CN107488576A - A kind of preparation method for the micro-fluidic chip for embedding oriented nanofibers film - Google Patents
A kind of preparation method for the micro-fluidic chip for embedding oriented nanofibers film Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method for the micro-fluidic chip for embedding oriented nanofibers film, including：By PEI/PVA spinning solutions, electrostatic spinning, vapor crosslinking, obtain being orientated PEI/PVA nano fibrous membranes；Surface modification amphion MPC and targeting ligand Cys PEG FA obtain PEI/PVA PMPC FA；It is bonded with fishbone PDMS microfluidic channel cover plate plasmas, obtains embedding the micro-fluidic chip of oriented nanofibers film, sorted available for circulating tumor cell.The present invention is simple to operate, high-purity capture and the quick release of circulating tumor cell in blood can quickly be realized, the excellent anti-albumen of nano fibrous membrane and anti-haemocyte adhesion property are imparted with amphion modification, improve the purity of capture cell, oriented nanofibers film reduces the non-specific adhesion of haemocyte, capture purity is further increased, is had broad application prospects.
Description
Technical field
The invention belongs to micro-fluidic chip and cell sorting techniques field, more particularly to a kind of embedding oriented nanofibers film
Micro-fluidic chip preparation method.
Background technology
Early diagnosis of the detection of circulating tumor cell (circulating tumor cells, CTC) for tumour, disease
Sick development prediction, curative effect evaluation, Index for diagnosis and individualized treatment are extremely important.But CTC numbers pole in blood
It is few, it is difficult to capture and separates using conventional means.For example, the difference according to density, using the method for density gradient centrifugation
Various components are methods the most classical in separation blood.But CTC may be migrated to plasma layer or stayed in red blood cell or neutrality
In granulocyte, loss [Lalmahomed ZS, the et al.Circulating tumor cells of CTC in separation process are caused
and sample size:the more,the better.Journal of Clinical Oncology.2010,28:
e288-e289.].Flow cytometry can fast and accurately screen cell, but complex operation, and the number of dependency analysis cell
For amount, it is necessary to which a large amount of peripheral bloods can effectively examine tumor cell burden, therefore, it is difficult to the CTC changes in blood are supervised for a long time
Control.There are some researchs by the way of membrane filtration, separated from whole blood CTC using the difference of cell size [Desitter I,
et al.A new device for rapid isolation by size and characterization of rare
circulating tumor cells.Anticancer Research.2011；31:427-441.], but general purity is not high.
Therefore, it is the emphasis studied at present to explore a kind of efficient, easy, CTC capture techniques of high-purity.
In recent years, the detection platform based on microflow control technique and nano material is of great interest.Micro-fluidic chip
It is the device tested by designing and producing various structure and size in the pipeline of micron dimension.The spy of microfluidic channel
Sign yardstick matches with cell size, by the precise controlling to cell peripheral flow field, or is processed on chip various miniature
Structure, various operations can be carried out to cell.In addition, micro-fluidic chip has, device volume is small, sample requirements are few and right
The advantages of micro liquid precisely operates, it is highly suitable for the sorting of CTC in blood.Electrostatic spinning nano fiber, which has, greatly compares table
Area, substantial amounts of cells contacting site can be provided, make the quantity increase of capture cell in unit volume, thus Static Spinning Nanowire
Dimension is more and more applied capture and the detection field in CTC.In addition, electrostatic spinning nano fiber also has preparation technology letter
List, raw material sources are extensive, are easy to many advantages, such as follow-up multifunction modification.For example, polyethyleneimine/polyvinyl alcohol PEI/
PVA electrostatic spinning nano fibers are a kind of Preen nono fibers using water as solvent, and PEI/PVA nanofiber surfaces are with substantial amounts of
Amino and hydroxyl are easy to follow-up functional modification, can modify targeting ligand and be used for the special of cancer cell in nanofiber surface
Property capture.Found in the research of this seminar early stage, random PEI/PVA nanofiber porous network structures are likely to result in carefully
The non-specificity of born of the same parents is embedded, and the cancer cell purity for causing to capture reduces [Zhao Y, et al.Hyaluronic acid-
functionalized electrospun polyvinyl alcohol/polyethyleneimine nanofibers for
cancer cell capture applications.Adv.Mater.Interfaces,2015,2,1500256.].Therefore,
The nanofiber of orientation can be attempted to prepare, to improve the purity of final capture cancer cell.
Research shows, preferably separation effect can be obtained using micro-nano matrix or containing the micro-fluidic chip that specific structure designs
Rate, but universal relatively low [Sheng W, the et al.Multivalent DNA nanospheres for of separation purity that CTC is current
enhanced capture of cancer cells in microfluidic devices.ACS Nano,2013,7(8):
7067.].Therefore by invention new material or development new method, while ensureing CTC efficient captures, effective means is taken to improve
CTC separation purity is a current important research direction.Wang et al. utilizes the nanometer of amphion cysteine modified
Particle is used for the magnetic resonance imaging of tumour, it is found that amphion has good antifouling property, can be assigned using amphion modification
The anti-albumen Adhesion property that nano material is excellent is given, while amphion modification can also reduce the phagocytosis of macrophage, extension is received
Circulation time [Wang P, the et al.Antifouling manganese oxide of rice material in blood
nanoparticles:synthesis,characterization,and applications for enhanced mr
imaging of tumors.Acs Applied Materials&Interfaces.2017,9:47-53.].Meanwhile consult text
Discovery is offered, blood inactive surfaces can be obtained by modifying zwitterionic nano material, reduced haemocyte and sticked [Chang Y, et
al.Blood-inert surfaces via ion-pair anchoring of zwitterionic copolymer
brushes in human whole blood.Adv Funct Mater.2013,23:1100-1110.].Therefore, Ke Yitong
Cross and modify amphion in material surface, its is obtained blood inactive surfaces, so as to reduce protein and haemocyte in blood
Non-specificity is sticked, and improves the CTC finally captured purity.
At present, common CTC sorting technologies are that the CTC in blood is captured or is enriched with mostly, swollen after capture
Oncocyte continues to remain in matrix, is unfavorable for follow-up detection and analysis, and such as cell culture, PCR are analyzed.Yu et al. is in 2014
Year is published in《Science》On achievement in research show, if can by CTC from capture matrix effectively dissociate and it is trained in vitro
Support, with reference to gene sequencing and In vitro chemo-drug sensitive test, it will help establish tumour tailored diagnostics technology [Yu M, et based on CTC
al.Ex vivo culture of circulating breast tumor cells for individualized
testing of drug susceptibility.Science,2014,345(6193):216-220.].Therefore, development is effective
Technological means, meet to realize CTC lossless releases while CTC efficient captures, obtain with high biologos CTC, be work as
Another focus of preceding CTC researchs.Currently, the existing reversible delivery systmes of part CTC achieve encouraging progress, but are deposited during discharging
The cellular damage and relatively low release efficiency the problems such as.Therefore gentle CTC delivery systmes are must be set up, obtain purity height, vigor
Good CTC is convenient to carry out follow-up cultivation to it and analyze again.Disulfide bond is a kind of chemical group of reduction-sensitive, is being reduced
Property material (such as dithiothreitol (DTT) DTT, glutathione GSH, sodium borohydride) effect under can fast fracture.Can be real using disulfide bond
The control release of existing nanomaterial loadings medicine, the fast fracture of disulfide bond, the medicine for tumour are realized by reducing substances
Thing and gene therapy.[Zhao D,et al.Redox-sensitive mPEG-SS-PTX/TPGS mixed micelles:An
efficient drug delivery system for overcoming multidrug
resistance.International Journal of Pharmaceutics,2016,515,281-292].There is researcher
Intermediate connector using disulfide bond as cancer cell targeting ligand, 30min is handled using DTT solution at room temperature, realize that capture is thin
Quick release [Huang C, et al.Multifunctional " smart " the particles engineered from of born of the same parents
live immunocytes:toward capture and release of cancer cells.Adv.Mater.2015,
27,310-313]。
The content of the invention
The technical problems to be solved by the invention are to provide a kind of system for the micro-fluidic chip for embedding oriented nanofibers film
Preparation Method and its application in circulating tumor cell sorting, micro-fluidic chip can realize CTC high-purity made from this method
Capture and quick nondestructive release, the preparation of the micro-fluidic chip to be sorted for circulating tumor cell provide a kind of new method,
Have a extensive future.
A kind of preparation method of the micro-fluidic chip of embedding oriented nanofibers film of the present invention, including：
(1) end is dissolved in deionized water for the PEGylation folic acid SH-PEG-FA of sulfydryl, adds cysteine
Cysteine and hydrogen peroxide, the reaction of ice bath magnetic agitation, then dialysis, vacuum freeze drying obtain concentration as 5~15mg/mL's
The Cys-PEG-FA aqueous solution；Wherein SH-PEG-FA, cysteine, the mol ratio of hydrogen peroxide are 1：1.5~2：2~4；Deionization
The volume ratio of water and hydrogen peroxide is 5mL：8~12 μ L；
(2) polyethyleneimine PEI and PVAC polyvinylalcohol are added in deionized water, magnetic agitation, obtaining mass fraction is
8~12wt% PEI/PVA spinning solutions, electrostatic spinning, obtain PEI/PVA nano fibrous membranes, vapor crosslinking, obtain crosslinking Treatment
Orientation PEI/PVA nano fibrous membranes afterwards；Wherein PEI and PVA mass ratio is 0.5：1~2；
(3) the orientation PEI/PVA nano fibrous membranes after the crosslinking Treatment for obtaining step (2) are immersed in anhydrous methylene chloride
In solution, triethylamine Et is added3N, then 2- bromine isobutyl acylbromide BBIB are added dropwise, ice bath reaction, then react at room temperature, through ultrasound
Cleaning obtains the PEI/PVA nano fibrous membranes PEI/PVA-Br of surface bromination；Wherein Et3N and BBIB mol ratio is 1~3：1,
BBIB anhydrous methylene chloride solution concentration is 20~50mg/mL；
(4) mixing that the PEI/PVA-Br that step (3) obtains is added to methanol and deionized water under nitrogen atmosphere is molten
Liquid, continuously adds cuprous bromide CuBr, bipyridyl BPY, 2- methylacryoyloxyethyl phosphocholine MPC monomers, polymerisation,
Then through being cleaned by ultrasonic, vacuum drying obtains the PEI/PVA nano fibrous membranes PEI/PVA- of amphion MPC functional modifications
PMPC-Br；Wherein MPC concentration is 50~300mg/mL, and CuBr, BPY, MPC mol ratio are 1：2：15~17；
(5) Cys-PEG-FA that step (1) obtains is dissolved in solvent, magnetic agitation fully dissolves, and adds acid binding agent
The PEI/PVA-PMPC-Br obtained with step (4), temperature reaction, is then cleaned by ultrasonic, amphion is obtained after vacuum drying
MPC and targeting ligand Cys-PEG-FA functional modifications PEI/PVA nano fibrous membranes PEI/PVA-PMPC-FA；Wherein solvent
Dosage be 18~22mL, the mol ratio of Cys-PEG-FA and acid binding agent is 1：1~1.2；
(6) slide for the PEI/PVA-PMPC-FA for obtaining load step (5) is as substrate, with the poly- diformazan of fishbone
Radical siloxane PDMS microfluidic channel cover plates, are bonded by plasma, obtain embedding the micro-fluidic chip of oriented nanofibers film.
The mass fraction of hydrogen peroxide in the step (1) is 30%.
The ice bath magnetic agitation time is 1~2h in the step (1).
The technological parameter of dialysis is in the step (1)：Using molecular cut off as 1000 bag filter, in deionized water
Dialysis 3 days.
The technological parameter of vacuum freeze drying is in the step (1)：Vacuum is 0.085~0.1mBar, freeze-drying
Temperature is -40~-50 DEG C, and sublimation drying is 48~72h.
The technological parameter of magnetic agitation is in the step (2)：Whipping temp be 80~100 DEG C, mixing time be 3~
5h。
The technological parameter of electrostatic spinning is in the step (2)：From No. 16 syringe needles, spinning voltage 25kV, flow velocity
0.6mL/h, receive distance 10cm, 20~25 DEG C of environment temperature, humidity 40~50%, to be coated on rotating speed as 2000~2500r/
Aluminium-foil paper in min rotating shafts is reception device.
The technological parameter of vapor crosslinking is in the step (2)：Penta 2 that mass fraction is 20~30% are placed with bottom
Vacuum is evacuated in the vacuum desiccator of aldehyde solution and reaches 0.08~0.09Mpa, 11~13h of crosslinking Treatment.
The ice bath reaction time is 3~5h in the step (3).
The room temperature reaction time is 8~12h in the step (3).
The technological parameter of ultrasonic cleaning is in the step (3)：It is cleaned by ultrasonic 3 successively with dichloromethane and deionized water~
8min。
The volume ratio of methanol and deionized water is 1 in mixed solution in the step (4)：1.
Polymerization reaction time is 1~6h in the step (4).
The technological parameter of ultrasonic cleaning is in the step (4)：It is cleaned by ultrasonic 3 successively with methanol and deionized water~
8min。
Vacuum drying technological parameter is in the step (4)：Vacuum is 0.09~0.1MPa, drying temperature is 40~
50 DEG C, drying time is 22~26h.
Solvent is dimethyl sulfoxide (DMSO) DMSO in the step (5).
Acid binding agent is potassium carbonate or triethylamine in the step (5).
The magnetic agitation time is 0.5~1h in the step (5).
The technological parameter of temperature reaction is in the step (5)：Reaction temperature is 70~80 DEG C, and the time is 5~7h.
The technological parameter of ultrasonic cleaning is in the step (5)：5~10min is cleaned by ultrasonic with methanol and deionized water.
Vacuum drying technological parameter is in the step (5)：Vacuum is 0.09~0.1MPa, drying temperature is 35~
45 DEG C, drying time is 46~50h.
Fishbone PDMS microfluidic channel cover plates in the step (6) be by design include an entrance, one
Outlet, the fishbone microfluidic channel structure of four parallel fishbone passages, then print mask plate, then in silicon chip glazing
Scribe for mould is gone out, then reverse mould obtains.
The microfluidic channel of the fishbone microfluidic channel structure is highly 40 μm, and fish-bone is highly 30 μm, and passage enters
Total length mouthful to outlet is 65mm, and fishbone passage length is 45.5mm, and passage overall width is 20mm, wall scroll fishbone passage
Width is 4mm.
In the step (6) plasma bonding technological parameter be：Vacuum is 20~26Pa, in air bonding handle
40~50s.
The micro-fluidic chip for the embedding oriented nanofibers film that the step (6) obtains is used for CTC points of circulating tumor cell
Choosing, includes the capture and release of cancer cell.
The technological parameter of capture and the release of the cancer cell is：First to the micro-fluidic core of the embedding oriented nanofibers
Be passed through phosphate buffer PBS in piece, ensure that passage and nano fibrous membrane fully infiltrate, by the cell suspension containing cancer cell or
Cancer patient's blood is passed through in micro-fluidic chip, is then passed through phosphate buffer PBS microfluidic channel again, is completed to catch
Obtain process；The good 30mM of configured in advance glutathione GSH solution is passed through into micro-fluidic chip with 8mL/h flow velocity, is continued
After being passed through certain time, and recovered liquid is collected, complete the release of capture cancer cell.
The comprehensive nanofiber of the present invention and microflow control technique, by oriented nanofibers surface modification amphion MPC,
The anti-protein adsorption of nano fibrous membrane and the ability of anti-haemocyte adhesion are assigned, so as to improve the purity of the cancer cell captured；
Targeting ligand FA is further modified on nano fibrous membrane surface so that nano fibrous membrane specifically can cross table with reference to folacin receptor
The cancer cell reached, realize the specificity capture of tumour cell；Targeting ligand and nanometer are used as using the disulfide bond of the sensitive fracture of reduction
The intermediate connector of fiber, realize the quick nondestructive release of circulating tumor cell；Utilize turbulent caused by fishbone passage, increasing
Add the making contact probability of cancer cell and nano fibrous membrane substrate, improve the efficiency of capture.Embedding orientation prepared by the present invention
The micro-fluidic chip of nanofiber realizes CTC efficient capture and quick nondestructive release, to be sorted for circulating tumor cell
The exploitation of micro-fluidic chip provide a kind of new method.
Beneficial effect
(1) material of main part is orientation PEI/PVA nano fibrous membranes and the micro-fluidic cover plates of PDMS in the present invention, and material processes work
Skill is simple, and cost is cheap, has the prospect of industrialized implementation.
(2) functional modification of the present invention by amphion to nano fibrous membrane, assign the anti-albumen of nano fibrous membrane and inhale
The ability of anti-haemocyte adhesion is echoed, so as to improve the purity of the cancer cell captured.
(3) present invention by preparing oriented nanofibers, stick, and further increases capture by the non-specificity for reducing cell
The purity of cancer cell.
(4) for the present invention using folic acid as targeting ligand, folic acid is cheap and easy to get, and the cancer cell that folacin receptor is overexpressed is targetted
High specificity, high sensitivity.
(5) present invention connects using the disulfide bond of reduction-sensitive fracture as targeting ligand and the middle of nanofiber substrate
Body, the quick nondestructive release of circulating tumor cell is realized, is easy to the detection and analysis subsequently to CTC.
(6) functionalization oriented nanofibers and microflow control technique are combined by the present invention, and the embedding orientation nano of preparation is fine
The micro-fluidic chip of dimension can realize CTC high-purity capture and quick nondestructive release, micro- to be sorted for circulating tumor cell
The exploitation of fluidic chip provides a kind of new method, has wide answer in terms of the separation and detection and analysis of circulating tumor cell
Use prospect.
Brief description of the drawings
Fig. 1 is the infrared spectrum comparison diagram of SH-PEG-FA and Cys-PEG-FA in the present invention；
Fig. 2 is the scanning electron microscope (SEM) photograph and size distribution plot that PEI/PVA nano fibrous membranes are orientated in the present invention；Wherein (a) is
Scanning electron microscope (SEM) photograph, (b) are size distribution plot；
Fig. 3 is the synthesis schematic diagram of functionalized nano-fiber PEI/PVA-PMPC-FA in the present invention；
Fig. 4 is that fiber film modified front and rear infrared spectrogram and thermogravimetric analysis figure are orientated in the present invention, wherein (a) is infrared
Spectrogram, (b) are thermogravimetric analysis figure；
Fig. 5 is that fiber film modified front and rear hemolytic test result and dynamic coagulation result are orientated in the present invention, wherein
(a) it is hemolytic test result, (b) is dynamic coagulation result；
Fig. 6 is film modified front and rear to the anti-adsorption test result of protein and to haemocyte for oriented nanofibers in the present invention
Anti- adhesion assay result, wherein (a) is to the anti-adsorption test result of protein, (b) is the anti-adhesion assay to haemocyte
As a result；
Fig. 7 is capture effect of the micro-fluidic chip to cancer cell that oriented nanofibers are embedded in the present invention, wherein (a) is
Micro-fluidic chip is to cancer cell HeLa capture rate under the conditions of different in flow rate, and (b) is micro-fluidic chip under the conditions of different in flow rate
Capture purity to cancer cell HeLa；
Fig. 8 be the micro-fluidic chip that oriented nanofibers are embedded in the present invention to the releasing effect of the cancer cell captured,
(a) it is the release efficiency of cancer cell after the different disposal time, (b) is the activity of release cancer cell after the different disposal time；
Fig. 9 is fishbone microfluidic channel structural representation in the present invention.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
(1) 37.2mg SH-PEG-FA are dissolved in 5mL deionized waters, add 2.42mg cysteines Cysteine and
10 μ L mass fractions be 30% hydrogen peroxide, ice bath magnetic agitation reaction 1h, then using molecular cut off as 1000 bag filter,
Dialysis three days, are 0.09mBar in vacuum, are freeze-dried under -45 DEG C of cryogenic conditions in deionized water (9 times, 2L/ times)
48h, obtain the Cys-PEG-FA aqueous solution.
(2) 0.5g polyethyleneimine PEI and 1.5g PVAC polyvinylalcohol are added in 18g deionized waters, magnetic force stirs at 90 DEG C
4h is mixed, obtains the PEI/PVA spinning solutions that mass fraction is 10wt%, PEI/PVA spinning solutions, injection are drawn with 10mL syringe
Device connects No. 16 syringe needles, and it is 0.6mL/h to set injection flow rate pump, to be coated on rotating speed as the aluminium-foil paper in 2500r/min rotating shafts
For reception device, it is 20cm that regulation, which receives distance, and setting spinning voltage is 25kV, in 25 DEG C of environment temperature, the condition of humidity 50%
Lower progress electrostatic spinning processing, obtains PEI/PVA nano fibrous membranes, is put into vacuum desiccator and carries out vapor crosslinking, vacuum is done
Dry device bottom is placed with the glutaraldehyde solution that mass fraction is 25%, is then evacuated to vacuum and reaches 0.085Mpa, at crosslinking
12h is managed, obtains the PEI/PVA nano fibrous membranes after crosslinking Treatment.
(3) the PEI/PVA nano fibrous membranes after the crosslinking Treatment for obtaining step (2) are immersed in 20mL anhydrous dichloromethane
In alkane solution, 500 μ L triethylamine is added, then 412 μ L 2- bromine isobutyl acylbromide BBIB are added dropwise, ice bath reaction 4h, then
8h is reacted at room temperature, 5min is cleaned by ultrasonic with dichloromethane and water successively after the completion of reaction, obtains the PEI/PVA nanometers of surface bromination
Tunica fibrosa PEI/PVA-Br.
(4) PEI/PVA-Br for obtaining step (3) is added in round-bottomed flask, and nitrogen charging catches up with oxygen 5 times, then in nitrogen atmosphere
Mixed solution (the v/v=1 of lower addition 20mL methanol and deionized water:1) 107mg cuprous bromides CuBr, 234mg, are continuously added
The 2- methylacryoyloxyethyl phosphocholine MPC monomers of bipyridyl, 1.77g, polymerisation 2h, then take out nanofiber
Film, 5min is cleaned by ultrasonic with methanol and deionized water successively, is 0.095MPa in vacuum, 24h is dried in vacuo under the conditions of 45 DEG C,
Obtain the PEI/PVA nano fibrous membranes PEI/PVA-PMPC-Br of amphion MPC functional modifications.
(5) Cys-PEG-FA that step (1) obtains is dissolved in 20mL dimethyl sulfoxide (DMSO) DMSO solvents, magnetic agitation 1h
Fully dissolving, the PEI/PVA-PMPC-Br that 2.1mg potassium carbonate obtains as acid binding agent and step (4) is added, then raises temperature to 75
DEG C, keeping temperature back flow reaction 6h, 8min is then cleaned by ultrasonic with methanol and deionized water, is 0.095MPa in vacuum, 40
Amphion MPC is obtained after being dried in vacuo 48h under the conditions of DEG C and the PEI/PVA of targeting ligand Cys-PEG-FA functional modifications receives
Rice tunica fibrosa PEI/PVA-PMPC-FA.
(6) fishbone passage is used, is wrapped using Auto CAD softwares design microfluidic channel structure, microfluidic channel design
Include：One entrance, one outlet, four parallel fishbone passages, 40 μm of channel height, 30 μm of fish-bone height, feeder connection
Total length to outlet is 65mm, fishbone passage length 45.5mm, and passage overall width is 20mm, wall scroll fishbone channel width
For 4mm (as shown in Figure 9)；Then designed chip is printed mask plate using high-resolution printer, recycles light
Lithography prepares Micro-fluidic chip die on silicon chip, finally goes out corresponding poly- diformazan using the Chip mold reverse mould prepared
Radical siloxane PDMS microfluidic channel cover plates.
(7) by plasma bonding techniques, setting vacuum is 21Pa, 50s is handled under air atmosphere, by load step
(5) PEI/PVA-PMPC-FA obtained slide is as substrate, the PDMS microfluidic channel cover plate keys obtained with step (6)
Close, obtain embedding the micro-fluidic chip of amphion functionalized nano-fiber film.
Embodiment 2
The present invention is with SEM (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric (TG) and molten
Blood-thrombotest, anti-albumen and haemocyte adhesion assay, the capture of cancer cell and release test characterize the work(prepared in the present invention
The properties of oriented nanofibers film can be changed and its combine application potential of the micro-fluidic chip in circulating tumor cell sorting.
Fourier transform infrared spectroscopy is tested：
Characterize whether the step of embodiment 1 (1) has synthesized the Cys-PEG-FA containing disulfide bond, infrared light stave using FTIR
Analysis result is levied as shown in figure 1, in 2591cm-1Locate as the infrared signature absorption peak of sulfydryl SH in SH-PEG-FA, and Cys-PEG-
2591cm in FA infared spectrums-1The infrared signature absorption peak for locating sulfydryl SH disappears, in 456cm-1There is disulfide bond-S-S- in place
Infrared absorption peak, illustrate that cysteine and SH-PEG-FA have obtained the Cys- containing disulfide bond by the oxidation reaction of sulfydryl
PEG-FA。
Amphion MPC and targeting are matched somebody with somebody in the PEI/PVA-PMPC-FA obtained using the FTIR sign steps of embodiment 1 (5)
Whether body Cys-PEG-FA is successfully modified on its surface, and as a result such as Fig. 4 (a) is shown：(1) curve is PEI/PVA nano fibrous membranes
Infrared curve, (2) curve is in 1275cm-1There is the characteristic absorption peak for the phosphorus oxygen double bond for belonging to amphion MPC, explanation in place
Amphion MPC is successfully modified in nanofiber surface；In addition, comparing (1), (2) curve, curve (3) is in 1570cm-1With
1485cm-1There is the infrared signature absorption peak of phenyl ring in folic acid FA, illustrate that targeting ligand FA is successfully modified in nanofiber
Surface.
Scanning electron microscope test：
The pattern and diameter of step (2) orientation PEI/PVA nano fibrous membranes are characterized using SEM.SEM results such as Fig. 2
(a) shown in, shown in diameter such as Fig. 2 (b), smooth surface, size uniformity has been prepared by electrostatic spinning technique, have been averaged directly
Footpath is 357nm orientation PEI/PVA nano fibrous membranes, and fiber-wall-element model is good, and mesh is less.
Thermal weight loss is tested：
Two in the oriented nanofibers film PEI/PVA-PMPC-FA that the step of embodiment 1 (5) obtains are detected by TGA
Property ion MPC and targeting ligand Cys-PEG-FA mass percent.Shown in TGA test results such as Fig. 4 (b)：In comparison diagram
PEI/PVA and PEI/PVA-PMPC thermogravimetric curve is understood, modifies the amphion on nanofiber PEI/PVA-PMPC surfaces
Shared content is 20.36%；By analyzing PEI/PVA-PMPC and PEI/PVA-PMPC-FA thermogravimetric curve and calculating,
In functionalized nano-fiber film PEI/PVA-PMPC-FA, content shared by Cys-PEG-FA is about 28.03%.So as to draw function
Change in nano fibrous membrane PEI/PVA-PMPC-FA, amphion MPC and targeting ligand Cys-PEG-FA mass percent difference
For 14.65% and 28.03%.
Embodiment 3
Hemolytic test is tested：
Pass through the blood compatibility of nano fibrous membrane before and after hemolytic test research modification.Take fresh normal adults whole blood
Then 1mL, 2000r/min centrifugal treating 5min are washed 3 times with phosphate buffer PBS, obtain red blood cell HRBCs.By HRBCs
30 times are diluted with PBS solution, 0.2mL HRBCs dilutions is taken out and is transferred in the 1.5mL centrifuge tubes containing 0.8mL PBS, make
For negative control.0.2mL HRBCs dilutions are taken to be transferred in the 1.5mL centrifuge tubes containing 0.8mL distilled water, as positive right
According to.Then, the HRBCs dilutions configured are diluted 5 times with PBS again, after the crosslinking Treatment of the step of Example 1 (2)
PEI/PVA nano fibrous membranes, the PEI/PVA-PMPC-Br nano fibrous membranes of step (4), the PEI/PVA-PMPC-FA of step (5)
Each 4mg of nano fibrous membrane is immersed in the HRBCs suspensions that 1mL is diluted after 5 times, and each sample sets 5 Duplicate Samples, in 37 DEG C of rings
2h is incubated under border.Finally, tunica fibrosa is taken out, control group and the HRBCs suspensions for soaking tunica fibrosa are turned with 10000r/min
Speed centrifugation 1min, takes pictures after centrifugation, and takes suction of the supernatant ultraviolet specrophotometer test supernatant at 450~800nm
Light value, and calculate hemolysis rate using formula.
Shown in the test result of hemolytic test such as Fig. 5 (a)：Hemolytic test result understands PBS negative control groups and 3 kinds of nanometers
Tunica fibrosa does not occur obvious haemolysis, and the hemolysis rate of 3 kinds of nano fibrous membranes is respectively less than 5%, illustrates nano fibrous membrane
With good blood compatibility.
Dynamic coagulation is tested：
The anticoagulation function of nano fibrous membrane is evaluated using dynamic coagulation.First by a diameter of 14mm circle
3 kinds of nano fibrous membranes of shape cover glass load, are put into 12 well culture plates, each sample takes 4 Duplicate Samples, a diameter of 14mm's
Cover glass is as control.Then, the stable health of 20 μ L heparin lithiums is added dropwise on fibrofelt and control group cover glass into every hole
Adult's whole blood, while add the calcium chloride CaCl that 10 μ L concentration are 0.2mol/L2Solution, be placed under 37 DEG C of environment be incubated 5,
10、20、30、40、60min.After each incubation time terminates, 5mL distilled water and 37 DEG C are added to be incubated 5 minutes to every hole, then
The light absorption value in supernatant at 540nm is tested with UV-Vis.
Shown in the test result of dynamic coagulation such as Fig. 5 (b)：Dynamic coagulation result understand by amphion and
The modification of targeting ligand, functionalized nano-fiber film obtain certain anticoagulation function.
Embodiment 4
Anti-adhesive properties are tested：
Absorption situation of the nano fibrous membrane to albumen before and after modifying is characterized from bovine serum albumin(BSA) BSA.Configuration ladder first
The PBS solution of concentration cellulose proteinogen is spent, using UV-Vis spectrophotometers test various concentrations BSA solution at 280nm
Light absorption value, obtain concentration-absorbance standard curve of BSA solution.The circular cover glass for being loaded with 3 kinds of nano fibrous membranes is put
Enter in 24 orifice plates, the PBS solution that 500 μ L are added per hole balances 4h at room temperature, then suctions out PBS solution, then add into every hole
1mL concentration is 1mg/mL BSA solution, and each sample setting 5 is parallel, and orifice plate is put into 37 DEG C of constant-temperature tables and is incubated 1h,
The absorbance of albumen in supernatant before and after nano fibrous membrane adsorbs is tested, and then calculates the adsorbance of protein.
Shown in anti-albumen adhesion assay result such as Fig. 6 (a)：Compared with the PEI/PVA nano fibrous membranes before modification, PEI/
The adsorption rate of albumen is obviously reduced PVA-PMPC and PEI/PVA-PMPC-FA, shows significant difference, illustrates to pass through both sexes
After ion functional modification, nano fibrous membrane obtains excellent anti-albumen adhesion property.
Take the healthy human blood that 5mL is fresh, using erythrocyte cracked liquid remove blood in red blood cell, 1500r/min from
The heart handles 5min, obtains leukocyte cell pellet, with calcein (Calcein-AM) dyestuff dialogue cell dyeing 15min, then uses PBS
Cleaning 3 times, then adds 5mL cell culture fluids into prestained leukocyte cell pellet, and piping and druming uniformly obtains leukocyte suspension.Take
Go out 10 μ L leukocyte suspensions, 1mL is diluted to nutrient solution, counted with cell count rifle, then to adding one in leukocyte suspension
Quantitative fresh culture, white blood cell concentration is obtained as 106/ mL leukocyte suspension.The circle of 3 kinds of nano fibrous membranes will be loaded with
Shape cover glass is put into 24 orifice plates, and the PBS solution that 500 μ L are added per hole balances 4h at room temperature.PBS solution is suctioned out, is added per hole
500 μ L concentration is 106/ mL leukocyte suspension, it is placed in 37 DEG C of constant incubators and co-cultures 2h.Suction out leukocyte suspension,
With the cleaning tunica fibrosa 3 times of 500 μ L PBS solution, then it is attached on using fluorescence microscope white on nano fibrous membrane
Cell simultaneously counts.
Shown in anti-haemocyte adhesion assay result such as Fig. 6 (b)：Unit area on PEI/PVA nano fibrous membranes before modification
Haemocyte adhere to number close to 300/mm2, the nano fibrous membrane haemocyte adhesion number after amphion is modified is only 0
~8/mm2, this absolutely proves that the leukocyte count of the nano fibrous membrane surface adhesion after amphion is modified significantly reduces, several
There is no leukocyte, there is excellent anti-haemocyte adhesion property.
Embodiment 5
Cancer cell captive test is tested：
The micro-fluidic chip of the embedding oriented nanofibers obtained using embodiment 1 studies micro-fluidic chip to cancer cell
Capture effect.First, capture rate of the micro-fluidic chip to cancer cell under the conditions of research is different in flow rate.Take fresh Healthy People blood
Liquid, leukocyte cell pellet is obtained after splitting erythrocyte, leucocyte is dyed with calcein orange, then with cell culture fluid weight
Suspend and counted with cell counter, a number of HeLa Cells are then incorporated into leukocyte suspension
In, white blood cell concentration is obtained as 106/ mL, the mixed cell suspension that cancer cell concentration is 200/mL, are used HeLa cells before incorporation
The red carry out fluorescent staining of calcein, in order to which later observation counts.Phosphate buffer PBS first is passed through to micro-fluidic chip,
Ensure that passage and nano fibrous membrane fully infiltrate, then take 1mL mixed cell suspensions (1mL/h, 2mL/ at different flow rates respectively
H, 4mL/h, 6mL/h, 8mL/h, 10mL/h) it is passed into micro-fluidic chip, capture is passed through phosphate-buffered again after completing
Liquid (PBS) clean microfluidic channel, then using fluorescence microscope statistics nano fibrous membrane on cancer cell (red) and
Leucocyte (green) number, calculate the capture rate of cancer cell.
Capture rate test of the micro-fluidic chip to cancer cell is as shown in Fig. 7 (a) under the conditions of different in flow rate：With entering field flow
The increase of speed, micro-fluidic chip gradually reduce to HeLa capture rate, and when flow velocity is 6mL/h, capture rate remains to reach
More than 90%, illustrate micro-fluidic chip in the present invention under conditions of high flow rate, remain to keep higher capture rate, can be short
The efficient capture of cancer cell can be completed in time.
Under the conditions of different in flow rate shown in the capture purity test such as Fig. 7 (b) of micro-fluidic chip to cancer cell：Bar different in flow rate
Under part, the capture purity of HeLa cells can reach more than 60%, and when flow velocity is 6mL/h, capture purity is up to 73.2%,
The significantly larger than capture purity (0.1%~1%) of cancer cell method for separating common at present, illustrates the micro-fluidic core in the present invention
The high-purity capture of cancer cell can be achieved in piece.
Embodiment 6
The release test test of cancer cell：
The micro-fluidic chip research micro-fluidic chip research of the embedding oriented nanofibers obtained using embodiment 1 is to capture
The releasing effect of the cancer cell arrived.First choice digests HeLa cells with pancreatin, with the culture medium resuspension cell of serum-free, then
Calcein (Calcein-AM) dyestuff is added to HeLa cell dyeing 15min, is then centrifuged for and is removed twice with PBS solution cleaning
Excess dyestuff is gone, finally uses serum free medium suspension cell again, it is dense using serum free medium regulation HeLa cells after counting
Spend for 104/mL.Phosphate buffer PBS first is passed through to micro-fluidic chip, it is 10 then to take 1mL concentration4/ mL HeLa cells hang
The floating flow velocity with 4mL/h is passed through micro-fluidic chip, and it is micro-fluidic logical to be passed through phosphate buffer PBS after completion capture again
Road, with fluorescence microscope and count.Then using 8mL/h flow velocity be passed through into micro-fluidic chip the good concentration of configured in advance as
30mM glutathione GSH solution, after being passed through different time, the cancer that is retained using fluorescence microscope nano fibrous membrane surface
Cell simultaneously counts, so as to calculate processing different time after cancer cell release efficiency.
After the different disposal time shown in release efficiency test result such as Fig. 8 (a) of cancer cell：With prolonging for processing time
Long, the release efficiency of cancer cell gradually increases, and is continually fed into GSH solution 15min, and the release efficiency of cancer cell can reach
91.2%, illustrate that the quick release of capture cancer cell can be realized by being passed through the processing of GSH solution.
HeLa cells are digested with pancreatin, with fresh culture resuspension cell, culture medium regulation HeLa is utilized after counting
Cell concentration is 104/mL.Phosphate buffer PBS first is passed through to micro-fluidic chip, it is 10 then to take 1mL concentration4/ mL HeLa
Cell suspends is passed through micro-fluidic chip with 4mL/h flow velocity, and phosphate buffer PBS miniflow is passed through again after completing capture
Control passage.Then the good 30mM of configured in advance glutathione GSH solution is passed through into micro-fluidic chip with 8mL/h flow velocity,
Recovered liquid is collected, and dead cell stain of living is carried out to being passed through the cancer cell after different time in recovered liquid, 5 are added into recovered liquid
The Calcein-Safranine T solution that the propidium iodide PI solution and 5 μ L concentration that μ L concentration is 8 μM are 2 μM, dyes 20min at room temperature, from
Heart processing removes dyeing liquor, then with fluorescence microscope and counts the number of living (green) extremely (red) cell, Jin Erji
The activity of cancer cell is discharged after calculating the different disposal time.
Shown in active testing result such as Fig. 8 (b) that cancer cell is discharged after the different disposal time：Handled by GSH solution
After 60min, the activity for discharging cancer cell still can reach more than 85%, illustrate that the micro-fluidic chip in the present invention can realize capture
The lossless release of cancer cell.
Claims (10)
1. a kind of preparation method for the micro-fluidic chip for embedding oriented nanofibers film, including：
(1) end is dissolved in deionized water for the PEGylation folic acid SH-PEG-FA of sulfydryl, adds cysteine Cysteine
And hydrogen peroxide, the reaction of ice bath magnetic agitation, then dialysis, vacuum freeze drying obtain the Cys-PEG- that concentration is 5~15mg/mL
The FA aqueous solution；Wherein SH-PEG-FA, cysteine, the mol ratio of hydrogen peroxide are 1：1.5~2：2~4；Deionized water and dioxygen
The volume ratio of water is 5mL：8~12 μ L；
(2) polyethyleneimine PEI and PVAC polyvinylalcohol are added in deionized water, magnetic agitation, obtain mass fraction for 8~
12wt% PEI/PVA spinning solutions, electrostatic spinning, obtain PEI/PVA nano fibrous membranes, vapor crosslinking, after obtaining crosslinking Treatment
Orientation PEI/PVA nano fibrous membranes；Wherein PEI and PVA mass ratio is 0.5：1~2；
(3) the orientation PEI/PVA nano fibrous membranes after the crosslinking Treatment for obtaining step (2) are immersed in anhydrous methylene chloride solution
In, add triethylamine Et3N, then 2- bromine isobutyl acylbromide BBIB are added dropwise, ice bath reaction, then react at room temperature, through being cleaned by ultrasonic
Obtain the PEI/PVA nano fibrous membranes PEI/PVA-Br of surface bromination；Wherein Et3N and BBIB mol ratio is 1~3：1, BBIB
Anhydrous methylene chloride solution concentration be 20~50mg/mL；
(4) PEI/PVA-Br that step (3) obtains is added to methanol and the mixed solution of deionized water under nitrogen atmosphere, after
Continuous addition cuprous bromide CuBr, bipyridyl BPY, 2- methylacryoyloxyethyl phosphocholine MPC monomers, polymerisation, then
Through being cleaned by ultrasonic, vacuum drying obtains the PEI/PVA nano fibrous membranes PEI/PVA-PMPC- of amphion MPC functional modifications
Br；Wherein MPC concentration is 50~300mg/mL, and CuBr, BPY, MPC mol ratio are 1：2：15~17；
(5) Cys-PEG-FA that step (1) obtains is dissolved in solvent, magnetic agitation fully dissolves, and adds acid binding agent and step
Suddenly the PEI/PVA-PMPC-Br that (4) obtain, temperature reaction, is then cleaned by ultrasonic, obtained after vacuum drying amphion MPC and
The PEI/PVA nano fibrous membranes PEI/PVA-PMPC-FA of targeting ligand Cys-PEG-FA functional modifications；The wherein dosage of solvent
For 18~22mL, the mol ratio of Cys-PEG-FA and acid binding agent is 1：1~1.2；
(6) slide for the PEI/PVA-PMPC-FA for obtaining load step (5) is as substrate, with fishbone poly dimethyl silicon
Oxygen alkane PDMS microfluidic channel cover plates, are bonded by plasma, obtain embedding the micro-fluidic chip of oriented nanofibers film.
2. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：The mass fraction of hydrogen peroxide in the step (1) is 30%；The ice bath magnetic agitation time is 1~2h；The technique of dialysis
Parameter is：Using molecular cut off as 1000 bag filter, dialyse 3 days in deionized water；The technological parameter of vacuum freeze drying
For：Vacuum is 0.085~0.1mBar, and freeze-drying temperature is -40~-50 DEG C, and sublimation drying is 48~72h.
3. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：The technological parameter of magnetic agitation is in the step (2)：Whipping temp is 80~100 DEG C, and mixing time is 3~5h；It is quiet
The technological parameter of Electrospun is：From No. 16 syringe needles, spinning voltage 25kV, flow velocity 0.6mL/h, distance 10cm, environment are received
20~25 DEG C of temperature, humidity 40~50%, it is to receive dress to be coated on rotating speed as the aluminium-foil paper in 2000~2500r/min rotating shafts
Put；The technological parameter of vapor crosslinking is：The vacuum desiccator for the glutaraldehyde solution that mass fraction is 20~30% is placed with bottom
In be evacuated to vacuum and reach 0.08~0.09Mpa, 11~13h of crosslinking Treatment.
4. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：The ice bath reaction time is 3~5h in the step (3)；The room temperature reaction time is 8~12h；The technological parameter of ultrasonic cleaning
For：3~8min is cleaned by ultrasonic with dichloromethane and deionized water successively.
5. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：The volume ratio of methanol and deionized water is 1 in mixed solution in the step (4)：1；Polymerization reaction time is 1~6h；
The technological parameter of ultrasonic cleaning is：3~8min is cleaned by ultrasonic with methanol and deionized water successively；Vacuum drying technological parameter
For：Vacuum is 0.09~0.1MPa, and drying temperature is 40~50 DEG C, and drying time is 22~26h.
6. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：Solvent is dimethyl sulfoxide (DMSO) DMSO in the step (5)；Acid binding agent is potassium carbonate or triethylamine；The magnetic agitation time is
0.5~1h；The technological parameter of temperature reaction is：Reaction temperature is 70~80 DEG C, and the time is 5~7h；The technique ginseng of ultrasonic cleaning
Number is：5~10min is cleaned by ultrasonic with methanol and deionized water；Vacuum drying technological parameter is：Vacuum be 0.09~
0.1MPa, drying temperature are 35~45 DEG C, and drying time is 46~50h.
7. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：Fishbone PDMS microfluidic channel cover plates in the step (6) are to be gone out by designing comprising an entrance, one
Mouthful, the fishbone microfluidic channel structure of four parallel fishbone passages, then print mask plate, then the photoetching on silicon chip
Mould is prepared, then reverse mould obtains；Wherein the microfluidic channel of fishbone microfluidic channel structure is highly 40 μm, and fish-bone is high
Spend for 30 μm, the total length of feeder connection to outlet is 65mm, and fishbone passage length is 45.5mm, and passage overall width is
20mm, wall scroll fishbone channel width are 4mm.
8. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：In the step (6) plasma bonding technological parameter be：Vacuum is 20~26Pa, and processing 40 is bonded in air
~50s.
9. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 1, its feature
It is：The micro-fluidic chip for the embedding oriented nanofibers film that the step (6) obtains sorts for circulating tumor cell CTC,
Capture and release including cancer cell.
10. a kind of preparation method of micro-fluidic chip for embedding oriented nanofibers film according to claim 9, its feature
It is：The technological parameter of capture and the release of the cancer cell is：First to the micro-fluidic core of the embedding oriented nanofibers film
Phosphate buffer PBS is passed through in piece, then passes to the cell suspension containing cancer cell or cancer patient's blood, then be passed through phosphoric acid
Salt buffer PBS, complete acquisition procedure；Gluathione is continually fed into the micro-fluidic chip of the embedding oriented nanofibers film
Peptide GSH solution, and recovered liquid is collected, complete the release of capture cancer cell.
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