CN107085107A - A kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell and its application - Google Patents
A kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell and its application Download PDFInfo
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Abstract
The invention discloses a kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell, prepared using following steps：First, the PDMS microfluidic channel systems for being divided into from porch and several fish bone structures being provided with more than 2 parallel microfluid channels, microfluid channel are made；2nd, the etching of silicon nanowires substrate；3rd, the surface modification of silicon nanowires and biotin biological functional, four, the modification of silicon nanowires substrate and the assembling of PDMS micro-fluid chips.Sample to be tested cell and p EpCAM and the p cKit of biotin modification 200 μ L PBS solutions are incubated 1 hour by the system altogether when in use, are then injected into being detected in microfluid system.The microfluid system of the present invention can recognize and combine out the extremely low CTC of content, traditional immunofluorescence dyeing can be carried out, detection result and phenotypic analysis result is obtained, further specificity can also be discharged, CTC purifying is realized, improves purity to adapt to the requirement of genetic test limit.
Description
Technical field
It is specially a kind of detection esophageal squamous cell the present invention relates to the microfluid system detected for circulating tumor cell
The microfluid system based on silicon nanowires substrate of cancer circulating tumor cell.
Background technology
With the increase of pollution level, the ill probability of tumour is already higher than 1/3 ratio.For the diagnostic method of tumour
Mainly include：Pathology (biopsy slice)；Iconography (ultrasound, X-ray, CT or PET etc.) and serology (the related egg of blood serum tumor
In vain, such as CA-125, CA-199 or CEA).But these methods to exist the defect of itself, it is impossible to for be continually changing tumour and
Diagnosis early warning provides help.Clinical at present generally acknowledged human peripheral circulating tumor cell (Circulating Tumor Cell, letter
Claim CTC) detection be acknowledged as one of best detection means at present.
In 2004, United States drug was approved first relevant CTC enrichment with food Surveillance Authority and counted first
Several technical products.This product be using the epithelial cell adhesion molecule (EpCAM) expressed by cell surface and stem cell because
Sub- acceptor (cKit) completes the sorting of cell.EpCAM on most of epithelial cell more expression, therefore in source
Also there is expression in the tumour cell of epithelial cell.C-Kit is typical III receptor EGFR-TK, is sent out in tumour
Played a very important role in exhibition and invasion and attack, migration and relapsing course, be the popular target of current tumor cells targeted therapy
One of mark.It is modified with anti-EpCAM or anti-cKit magnetic bead to interact with the blood cell of solidification, so owns
Expression EpCAM or cKit cell surface can all adhere to some magnetic beads, finally be sorted again by magnetic field.Further to recognize
Double positive CTC, all captured cells can be disintegrated down from magnetic bead first, then with cytokeratin Krt19 and in vain
The fluorescence antibody of cellular antigens (CD45) is marked.In this system, the technical definition for CTC is EpCAM+/cKit
The nucleolate objects of +/CD45-.The apparatus and method are applied in several treatments as an embedded bio mark
In related clinical test.The first clinical trial about CTC count detections was carried out in 2004, mainly for
Have occurred and that the breast cancer patients of transfer.CTC testing results before the drawn treatment of current experiment and during treatment are simultaneously pre-
Progression free survival phase (PFS) and Overall survival (OS) are surveyed.In the very short time, also carried out in a variety of different cancer researches
Similar CTC counts test, wherein just including intestinal cancer, cutaneum carcinoma, lung cancer and prostate cancer, and has drawn similar hair
It is existing.It is worth noting that, CTC count detection results are divided into two classes by these researchs mostly：Qualitatively as (for evaluating patient
The state of an illness) it is favourable or unfavorable, rather than using they as one can consecutive variations quantitative data.
The various problems that the limitation of current CTC technologies is brought.First, detection sensitivity is still extremely in CTC researchs
Close important influence factor.The CTC technologies that FDA is approved can not all be detected in 50% esophageal squamous cell carcinoma patient
CTC.Secondly, the CTC beneficiation technologies based on the independent antibody of EpCAM can not detect occurring the swollen of Epithelial and stromalization conversion
Oncocyte.The CTC that the CTC of early stage studies some verified patients has interstitial feature, so it has been generally acknowledged that Epithelial and stromal
Change phenomenon and very important effect is played during CTC generation.3rd, only CTC is counted, ignored in CTC
The significance of subgroup.Shown in nearest research, special shape characteristic and the table of splicing variants that some CTC have
Up to relevant with tumour visceral metastases and the cancer resistance to the action of a drug.These discoveries all point out that CTC, as tumor markers, is not only used for
Count.4th, the analysis of molecules that the method that the first immobilization of blood sample is detected again can limit downstream after separation CTC is ground
Study carefully.
The content of the invention
The technical problem to be solved in the present invention is that purity and sensitivity are not high enough when overcoming existing CTC detections, while capture
CTC can not continue on for postorder research defect there is provided it is a kind of detect esophageal squamous cell carcinoma circulating tumor cell based on silicon
The microfluid system of nanowire base.
In order to solve the above-mentioned technical problem, the invention provides following technical scheme：
A kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell, is prepared using following steps：
First, the making of PDMS micro-fluid chips
PDMS microfluidic channels system includes an entrance and one outlet, from porch be divided into more than 2 it is parallel micro-
Several fish bone structures are provided with fluid channel, microfluid channel；It is preferred that fish bone structure be by PDMS be printed as it is some
V-shaped groove is parallel to each other in individual V-shaped groove, single fish bone structure, and the drift angle of the V-shaped groove of adjacent fish bone structure interlocks up and down；Entering
Mouth and exit are provided with PDMS holes；
It is preferred that the PDMS microfluidic channels system be divided into 2 parallel microfluid channels from porch, each channel is long
For 25mm, wide position 2mm；A width of 50 microns of fish bone structure in the microfluid channel, 50 microns of spacing is high 30 microns.It is this
Fish bone structure adds Cell capture efficiency, and obvious vortex can be produced in runner, can batch low cost production.
It is preferred that, fillet is provided with each shunting of the PDMS microfluidic channels, on the one hand increase sample water conservancy diversion, another
Aspect lowers injury of the cell caused by shearing force.Just start the whirlpool that generation is produced by fish bone structure at channel 0.5mm entering
Effect is flowed, fish bone structure is stirred to cell solution, increases the chance of cells contacting silicon nanowires；
The design that PDMS microfluidic channels system is singly gone out using singly entering, and larger runner design realize high flux
CTC purification process, compared with conventional method, the behaviour that can reduce half does the time, and may be used as shadow after collocation analysis software
As analysis.
2nd, the etching of silicon nanowires substrate
1), the rotary coating SU-8 photoresistance glue on silicon chip, limits etch areas by standard photolithographic methods, only etches parallel
Microfluid channel need cover region；It is preferred that, during the etching of silicon nanowires substrate from thickness be 0.7 millimeter, it is a length of
50 millimeters and a width of 25 millimeters of silicon chip, sodium rice line length is 2~5 microns.
2) Ag nanoparticle depositions, are introduced by NH4F/AgNO3 reagents, in silicon chip surface formation nanoparticle film；It is preferred that
, Ag nanoparticle depositions are less than or equal to 50nm；It is preferred that, the deposition of Ag nano particles is aided in using ultrasonic oscillation；More can
Uniformly etch uniform nano wire, there is provided more preferable Cell capture ring than more uniform, smooth for the nano wire of even thickness
Border, is conducive to increasing the survival rate of cell after purification；
3), using H2O2/ NH4F is performed etching, and obtains the nanowire corresponding with parallel microfluid channel；
4) Ag nano particles first, are washed away using concentrated nitric acid, then using the concentrated sulfuric acid/hydrogen peroxide mixed solution cleaning silicon chip table
Face, then deionized water washes clean；The volume ratio of the concentrated sulfuric acid/hydrogen peroxide is 3:1；
It is preferred that, from thickness it is 0.7 millimeter, a length of 50 millimeters and a width of 25 millimeters during the etching of silicon nanowires substrate
Silicon chip, sodium rice line length is 2~5 microns, optimal 3 μm of CTC capture rates sodium rice line length；
The etching of the silicon nanowires substrate of the present invention, it is different from traditional silicon nanowires, do not introduce HF this to environment
There is the reagent of very big harm with operator, can be by whole operation and preparation process in generalization while environmental protection
Learn in laboratory and complete, easy manufacturing conditions are provided for later product production, while ensure that the bio-safety of material surface
Property, it is more suitable for Cell capture and is used.The silicon nanowires base chip being made can be used with pathological glass slide collocation, Ke Yiyong
In fluorescence analysis, can also equally it be stored with the pathological analysis section of standard.
3rd, the surface modification and biological functional of silicon nanowires
1), with washes of absolute alcohol silicon nanowires substrate, N2 air-blowings are done under super-clean environment；
2), freshly prepared mass concentration is the toluene solution of 1.0% APTES, and will be clear
Wash the dried silicon chip with silicon nanowires and soak half an hour wherein；It is preferred that, the concussion that rotating speed is 60rpm can be used
Groove increases reaction rate；
3) silicon chip of surface bond APTES, is taken out, is cleaned with technical grade ethanol, N2 air-blowings
It is dry；
4), silicon chip is stood in freshly prepared 50ng/mL polyethylene glycol 2- amino-ethyl ether biotin PBS solutions
3 hours, cleaned, be stored in after N2 air-blowings are dry standby under 4 DEG C of dry environments with technical grade ethanol；
5), before sample test, with PBS chip surface twice after, add 200 μ L, 5.0 μM of Streptavidins it is molten
Liquid, 37 DEG C are incubated 1 hour；After PBS, then and two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit 200 μ L
PBS solution is incubated 1 hour altogether, to be used for CTC capture after PBS；
Surface of silicon nanowires is modified upper biotin by this step by the chemical bond and method of safety, then affine by strepto-
The introducing of element, the peptide molecule with CTC identification functions is further modified on its surface, be easy to the cell of later stage selectivity to catch
Shearing and release after obtaining；, can be for reducing non-specific adsorption because PEG molecular weight is 2300, PEG water absorbing capacity can
With the cell death for significantly avoiding cell rupture from causing, increase the feasibility in follow-up CTC analysis of molecules.
4th, the modification of silicon nanowires substrate and the assembling of PDMS micro-fluid chips
1), the μ L of Streptavidin PBS solution 200 of 5.0 μM of preparation, are covered on PDMS micro-fluid chips, quiet in 37 DEG C
Put 1 hour, PBS three times；After PBS, then and the p-EpCAM containing biotin modification and p-cKit two kinds of polypeptides
200 μ L PBS solutions are incubated 1 hour altogether, to be used for CTC capture after PBS；
2), the chip of silicon nanowires substrate is placed on chip microscope carrier again, and by parallel microfluid channel and sodium rice
Line is corresponding, then pushes down micro-fluid chip with the upper lid of microscope carrier；
3) the PDMS holes connection at entrance and exit, is connected into Tygon fluid delivery tubes respectively, and and is already installed on
Syringe link on syringe pump.
The application method of the microfluid system of the detection esophageal squamous cell carcinoma circulating tumor cell of the present invention, including it is following
Step：
1), by sample to be tested cell and the 200 μ L PBS of two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit
Solution is incubated 1 hour altogether, has isolated two kinds of biotin modification p-EpCAM and p-cKit afterwards with 1000rpm centrifugation
The cell of polypeptide is detected；P-EpCAM, p-cKit are the high peptide molecules of two species specificity, either to Artificial sample and
Blood sample all has higher specificity；
2), cell sample to be tested is injected into microfluid system by syringe and detected.
Surface modification and biotin functionalized modification and PDMS microfluidic channel systems that the present invention passes through silicon nanowires substrate
It is engaged, after sample to be tested cell is incubated altogether using the two kinds of peptide molecules of p-EpCAM and p-cKit for having biotin modification, Ke Yishi
Other and combination goes out the extremely low CTC of content, is attracted to surface of silicon nanowires with reference to the CTC after capture, can carry out traditional exempt from
Epidemic disease fluorescent staining, obtains detection result and phenotypic analysis result, can also further discharge, and realizes CTC purifying, improves purity
To adapt to the requirement of genetic test limit, it is suitable for molecule diagnosis and treatment and the precisely requirement of medical treatment, because the examination for discharging CTC
Agent is primarily directed to the peptide molecule combined with CTC, so release has specificity, the CTC samples finally collected have very
High purity, possibility is provided for the genetic test in later stage.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for specification, the reality with the present invention
Applying example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the structural representation of the PDMS microfluidic channel systems of the embodiment of the present invention；
Fig. 2 is the mplifying structure schematic diagram at A in Fig. 1；
Fig. 3 is the flow simulating of cell when being not provided with fish bone structure in the parallel channel of PDMS microfluidic channel systems
Figure；
Fig. 4 is the flow simulating figure of cell when fish bone structure is set in the parallel channel of PDMS microfluidic channel systems；
Fig. 5 is cell flow rate simulation drawing when fillet is set at PDMS microfluidic channel system shuntings.
Embodiment
The preferred embodiments of the present invention are illustrated below in conjunction with accompanying drawing, it will be appreciated that preferred reality described herein
Apply example to be merely to illustrate and explain the present invention, be not intended to limit the present invention.
Embodiment 1
A kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell, is prepared using following steps：
First, the making of PDMS micro-fluid chips
PDMS microfluidic channels system includes an entrance and one outlet, and PDMS microfluidic channels system is punished from entrance
For 2 parallel microfluid channels, fillet, each a length of 25mm of channel, wide position 2mm are provided with each shunting；The microfluid letter
A width of 50 microns of fish bone structure in road, 50 microns of spacing is high 30 microns, as shown in figure 1, wherein 1 be fish bone structure, 2 be into
Mouthful, 3 be outlet, and 4 be microfluid channel.Fig. 5 shows influence of the fillet design to flow velocity at shunting.In Figure 5, the longitudinal axis and horizontal stroke
Axle is all runner size, and unit is millimeter, and the right legend shows the size of flow velocity；As can be known from Fig. 5, fillet diversion design
When, can producing one layer, layer is at fillet shunting at a slow speed, and such as figure circle bit selecting puts shown, and the layer at a slow speed produced at the fillet shunting can
Effectively to reduce cell in flowing, therefore the destruction produced because of hitting also improves the biological integrity of cell.
Fish bone structure is that V-shaped groove is parallel to each other in several V-shaped grooves being printed as by PDMS, single fish bone structure, adjacent
The drift angle of V-shaped groove of fish bone structure interlock up and down, as shown in Figure 2；PDMS holes are provided with entrance and exit；
The longitudinal axis and transverse axis are all runner sizes in figs. 3 and 4, and unit is millimeter, and the right legend shows the big of flow velocity
It is small；From Fig. 3 and Fig. 4, when not having fish bone structure, fluid is stable laminar condition, represents that fluid is not disturbed,
Cell also thus will not increase the touch opportunity with bottom；And be provided with after fish bone structure, fluid has obvious flow velocity difference, with
And produce many phenomenons for upsetting flowing.Compared with no fish-bone, the situation that the particle (or cell) in fluid flows at this
Under, added with the chance of beneath contact area.
The mask plate of microfluidic channel structure and fish bone structure is printed respectively；
2nd, the etching of silicon nanowires substrate
1), from the silicon chip that thickness is 0.7 millimeter, a length of 50 millimeters and a width of 25 millimeters, the rotary coating SU- on silicon chip
8 photoresistance glue, rotary coating rotating speed is 3000rpm, using fluid channel mask plate illumination channel design region, is led to after imaging
Road structure；
2) the Ag nanoparticle depositions less than or equal to 50nm, are introduced by NH4F/AgNO3 reagents, formed in silicon chip surface
Nanoparticle film；
3), using H2O2/ NH4F is performed etching, and obtains the nanowire corresponding with parallel microfluid channel, sodium rice line length
Spend for 2~5 microns；
4) Ag nano particles first, are washed away using concentrated nitric acid, then using the concentrated sulfuric acid/hydrogen peroxide mixed solution cleaning silicon chip table
Face, then deionized water washes clean；The volume ratio of the concentrated sulfuric acid/hydrogen peroxide is 3:1；
3rd, the surface modification and biological functional of silicon nanowires
1), with washes of absolute alcohol silicon nanowires substrate, N2 air-blowings are done under super-clean environment；
2), freshly prepared mass concentration is the toluene solution of 1.0% APTES, and will be clear
Wash the dried silicon chip with silicon nanowires and soak half an hour wherein；It is preferred that, the concussion that rotating speed is 60rpm can be used
Groove increases reaction rate；
3) silicon chip of surface bond APTES, is taken out, is cleaned with technical grade ethanol, N2 air-blowings
It is dry；
4), silicon chip is stood in freshly prepared 50ng/mL polyethylene glycol 2- amino-ethyl ether biotin PBS solutions
3 hours, cleaned, be stored in after N2 air-blowings are dry standby under 4 DEG C of dry environments with technical grade ethanol；
5), before sample test, with PBS chip surface twice after, add 200 μ L 5.0 μM of Streptavidins it is molten
Liquid, 37 DEG C are incubated 1 hour；After PBS, then and two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit 200 μ L
PBS solution is incubated 1 hour altogether, and CTC capture is used for after cleaning；
4th, the modification of silicon nanowires substrate and the assembling of PDMS micro-fluid chips
1), the μ L of Streptavidin PBS solution 200 of 5.0 μM of preparation, are covered on PDMS micro-fluid chips, quiet in 37 DEG C
Put 1 hour, PBS three times；After PBS, then and the p-EpCAM containing biotin modification and p-cKit two kinds of polypeptides
200 μ L PBS solutions are incubated 1 hour altogether, to be used for CTC capture after PBS；
2), the chip of silicon nanowires substrate is placed on chip microscope carrier again, and by parallel microfluid channel and sodium rice
Line is corresponding, then pushes down micro-fluid chip with the upper lid of microscope carrier；
3) the PDMS holes connection at entrance and exit, is connected into Tygon fluid delivery tubes respectively, and and is already installed on
Syringe link on syringe pump.
The application method of the microfluid system of the detection esophageal squamous cell carcinoma circulating tumor cell of the present invention, including it is following
Step：
1), by sample to be tested cell and the 200 μ L PBS of two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit
Solution is incubated 1 hour altogether；Two kinds of biotin modification p-EpCAM and p-cKit has been isolated with 1000rpm centrifugation afterwards
The cell of polypeptide is detected；
2), 200 μ L analog samples are fitted into 1mL syringes, injecting systems are connected；
3) after, injection is finished, PFA (paraformaldehyde) solution that 100 μ L concentration are 1% is loaded in syringe, with flow velocity
1.0mL/h flows through chipset, and the cell of capture is fixed；
4), after dismounting chipset, silicon nanowires substrate slide is taken out；
5), after PBS, by the primary antibody of preparation：5 μM of anti-cytokeratin Ab anti-Krt19 and 5 μM of CD45 antibody
(anti-CD45) the μ L of mixed solution 200 are covered at silicon nanowires region, 4 DEG C, stand 8 hours；
6), clean afterwards, and standard method carries out the dyeing of secondary antibody.Correspondence CK antibody marks for Alex488, correspondence CD45
Antibody marks for Alex555, and two anti-dye are 40 minutes at room temperature；
7), with PBS it is secondary after, as far as possible draw remove surface solution residual, and with contain Hoechest (DAPI)
The μ L of sealing liquid 60 encapsulation of core reagent is contaminated, cover glass is capped；
8), carry out fluorescence imaging and counted.
9), by the cell count of Krt19+/CD45-/DAPI+ in imaging results for capture cancer cell, by compared with
Begin the cancer cell count 200 added, calculates corresponding capture rate.
Interference experiment
The leucocyte extracted in healthy human blood is used as interference cell 2.0 × 106/ mL, prepares analog sample, and compare
For different types of cancer cell (KYSE-30, KYSE-150 and KYSE-180) capture ability, it was demonstrated that with it is white in actual blood
The interference of cell equivalent, does not interfere with the efficiency of detection.
Cancer patient's blood sample applicating example
First, blood of cancer patients is gathered with BD Vacutainer Glass ACD Solution A tube, in two times
Collecting (to avoid EDTA anti-freezings from being damaged for the antigen of cell surface in blood, causes the combination of influence capture antibody, reduction is caught
Obtain efficiency)
First pipe of blood sampling only extracts 2mL, is not used to be tested, and is taken off because depositing in blood when there is syringe needle piercing
, there is the reading of false positive cell in the epithelial cell fallen.Second pipe, which extracts 4ml, is used for subsequent experimental.
2nd, gradient density centrifugal is taken to carry out preliminary purification to the second pipe blood sample of collection
(a) 4mL PBS solutions are added, are diluted in equal volume for blood sample
(b) after being well mixed, it is slowly added in the 15mL centrifuge tubes for having been added to 4mL gradient density centrifugals liquid (1077)
Blood sample after dilution.
(c) 300g is used, is centrifuged within 40 minutes.
(d) about 2-4mL mononuclear cell layer (PBMC, peripheral blood is collected in centrifugation after terminating
mononuclear cell)。
(e) mononuclear cell layer collected is centrifuged, 400g, 5 minutes, removes supernatant liquor
(f) cleaned with 2mL PBS.
(g) centrifugation is removed after supernatant liquor, add freshly prepared 5 μM of anti-cytokeratin Abs 19 (anti-Krt19) and
5 μM of μ L of CD45 antibody (anti-CD45) mixed solution 400, break up and are incubated altogether 45 minutes at cell aggregation, 37 DEG C.
(h) clean, and constant volume is in 400 μ L PBS, carries out subsequent experimental
As a result：
In 9 samples collected and studied (6 esophageal squamous cell carcinoma patients and 3 Healthy Peoples), it have collected CTC's
Cell suspending liquid.After neutralizing, cleaning, CTC cell depositions (cell pellet) are carried out using whole genome amplification kit
DNA is extracted and expanded.Afterwards, amplification has been carried out for the significant gene PIK3CA of esophageal squamous cell carcinoma and Sanger is sequenced.
As a result PIK3CA hypotypes have detection in 6 patients with esophageal squamous cell, and are not detected in 3 Healthy People samples.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's
Within protection domain.
Claims (7)
1. a kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell, it is characterised in that use following steps
Prepare：
First, the making of PDMS micro-fluid chips
PDMS microfluidic channels system includes an entrance and one outlet, is divided into more than 2 parallel microfluids from porch
Several fish bone structures are provided with channel, microfluid channel, PDMS holes are provided with entrance and exit；
2nd, the etching of silicon nanowires substrate
1), the rotary coating SU-8 photoresistance glue on silicon chip, etch areas is limited by standard photolithographic methods, is only etched parallel micro-
Fluid channel needs the region covered；
2) Ag nanoparticle depositions, are introduced by NH4F/AgNO3 reagents, in silicon chip surface formation nanoparticle film；
3), using H2O2/ NH4F is performed etching, and obtains the nanowire corresponding with parallel microfluid channel；
4) Ag nano particles first, are washed away using concentrated nitric acid, then using the concentrated sulfuric acid/hydrogen peroxide mixed solution cleaning silicon chip surface,
Then deionized water washes clean；The volume ratio of the concentrated sulfuric acid/hydrogen peroxide is 3:1；
3rd, the surface modification and biological functional of silicon nanowires
1), with washes of absolute alcohol silicon nanowires substrate, N2 air-blowings are done under super-clean environment；
2), freshly prepared mass concentration is the toluene solution of 1.0% APTES, and cleaning is done
The silicon chip with silicon nanowires after dry soaks half an hour wherein；
3) silicon chip of surface bond APTES, is taken out, is cleaned with technical grade ethanol, N2 air-blowings are done；
4), silicon chip is stood to 3 in freshly prepared 50ng/mL polyethylene glycol 2- amino-ethyl ether biotin PBS solutions small
When, cleaned, be stored in after N2 air-blowings are dry standby under 4 DEG C of dry environments with technical grade ethanol；
5), before sample test, with PBS chip surface twice after, add 200 μ L 5.0 μM of solution of streptavidin, 37
DEG C be incubated 1 hour；After PBS, then and two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit 200 μ L PBS
Solution is incubated 1 hour altogether, to be used for CTC capture after PBS；
4th, the modification of silicon nanowires substrate and the assembling of PDMS micro-fluid chips
1), the μ L of Streptavidin PBS solution 200 of 5.0 μM of preparation, are covered on PDMS micro-fluid chips, and it is small to stand 1 in 37 DEG C
When, PBS three times；After PBS, then and two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit 200 μ L
PBS solution is incubated 1 hour altogether, to be used for CTC capture after PBS；
2), the chip of silicon nanowires substrate is placed on chip microscope carrier again, and by parallel microfluid channel and nanowire phase
Correspondence, then micro-fluid chip is pushed down with the upper lid of microscope carrier；
3) the PDMS holes connection at entrance and exit, is connected into Tygon fluid delivery tubes respectively, and and is already installed on injection
Syringe link on pump.
2. the microfluid system of esophageal squamous cell carcinoma circulating tumor cell is detected as claimed in claim 1, it is characterised in that
Described fish bone structure is that V-shaped groove is parallel to each other in several V-shaped grooves being printed as by PDMS, single fish bone structure, adjacent
The drift angle of the V-shaped groove of fish bone structure interlocks up and down.
3. the microfluid system of esophageal squamous cell carcinoma circulating tumor cell is detected as claimed in claim 2, it is characterised in that
From the silicon chip that thickness is 0.7 millimeter, a length of 50 millimeters and a width of 25 millimeters, sodium rice line length during the etching of silicon nanowires substrate
For 2~5 microns.
4. the microfluid system of esophageal squamous cell carcinoma circulating tumor cell is detected as claimed in claim 1, it is characterised in that
Step 2 during the etching of silicon nanowires substrate) in Ag nanoparticle depositions be less than or equal to 50nm.
5. the microfluid system of esophageal squamous cell carcinoma circulating tumor cell is detected as claimed in claim 3, it is characterised in that
The PDMS microfluidic channels system is divided into 2 parallel microfluid channels, each a length of 25mm of channel, wide position 2mm from porch；
A width of 50 microns of fish bone structure in the microfluid channel, 50 microns of spacing is high 30 microns.
6. the microfluid system of esophageal squamous cell carcinoma circulating tumor cell is detected as claimed in claim 1, it is characterised in that
Fillet is provided with each shunting of the PDMS microfluidic channels.
7. the microfluid system of the detection esophageal squamous cell carcinoma circulating tumor cell as described in claim any one of 1-6 makes
With method, it is characterised in that comprise the following steps：
1), by 200 μ L PBS solutions of sample to be tested cell and two kinds of polypeptides of the p-EpCAM containing biotin modification and p-cKit
It is incubated 1 hour altogether, two kinds of polypeptides of biotin modification p-EpCAM and p-cKit has been isolated with 1000rpm centrifugation afterwards
The cell of specific adsorption is detected；
2), cell sample to be tested is injected into microfluid system by syringe and detected.
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