solution be passed through with 50 μ L/min flow velocity it is three-dimensional porous micro-fluidic
Activated 30 minutes in chip, the anti-Epithelial Cell Adhesion factor antibodies of 10 μ g/mL are passed through with 50 μ L/min flow velocity afterwards
（EBioscience companies, Cat. No.: 13-9326-82）, again with PBS solution with 50 μ L/min after being incubated 1 hour at room temperature
Flow velocity rinse 3 minutes, that is, obtain the three-dimensional porous micro-fluidic detection chip of anti-Epithelial Cell Adhesion factor antibody modification.Antibody
The secondary antibody that modifying can be marked by FITC is characterized, and its fluorescence picture is as shown in figure 3, represent the anti-Epithelial Cell Adhesion factor
Antibody is successfully modified on three-dimensional porous PDMS material surface.
（11）100 are taken with the prestained human breast cancer cells of DiI（MCF-7）It is scattered in 1mL PBS solutions, with 100 μ
L/min flow velocity is passed into the three-dimensional porous micro-fluidic detection chip of anti-Epithelial Cell Adhesion factor antibody modification, then with 100
μ L/min flow velocity is rinsed 3 minutes with PBS.Chip is placed in observe under inverted fluorescence microscope and counted, average capture rate reaches
More than 91.7%.Fig. 4 is the fluorescence micrograph for the MCF-7 cells being captured to.
Embodiment 2
（1）By nickel foam（1.2mm×4mm×20mm）Priority acetone, ethanol, ultra-pure water are cleaned by ultrasonic 30 points respectively
Clock, afterwards oven drying.
（2）Dried nickel foam is immersed in the 2mL centrifuge tubes for filling uncrosslinked PDMS liquid, 6000rpm from
The heart makes PDMS fully infiltrate nickel foam in 3 minutes.
（3）By step（2）The nickel foam of obtained PDMS infiltrations is transferred in the 2mL centrifuge tubes of sky, 3000rpm centrifugations 3
Minute, 75 DEG C of baking ovens heating afterwards solidified PDMS in 4 hours to remove PDMS unnecessary in nickel foam hole.
（4）By step（3）The nickel foam of obtained PDMS claddings is immersed in 14mol/L salpeter solution, is etched 5 hours
To remove nickel template, then with ultra-pure water the salpeter solution of residual is washed, obtain three-dimensional porous material.
（5）It is immersed at once containing 10% carboxyethylsilane after obtained three-dimensional porous material Plasma is handled 3 minutes
The phosphate buffer of three sodium alkoxides（PBS）In, it is incubated 4 hours at room temperature, then clean with ultra-pure water unnecessary carboxy ethyl silicon
The phosphate buffer of the sodium alkoxide of alkane three, obtains the three-dimensional porous material of carboxylated.
（6）The three-dimensional porous material of carboxylated, which is immersed in 10% gelatin solution, makes it be totally submerged filling space, takes
Go out three-dimensional porous material be placed in less than 35 DEG C treat gelatin solidify, obtain gelatin closure three-dimensional porous material.
（7）By step（6）The three-dimensional porous material of obtained gelatin closure is transferred to the silicon chip surface of cleaning, and addition is not handed over
The PDMS liquid of connection, place makes the full cross-linked solidifications of PDMS for 24 hours at room temperature.
（8）The part that solidification PDMS three-dimensional porous material will be covered is cut, and inlet is beaten respectively at two ends with card punch
And liquid outlet, Plasma handle 3 minutes after be bonded with slide, obtain gelatin close micro-fluidic chip.
（9）The micro-fluidic chip that gelatin is closed is heated to 50 DEG C, and be passed through warm water with 100 μ L/min flow velocity fills for 40 minutes
Divide and rinse out remaining gelatin, obtain three-dimensional porous micro-fluidic chip.
（10）Solution containing 10mmol/L EDCHCl and 10mmol/L NHS is passed through three with 50 μ L/min flow velocity
Tie up in porous micro-fluidic chip and activate 30 minutes, 100 μ g/mL Streptavidins are passed through with 50 μ L/min flow velocity afterwards
（Sigma-Aldrich CAS. 9013-20-1）, it is incubated 4 hours at room temperature, then 10 μ g/mL are passed through with 50 μ L/min flow velocity
Biotinylated anti-Epithelial Cell Adhesion factor antibody（EBioscience companies, Cat. No.: 13-9326-82）, at room temperature
Rinsed 3 minutes with PBS with 50 μ L/min flow velocity again after being incubated 1 hour, that is, obtain anti-Epithelial Cell Adhesion factor antibody modification
Three-dimensional porous micro-fluidic detection chip.The secondary antibody that antibody modification can be marked by FITC is characterized.
（11）Mixed solution containing 5% bovine serum albumin(BSA) and 0.2% tween is passed through anti-epithelium with 50 μ L/min flow velocity
The three-dimensional porous micro-fluidic detection chip of CAF antibody modification, uses PBS with 50 μ after closing being incubated at room temperature 1 hour
L/min flow velocity is rinsed 3 minutes.
（12）The blood 1mL that cancer patient is fresh is taken, hyclone is passed into 100 μ L/min flow velocity and tween is closed
In the three-dimensional porous micro-fluidic detection chip of anti-Epithelial Cell Adhesion factor antibody modification afterwards（Fig. 5）, afterwards with 100 μ L/min
Flow velocity with PBS rinse 3 minutes, remove haemocyte；Then 4% paraformaldehyde is passed through with 50 μ L/min flow velocity, is incubated at room temperature
Educate 30 minutes, rinse unnecessary paraformaldehyde with PBS with 50 μ L/min flow velocity；50 μ L/min flow velocity is passed through 0.2% afterwards
Triton X100,4 DEG C are incubated 30 minutes, rinse unnecessary Triton X100 with PBS with 50 μ L/min flow velocity；Again with 50 μ
4 DEG C of the bovine serum albumin(BSA) that L/min flow velocity is passed through 5% is closed 1 hour, is rinsed 3 minutes with PBS with 50 μ L/min flow velocity；Most
It is passed through afterwards with 50 μ L/min flow velocity containing CK-FITC（Green fluorescence）、CD45-PE（Red fluorescence）And DAPI（Blue-fluorescence）'s
4 DEG C of solution is incubated 2 hours, with 50 μ L/min flow velocity PBS 3 minutes, is placed under inverted fluorescence microscope and is observed.Circulation
Tumour cell is defined as can be while mark DAPI blue-fluorescences and CK-FITC green fluorescence（Size is more than 10 μm）, leucocyte
Being defined as can be while marks DAPI blue-fluorescences and PE-CD45 red fluorescence, and representational cell is as shown in Figure 6.More than
As a result illustrate that the three-dimensional porous micro-fluidic detection chip of the present invention can be used for the separation of circulating tumor cell.
Above-described embodiment shows that three-dimensional porous micro-fluidic detection chip prepared by the inventive method has preferably separation effect
Really, available for field of biomedical research.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (5)
1. a kind of preparation method of three-dimensional porous micro-fluidic chip, it is characterised in that comprise the following steps：
(1) foam metal is successively put into acetone, ethanol, water and be cleaned by ultrasonic respectively 1-60 minutes；
(2) foam metal after cleaning is immersed in liquid micro-fluidic, under 1000-8000rpm rotating speed from
The heart makes micro-fluidic fully infiltrate foam metal；
(3) foam metal for having infiltrated micro-fluidic is centrifuged under 1000-6000rpm rotating speed to remove in hole
Micro-fluidic, reheating solidify micro-fluidic；
(4) the coated foam metal of micro-fluidic that step (3) is obtained is immersed in 5-14mol/L salpeter solution
To remove foam metal, three-dimensional porous material is obtained within 1-12 hours；
(5) it is immersed in after three-dimensional porous material Plasma is handled 1-5 minutes in the silylating reagent solution that concentration is 1-50%
1-10 hours, obtain the three-dimensional porous material of silylating reagent processing；
(6) three-dimensional porous material of silanization treatment is immersed in the temperature sensing material solution that concentration is 1-50%, makes temperature sensitive material
Expect abundant impregnating porous structure, transformation temperature solidifies temperature sensing material, obtain the three-dimensional porous material of temperature sensing material closure；
(7) three-dimensional porous material of the temperature sensing material closure obtained step (6) is placed on the silicon chip of cleaning, pours into liquid miniflow
Control chip material makes it be totally submerged three-dimensional porous material, and placement makes micro-fluidic fully solidify in 1-24 hours；
(8) part for covering the three-dimensional porous material for solidifying micro-fluidic is cut, card punch is thrown at two ends respectively
Liquid mouthful and liquid outlet, Plasma are bonded after handling 1-5 minutes with slide, obtain the micro-fluidic chip of temperature sensing material closing；
(9) changing the temperature for the micro-fluidic chip that step (8) is obtained dissolves temperature sensing material, and being passed through ultra-pure water 1-1000 minutes will
Temperature sensing material, which is rinsed well, obtains three-dimensional porous micro-fluidic chip；
In above-mentioned steps, described foam metal is nickel foam, and described micro-fluidic is dimethyl silicone polymer, institute
The silylating reagent stated is the sodium alkoxide of carboxyethylsilane three, and described temperature sensing material is gelatin.
2. a kind of preparation method of three-dimensional porous micro-fluidic detection chip, it is characterised in that comprise the following steps：Right will be passed through
It is required that after the 1 three-dimensional porous micro-fluidic chip prepared is activated with chemical cross-linking agent, being passed through 1~500mg/mL biomolecule
It is incubated 0.5~10 hour, then obtain three-dimensional porous micro-fluidic detection chip after fully being washed through PBS.
3. the preparation method of three-dimensional porous micro-fluidic detection chip according to claim 2, it is characterised in that：Described life
Thing molecule includes antibody, Streptavidin, agglutinin, growth factor, aptamer.
4. a kind of method for separating circulating tumor cell in blood, it is characterised in that comprise the following steps：
(1) solution of bovine serum albumin(BSA) containing 1-10% and 0.1-10% surfactants is passed through specific recognition and capture is swollen
In the three-dimensional porous micro-fluidic chip of the biomolecule modification of oncocyte, closing 1-1000min is incubated, PBS is rinsed afterwards；It is described
Three-dimensional porous micro-fluidic chip obtained by the preparation method described in claim 1；
(2) the new blood 1-10mL of cancer patient is taken, the biology point that step (1) is obtained is passed through with 1-200 μ L/min flow velocity
In the three-dimensional porous micro-fluidic chip of son modification, so that the circulating tumor cell in blood is captured by the biomolecule.
5. method according to claim 4, it is characterised in that：Surfactant described in step (1) includes tween, taken off
Fat milk powder.
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| CN108753573A (en) * | 2018-06-07 | 2018-11-06 | 武汉大学 | The method for being captured in micro-fluidic chip and identifying fetal nucleated red blood |
| CN109053216B (en) * | 2018-10-09 | 2021-09-24 | 中国科学院宁波材料技术与工程研究所 | Block material with wetting anisotropy and preparation method thereof |
| CN109821134A (en) * | 2019-01-18 | 2019-05-31 | 武汉大学 | A kind of three-dimensional network bioprobe and the preparation method and application thereof for living body |
| CN114112605A (en) * | 2020-08-27 | 2022-03-01 | 王剑 | Method for making blood circulation tumor cell pathological chip |
| CN114196503A (en) * | 2020-09-17 | 2022-03-18 | 中国科学院大连化学物理研究所 | Exosome separation and enrichment method based on exosome enrichment chip |
| CN113013019A (en) * | 2021-02-03 | 2021-06-22 | 沈发明 | Silicon wafer surface base treatment method |
| CN112973813B (en) * | 2021-02-10 | 2023-03-14 | 齐鲁工业大学 | Micro-fluidic chip for separating and enriching exosomes and manufacturing method thereof |
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