CN107653575A - A kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film - Google Patents

A kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film Download PDF

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CN107653575A
CN107653575A CN201711003398.4A CN201711003398A CN107653575A CN 107653575 A CN107653575 A CN 107653575A CN 201711003398 A CN201711003398 A CN 201711003398A CN 107653575 A CN107653575 A CN 107653575A
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cbaa
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史向阳
王梦媛
肖云超
朱晓玥
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Donghua University
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Abstract

The present invention relates to a kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film, including：By chitosan/PEO spinning solutions, electrostatic spinning, it is dried in vacuo, crosslinking, obtains chitosan nano fiber membrane CNFs；Show to modify amphion CBAA and targeting ligand HA Cys MPTMS, obtain the nano fibrous membrane HA CBAA CNFs of HA functionalization；With PDMS microfluidic channel cover plates, it is bonded by plasma, obtains embedding the micro-fluidic chip of the nano fibrous membrane of HA functionalization, available for circulating tumor cell sorting and lossless release.The present invention has technique simple, it is easy to operate, the advantages that high specificity, the unmarked of tumour cell, high flux sorting and not damaged release can be completed in a short time, circulating tumor cell is efficiently separated so as to realize, there is good application prospect to the early detection of tumor patient.

Description

A kind of preparation for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film Method
Technical field
The invention belongs to micro-fluidic chip and cell sorting techniques field, more particularly to a kind of embedding hyaluronic acid functionalization The preparation method of the micro-fluidic chip of nano fibrous membrane.
Background technology
Cancer is to cause the primary factor of human death, according to the World Health Organization (WHO) scholarly forecast, the year two thousand twenty whole world cancer Disease number of the infected is up to 20,000,000 people, and death toll is up to 12,000,000 people, and cancer will turn into 21 century influence human survival With the chief threat of health.Research shows that the death of 90% cancer patient is all closely related with the transfer of tumour.As swollen Key link between knurl primary tumor and transfer stove, circulating tumor cell (Circulating tumor Cells, CTCs) is near Cause extensive concern both domestic and external within several years.At metastases initial stages, tumour cell comes off from primary tumor solid tumor, into blood Liquid, turn into CTCs.Although the demand of diagnosis and treatment of the people for cancer is very urgent, clinical treatment cancer it is normal Rule means are extremely limited.In addition, once cancer development is to late stage, almost without the possibility of healing.Therefore, the early stage of cancer It was found that, Clinics and Practices be effectively improve survival rate, reduce the death rate main method.
Because the number extremely rareness of CTC in tumor patient blood is (every about 106~107Just there is one in individual leucocyte to follow Ring tumour cell), it is difficult to separate and captures using conventional means.Such as：CellSearch is the currently the only acquisition U.S. Food and medicine Surveillance Authority approves and is used for the commercially produced product of CTC detections.But testing cost is expensive, is targetted and tied with magnetic bead Close CTC and be unfavorable for follow-up detection and analysis.Currently used separation method include physical partition method (based filtration, dielectrophoresis, Hydrodynamics) and biochemistry separating method (immunomagnetic beads, immune microsphere, surface adhesion), and biochemistry separating method is most normal Analysis method.
In recent years, the detection platform based on micro-fluidic chip and nano material receives people and widely paid close attention to.Static Spinning Nanofiber operates the features such as easy and efficient, and have than table by the nanofiber of its preparation because its equipment is simple Area is big, homogeneity is good, morphology controllable and receives much concern the advantages that be easy to functional modification, by electrostatic spinning nano fiber film For CTC separation and concentration, cell can be increased with targetting the making contact probability of material, and then improve the capture rate of cell (Sun N,Liu M,Wang J.N.,et al.Chitosan Nanofibers for Specific Capture and Nondestructive Release of CTCs Assisted by Pcbma Brushes[J].Small:2016.12 (36):5090-5097.).Micro-fluidic chip has the advantages that sample requirements are few, detection sensitivity is high and analyze speed is fast, non- Often it is suitably applied cell sorting (Chen J, Li J, Sun Y.Microfluidic approaches for cancer cell detection,characterization,and separation[J].Lab on a Chip:2012.12(10): 1753-1767.).In addition, very limited for the blood sample amount for sorting CTCs, micro-fluidic chip by chance compensate for this and lack Fall into, so receiving the favor of numerous researchers with micro-fluidic chip sorting CTCs technology.
Current most of capture CTCs method is all based on the specific binding of antibody-antigene, but antibody is as one Kind of protein matter, preserve difficult, easy inactivation and expensive.For antibody, natural polymer part and acceptor Albumen has stronger adhesion, and these natural polymers are cheap, are not easily decomposed, and preservation condition is simple.CD44 by Body has too high expression to include epithelioma, lymph cancer, breast cancer and lung cancer etc. in a variety of cancer cell surfaces, itself and tumour cell Transfer has close relationship.CD44 acceptors have very strong adhesion with hyaluronic acid, have had many researchs utilizing HA It is used for targeted therapy (Kim Y, the Kumar S.CD44-Mediated of tumour to macromolecular surface as targeted molecular modification Adhesion to Hyaluronic Acid Contributes to Mechanosensing and Invasive Motility[J].Mol.Cancer Res.:2014.12(10):1416-1429).Therefore, it is used as target instead of antibody by the use of HA Not only specific tumour cell can be captured to molecule, clinical application cost can also be substantially reduced.
The method discharged for the circulating tumor cell of subsequent captured has by light-sensitive material, pH response keys and two Sulfide linkage etc..Disulfide bond enjoys researcher to favor due to its efficient release efficiency, makes the conventional reduction of disulfide bonds at present Agent has glutathione GSH, dithiothreitol (DTT) DTT, sodium borohydride etc..
The content of the invention
The technical problems to be solved by the invention are to provide a kind of miniflow for embedding hyaluronic acid functionalized nano-fiber film The preparation method of chip is controlled, has that technique is simple, it is easy to operate, the advantages that high specificity, moreover it is possible to the circulating tumor cell of capture Lossless release is carried out, is advantageous to further do subsequent analysis, higher clinical reference is provided in terms of the early diagnosis of cancer Value.
A kind of preparation method of the micro-fluidic chip of embedding hyaluronic acid functionalized nano-fiber film of the present invention, including：
(1) it is 9 by mass ratio：0.5~1.5 chitosan is dissolved in solvent with PEO PEO, and stirring is anti-at room temperature Should, room temperature is cooled to, obtains chitosan/PEO spinning solutions, electrostatic spinning, vacuum drying obtains chitosan nano fiber membrane, with penta Dialdehyde is crosslinked, and obtains cross-linked chitosan nano fibrous membrane CNFs；
(2) by hyaluronic acid HA, 1- (3- dimethylamino-propyls) -3- ethyl diimmonium salt hydrochlorate EDCHCl, N- hydroxyls Succinimide NHS is dissolved in solvent, stirring reaction, the HA-COOH activated, is added dropwise to Ethitanin hydrochloric acid Continue stirring reaction in the salt H-Cys-OEt.HCl aqueous solution, through dialysing, being freeze-dried, obtain HA-Cys；Wherein HA, EDC HCl, NHS, H-Cys-OEt.HCl mass ratio are 1：2.4~2.5：1.4~1.5：0.8~0.9；
(3) HA-Cys that step (2) obtains is dissolved in solvent, oxydol H is added dropwise2O2, mercaptosilane coupling agents MPTMS, ice-water bath stirring reaction, through dialysing, being freeze-dried, obtain HA-Cys-MPTMS；Wherein HA-Cys, MPTMS, H2O2's Mol ratio is 1：3：5~8；
(4) CNFs for obtaining step (1) is added in methanol and the mixed solution of NaCl solution, adds amphion carboxylic acid Glycine betaine acrylamide CBAA solution, stirring reaction, is then washed, naturally dry at room temperature, obtains the shell of amphion modification Glycan nano fibrous membrane CBAA-CNFs；Wherein CNFs, CBAA mass ratio are 4~5：1；
(5) CBAA-CNFs that step (4) obtains is dissolved in solvent, adds the HA-Cys-MPTMS water that step (3) obtains Solution, stirring reaction, then washing, naturally dry, obtain the nano fibrous membrane HA-CBAA-CNFs of HA functionalization；Wherein CBAA-CNFs, HA-Cys-MPTMS mass ratio are 1：1.5~1.7；
(6) slide for the HA-CBAA-CNFs for obtaining load step (5) is as substrate, with dimethyl silicone polymer PDMS microfluidic channel cover plates, are bonded by plasma, obtain embedding the micro-fluidic chip of the nano fibrous membrane of HA functionalization.
Solvent in the step (1) is the acetic acid that concentration is 85%.
The concentration of chitosan is 2.5~3.5wt% in chitosan/PEO spinning solutions in the step (1).
Stirring in the step (1) is magnetic agitation, and the time of stirring reaction is 7~9h.
The technological parameter of electrostatic spinning is in the step (1)：Spinning voltage is 30kV, flow velocity 0.1mL/h, spinning distance 12~15cm, 20~30 DEG C of environment temperature, humidity 20~40%, using the board device covered with aluminium-foil paper as reception device.
The vacuum drying time is 12~24h in the step (1).
The time of crosslinking is 4~8h in the step (1).
The molecular weight of HA in the step (2) is 5830.
Solvent in the step (2) is dimethyl sulfoxide (DMSO) DMSO.
Stirring in the step (2) is magnetic agitation；The time of stirring reaction is 2.5~3h；Continue stirring reaction Time is 2~3d.
The process conditions of dialysis are in the step (2), (3)：Bag filter of the molecular cut off for 1000Da is used, in phosphorus After being dialysed 1 day in hydrochlorate PBS, it is replaced by ultra-pure water and dialyses 2 days.
Solvent in the step (3) is ethanol.
The concentration of hydrogen peroxide in the step (3) is 28~32%.
Stirring in the step (3) is magnetic agitation, and the time of stirring reaction is 3~5h.
Amphion CBAA in the step (4) is by the way that dimethylamino propylamine, beta-propiolactone are dissolved in into anhydrous third In ketone, polymerization inhibitor 1 is added, 1- diphenyl -2- trinitrophenyl-hydrazine DPPH, 2.5~3.5h of ice-water bath reaction, is passed through under nitrogen protection Purifying, vacuum drying are made；Wherein dimethylamino propylamine, beta-propiolactone, DPPH, the amount ratio of anhydrous propanone are 1.6g：1~ 1.1g：50mg：18mL.
The process conditions of the purifying are to be purified by flash with anhydrous propanone.
The volume ratio of methanol and NaCl solution is 1 in mixed solution in the step (4)：1.
The concentration of NaCl solution in the step (4) is 0.1~0.2M.
The concentration of CBAA solution in the step (4) is 0.07~0.09mg/mL.
The time of stirring reaction is 2~3d in the step (4).
Solvent in the step (5) is isopropanol.
The amount ratio of solvent, the HA-Cys-MPTMS aqueous solution in the step (5) is 200mL：800μL.
The technological parameter of stirring reaction is in the step (5)：Whipping temp is 70~80 DEG C, and the stirring reaction time is 7 ~9h.
The micro-fluidic chip for the embedding hyaluronic acid functionalized nano-fiber film that the step (5) obtains is used for circulating tumor Cell CTCs sorting and lossless release.
The sorting of the CTCs and the technological parameter of lossless release are：To the embedding hyaluronic acid functionalized nano-fiber The cell suspending liquid containing cancer cell or cancer patient's blood are passed through in the micro-fluidic chip of film, utilizes the specific table of cancer cell surfaces Up to CD44 antigen and HA adhesion, the sorting of cancer cell is completed；To the embedding hyaluronic acid functionalized nano-fiber film Micro-fluidic chip in be continually fed into glutathione GSH solution, and collect recovered liquid, complete the lossless release of cancer cell.
The chitosan nano fiber cost of the present invention is cheap, and largely abundant amino and oh group, Yi Xiu are contained in surface Decorations, amphion CBAA is modified first to reduce the non-specific adsorption of cell, improves the capture purity of cell；Two sulphur are modified again The compound of key is easy to subsequently discharge the tumour cell of capture, finally modifies targeted molecular HA in shell by disulfide bond On glycan nanofiber, according to the easy fracture property of disulfide bond, using reducing substances DTT by the circulating tumor cell of capture from Released on nano fibrous membrane, for target capture circulating tumor cell, realize the subsequent analysis to tumour cell.
Beneficial effect
(1) chitosan nano fiber membrane surface of the invention has abundant amino and hydroxyl, is easy to modify, with reference to miniflow Chip technology is controlled, there is the advantages of device is simple, reproducible.
(2) present invention is using hyaluronic acid decorated nano fibrous membrane specificity capture apparent height expression CD44 acceptors Circulating tumor cell, high specificity, operating process is easy, separative efficiency is high.
(3) nano fibrous membrane of functionalization is combined by the present invention with micro-fluidic chip, for circulating tumor cell tradition The problems such as efficiency existing for method for separating is low, purity is low, easy damaged cells, caught using surface-functionalized nanofiber specificity Circulating tumor cell is obtained, with reference to micro-fluidic sorting technology, realizes efficiently separating for circulating tumor cell.
(4) micro-fluidic chip of embedding hyaluronic acid functionalized nano-fiber film produced by the present invention, required blood sample Amount is few, and detection efficiency is high, has good application prospect.
Brief description of the drawings
Fig. 1 is that the SEM of chitosan nano fiber membrane prepared by the present invention schemes (left side) and diameter distribution profile (right side)；
Fig. 2 is CBAA prepared by present invention proton nmr spectra；
Fig. 3 is CBAA-CNFs prepared by present invention infrared spectrogram (a), HA-Cys infrared spectrogram (b) HA- Cys-MPTMS infrared spectrogram (c) and HA-CBAA-CNFs infrared spectrogram (d)；
Fig. 4 is the anti-protein adsorption rate of CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane prepared by the present invention As a result (a) and anti-leukocyte cell rate result (b)；
Fig. 5 is CNFs (a), CBAA-CNFs (b) prepared by the present invention and HA-CBAA-CNFs (c) nano fibrous membranes in 0s When water contact angle image；
Fig. 6 (a) is CNFs, the CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane of the invention prepared to blood compatibility Property evaluation ultraviolet absorpting spectrum；(b) it is enlarged drawing of (a) figure at 500-600nm；
Fig. 7 is CNFs, the CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane of the invention prepared at different time points Anticoagulation behavior characterization result；
Fig. 8 is CNFs (a), CBAA-CNFs (b) and HA-CBAA-CNFs (c) nano fibrous membranes and cancer prepared by the present invention To the shows fluorescent microscopy images of cell capture and in different incubation time section (10,20,40 and when cell is incubated 40min altogether 60min) to the static capture rate (d) of cancer cell；
Fig. 9 (a) is that 40min glutathione discharges to the cancer cell of capture and the cancer cell of release is labored under static conditions The shows fluorescent microscopy images extremely dyed；(b) it is the differential responses time interior release efficiency to cancer cell；(c) cancer to release The survival rate that cell work is extremely dyed；
Figure 10 (a) is trouble of the micro-fluidic chip to simulation of embedding HA-CBAA-CNFs nano fibrous membranes prepared by the present invention Person's blood under different in flow rate (0.5mL/h, 1.0mL/h, 2.0mL/h, 4.0mL/h and 6.0mL/h) Dynamical capture efficiency； (b) for the HA-CBAA-CNFs nanofibers micro-fluidic chip for preparing of the present invention under 1.0mL/h flow velocitys to the cancer of varying number The Dynamical capture efficiency of cell；
Figure 11 is stream of the micro-fluidic chip in 1.0mL/h of embedding HA-CBAA-CNFs nano fibrous membranes prepared by the present invention Under speed, to the Dynamical capture separative efficiency of the cancer cell of varying number；
Figure 12 is hyaluronic acid functionalized nano-fiber film HA-CBAA-CNFs prepared by present invention synthesis schematic diagram.
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) 180mg chitosans and 20mg PEOs PEO are dissolved in 85% acetum, magnetic force stirs at room temperature Reaction 8h is mixed, untill chitosan turns into the solution of stable homogeneous, is cooled to room temperature, the shell that concentration is 3.0wt% is obtained and gathers Sugar/PEO spinning solutions, it is stored in standby in 4 DEG C of refrigerators.Above-mentioned gained spinning solution is slowly drawn in syringe, utilizes high pressure Electrostatic spinning machine carries out electrostatic spinning, and spinning condition is：Spinning voltage is 30kV, flow velocity 0.1mL/h, spinning distance 12cm, ring 20~30 DEG C of border temperature, humidity 20~40%, using the board device covered with aluminium-foil paper as reception device, it is subsequently placed in vacuum and does 24h in dry case, obtains chitosan nano fiber membrane, and 6h is crosslinked in drier with glutaraldehyde, obtains crosslinking shell not soluble in water Glycan nano fibrous membrane CNFs.
(2) 200.94mg hyaluronic acids HA is dissolved in 8mL DMSO, by 492.67mg 1- (3- dimethylaminos third Base) -3- ethyl diimmonium salt hydrochlorates EDCHCl, 296.11mg N- hydroxysuccinimides NHS is dissolved in 1mL DMSO, Magnetic agitation reacts 3h, the HA-COOH activated；It is added dropwise to 10mL and contains 176.47mg ethylcysteine hydrochlorides In the H-Cys-OEt.HCl aqueous solution, continue magnetic agitation reaction 3d, use bag filter of the molecular cut off for 1000Da, in After being dialysed 1 day in phosphate PBS, it is replaced by ultra-pure water and dialyses 2 days, be then freeze-dried, obtains white powder production Thing HA-Cys, be stored in -20 DEG C it is standby.
(3) HA-Cys that 500mg steps (2) obtain is dissolved in 10mL ethanol, under magnetic stirring, concentration is added dropwise For 30% oxydol H2O2, then add 50 μ L mercaptosilane coupling agents MPTMS, ice-water bath magnetic agitation reaction 4h, using retention Molecular weight is 1000Da bag filter, after being dialysed 1 day in phosphate PBS, is replaced by ultra-pure water and dialyses 2 days, then Freeze-drying, obtain HA-Cys-MPTMS, be stored in -20 DEG C it is standby.
(4) 1.6g dimethylamino propylamines, 1.024g beta-propiolactones are dissolved in 18mL anhydrous propanones, add 50mg 1, 1- diphenyl -2- trinitrophenyl-hydrazine DPPH polymerization inhibitors, ice-water bath reaction 3h, obtains white precipitate, Ran Houyong under nitrogen protection Anhydrous propanone is purified by flash, and is obtained white powder, is finally dried in vacuo, and obtains amphion carboxylic acid glycine betaine acrylamide CBAA。
(5) CNFs for obtaining 72mg steps (1) adds methanol and the mixed solution (v/v=of 0.138M NaCl solution 1:1) in, the amphion CBAA solution (concentration 0.08mg/mL) that 16mg steps (4) obtain is added, at room temperature stirring reaction 2 ~3d, 2-3 times, naturally dry then are cleaned with ultra-pure water, obtain the chitosan nano fiber membrane CBAA- of amphion modification CNFs。
(6) CBAA-CNFs for obtaining 72mg steps (5) is immersed in 200mL isopropanols, is added 800 μ L steps (3) and is obtained The aqueous solution containing 118mg HA-Cys-MPTMS, 75 DEG C of stirring reaction 8h, then clean 2-3 times with ultra-pure water, dry in the air naturally It is dry, obtain the nano fibrous membrane HA-CBAA-CNFs of HA functionalization.
(7) slide for the HA-CBAA-CNFs for obtaining load step (6) is as substrate, with dimethyl silicone polymer PDMS microfluidic channel cover plates, are bonded by plasma, obtain embedding the micro-fluidic chip of the nano fibrous membrane of HA functionalization.
Embodiment 2
The present invention with SEM (SEM), decay total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), Proton nmr spectra (1H NMR), uv-visible absorption spectra (UV-vis), anti-protein adsorption experiment, water contact angle test, blood Liquid phase compatibility test, anti-freezing blood test, static state/Dynamical capture of cancer cell and release test and Immunostaining assay characterize this The properties and its combination micro-fluidic chip of the hyaluronic acid functionalized nano-fiber film prepared in invention are thin in circulating tumor Born of the same parents sort and the application potential in lossless release.
Scanning electron microscope test：
Pattern and the diameter distribution for the chitosan nano fiber that the step of embodiment 1 (1) obtains, SEM results are characterized using SEM As shown in figure 1, the chitosan nano fiber surface prepared by electrostatic spinning technique is smooth, uniform diameter, fiber is averagely straight Footpath is (150.9 ± 29.50) nm.
Proton nmr spectra is tested：
Using1H NMR spectras characterize the synthesis for the amphion CBAA that the step of embodiment 1 (4) obtains, as a result such as Fig. 2 institutes Show：Proton peak at 5.62~6.11ppm of chemical shift represents the proton peak in C=C double bonds in CBAA, in chemical shift Proton peak at 2.91ppm represents N- (CH3)2Proton peak, the proton peak at chemical shift 3.40ppm represents N-CH2- CH2- COO proton peak, the proton peak at chemical shift 3.2ppm and 1.89ppm represent NH-CH2-CH2-CH2Proton peak. As a result show, successfully prepare the amphion CBAA needed for experiment.
Decay total reflection-Fourier transform infrared spectroscopy test：
The structure of CNFs, CBAA-CNFs and HA-CBAA-CNFs in embodiment 1 are characterized using ATR-FTIR, checking is transparent Whether the nano fibrous membrane HA-CBAA-CNFs of matter acid functionalization successfully prepares, as a result as shown in Figure 3.
Curve (1) is in 3024cm in Fig. 3 (a)-1Place is the infrared signature absorption peak of-CH in C=C double bonds in CBAA, curve (2) in 1590cm-1Place is the characteristic absorption peak that-NH vibrates on amino, and curve (3) is in 1375cm-1There is C-N feature in place Absworption peak, illustrate that CBAA is successfully modified in nanofiber surface.
Curve (1) is in 1541cm in Fig. 3 (b)-1And 1580cm-1Place is that the infrared signature of amino in H-Cys-OEt.HC is inhaled Peak is received, curve (2) is in 3292cm-1Place is the infrared absorption peak of O-H in HA carboxyls, and curve (3) is in 1739cm-1Locate appearance-CONH Infrared signature absorption peak, show that H-Cys-OEt.HCl is successfully modified on HA.
Curve (1) is in 1739cm in Fig. 3 (c)-1Locate appearance-CONH infrared signature absorption peak, curve (2) is in 2562cm-1 Place is the characteristic absorption peak of-SH on MPTMS, and curve (3) is in 564cm-1There is the infrared signature absorption peak of S -- S in place, illustrates to close Into HA-Cys-MPTMS compounds in be successfully generated S -- S.
Curve (1) is in 2922cm in Fig. 3 (d)-1Place is-CH on HA-Cys-MPTMS2Infrared signature absorption peak, curve (2) in 2943cm-1Place is-CH on CBAA-CNFs2Infrared signature absorption peak, and curve (3) is 2933cm in wave number-1With 2878cm-1There is-the CH of enhancing in place2Infrared signature absorption peak, illustrate the nano fibrous membrane HA- of hyaluronic acid functionalization CBAA-CNFs is successfully prepared.
Embodiment 3
Anti- protein adsorption test：
Anti- albumen is carried out to CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiber in embodiment 1, and (BSA and fibre are even Albumen) adsorption assessment, for characterize CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane anti-BSA adsorption capacities and Anti- fibronectin adsorption capacity, first, with the ultraviolet BSA for measuring various concentrations and fine related white calibration curve equation.Herein On the basis of, the concentration for choosing BSA is 2mg/mL and fine related white concentration is that 1mg/mL is test concentrations.Take respectively CNFs, Each 10mg of CBAA-CNFs and HA-CBAA-CNFs nanofibers, each sample of sample four are put into 24 orifice plates, then respectively to Each orifice plate adds 1mL BSA and Fibronectin solution, is incubated 1h altogether at room temperature, takes out nanofiber, takes supernatant to be used in combination The type ultraviolet specrophotometers of Lamada 25 are tested light absorption value of the supernatant at 278nm, are existed according to different samples Light absorption value at 278nm calculates adsorption rate, and test result is as shown in Figure 4.
Fig. 4 (a) is anti-protein adsorption rate test result, it is known that compared with the CNFs nano fibrous membranes before modification, CBAA- The adsorption rate of albumen is obviously reduced CNFs and HA-CBAA-CNFs, shows significant difference, illustrates to pass through amphion work( After energyization modification, nano fibrous membrane obtains excellent anti-albumen adhesion property.Fig. 4 (b) tests for anti-leukocyte cell rate As a result, it is known that compared with the CNFs nano fibrous membranes before modification, the adsorption rate of albumen is obviously reduced the CNFs after modification, presents Go out significant difference, further prove that there is good anti-protein adsorption by the nano fibrous membrane of amphion functional modification Performance.
Embodiment 4
Water contact angle is tested：
The front and rear hydrophilicity of chitosan nano fiber modification have studied by water contact angle measuring instrument, for characterizing The hydrophilicity of CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane.The load of thickness even fiber will be loaded with Slide is placed on load sample platform, randomly selects different positions, and 3 μ L drops at instrument injection needle are dropped on fibrofelt, passed through Survey Software determines the size of contact angle, and shoots drop pattern water contact angle, as a result as shown in Figure 5, it can be seen that CNFs, The contact angle of CBAA-CNFs and HA-CBAA-CNFs nanofibers is respectively 46.9 ± 4.5 °, 35.0 ± 2.4 ° and 24.8 ± 2.4 ° as can be seen here, and the contact angle of the nanofiber after modification is in the trend being gradually reduced, and shows the nanofiber after modification Film has stronger hydrophily, further illustrates that CBAA and HA has successfully been modified on the surface of chitosan nano fiber.
Embodiment 5
Blood compatibility is tested：
Blood compatibility is the important indicator that can a kind of material of evaluation be applied to have CTCs in capture blood.By molten The blood compatibility of nano fibrous membrane prepared by blood experimental evaluation.Haemolysis viability experiment be used for study CNFs, CBAA-CNFs and The biocompatibility of HA-CBAA-CNFs nano fibrous membranes in vivo.
The blood of 1mL Healthy Peoples is taken, centrifugation 5min (rotating speed 150r/min), abandons supernatant, precipitation is washed 5 times with PBS, obtains Red blood cell.With PBS according to 1:10 proportional arrangement red blood cell suspension, it is standby in 4 DEG C of refrigerators.Control group is red thin by 0.2mL Born of the same parents' suspension is dissolved in 0.8mL PBS (negative control) and 0.8mL H respectively2In O (positive control).Then, by CNFs, CBAA-CNFs and HA-CBAA-CNFs chitosan nano fibers are after 4mg/mL is immersed in 10 times of dilution according to mass volume ratio In red blood cell suspension, each nanofiber sample takes 3 Duplicate Samples, and 2h is incubated under the conditions of 37 DEG C.Finally take out fiber Felt, control group and the red blood cell suspension for soaking fibrofelt are centrifuged into 1min (10000r/min), take supernatant to be used in combination The type ultraviolet specrophotometers of Lamada 25 are tested light absorption value of the supernatant at 450-800nm, are existed according to different samples Light absorption value at 540nm calculates hemolysis rate, as a result as shown in fig. 6, (a) is CNFs, CBAA-CNFs and HA-CBAA-CNFs nanometer The ultraviolet absorpting spectrum that fiber is evaluated blood compatibility；(b) it is enlarged drawing of (a) figure at 500-600nm.
As shown in Figure 6 in the range of 450-800nm, the light absorption value of supernatant is significantly higher in control group water, and this shows blood Lactoferrin content is higher, i.e., red blood cell rises brokenly completely in water, serious haemolysis occurs.But in CNFs, CBAA-CNFs and In HA-CBAA-CNFs nanofibers and PBS, supernatant light absorption value is very low, illustrates that red blood cell does not occur to rise brokenly.At 540nm Light absorption value, which calculates, to be understood, when the nanofiber of functionalization is with Human red blood cells in suspension volume ratio 4mg/mL, the haemolysis of material Rate is 0.98%, 0.82% and 0.64%, and its value is all much below critical value 5%.Show CNFs, CBAA-CNFs and HA- CBAA-CNFs nanofibers can not make human blood cell produce haemolysis, have good blood compatibility.
Embodiment 6
Anticoagulation is tested：
In order to further verify the blood compatibility of the chitosan nano fiber after modification, using dynamic blood coagulation time method Anticoagulation function evaluation is carried out to the nanofiber of functionalization.Anticoagulation is tested for characterizing CNFs, CBAA-CNFs and HA- The anticoagulation function of CBAA-CNFs nano fibrous membranes.
CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiber are cut into circular (φ=14mm) first, are put into 24 holes In plate, each sample takes 4 Duplicate Samples (control group be slide, cover slip).Then to every hole fibrofelt and control group 20 μ L healthy human bloods of upper dropwise addition and 10 μ L CaCl2Solution (0.2mol/L), under the conditions of being placed in 37 DEG C be incubated 5,10,20,40, 60min.After each incubation time terminates, 3mL distilled water is added to each orifice plate, 5min is then incubated again, uses ultraviolet spectrometry Photometric determination 540nm light absorption value, control group slide, CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiber anticoagulation The evaluation result of performance is as shown in Figure 7.
Because the haemocyte that the higher representative of the content of hemoglobin solidifies is fewer, i.e. the anticoagulation function of material is got over It is good.The hemoglobin in different time point, CBAA-CNFs and HA-CBAA-CNFs nanofiber supernatants as shown in Figure 7 Light absorption value is all remarkably higher than slide and CNFs, and the nanofiber after this explanation targeting modification has preferable anticoagulant active.
Embodiment 7
The static capture test of cancer cell：
In order to verify that targeting fibrous material has the effect of specificity capture cancer cell, with the high expression of CD44 acceptors Lung carcinoma cell (A549) is cell model to examine the static capture cancer of functionalized nano-fiber specificity that the HA of preparation is modified thin The effect of born of the same parents.It will be covered with the circular slide of CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiberOne The sample of formula four is put into 24 orifice plates to be fixed with steel loop, different time points (10,20,40,60min) sample to be placed on different trainings Support in plate, the radiation sterilization h under the conditions of ultraviolet.The serum free medium that 500 μ L are added in the orifice plate to have sterilized is soaked fiber 30min.Then culture medium is suctioned out, the cell suspending liquid contaminated in advance is added in orifice plate, leucocyte calcein dye is Green, the red dye of A549 cell calceins are red, white blood cell concentration 105Individual/mL, A549 cell per well add 300, Suspension dosage is 500 μ L.Be then placed in cell culture incubator and be incubated 10 altogether, 20,40,60min, to regular hour point after respectively Culture plate is taken out, is taken pictures using fluorescence microscope (20 ×) and observes counting with PBS three times, as a result such as Fig. 8 (a)-(c) It is shown, it is known that to have more leucocyte and cancer cell on unmodified nanofiber, on the nanofiber for having amphion modification There are less leucocyte and cancer cell, and have more cancer cell and less leucocyte on targeted nano fiber, absolutely prove The target capture cell effect of HA mediations.Capture rate result such as Fig. 8 (d) institute of three kinds of different materials to A549 cells Show, with the growth of incubation time, three kinds of different materials are in increase trend to the capture rate of A549 cells, in 60min Shi Jiben tends to balance state, but it is much big to the capture rate of cancer cell to target material HA-CBAA-CNFs nano fibrous membranes In non-targeted material C NFs and CBAA-CNFs capture rate.When incubation time is 60min, HA-CBAA-CNFs Nanowires Dimension film is up to 80% to A549 capture rate, and CNFs and CBAA-CNFs nano fibrous membranes are to A549 capture rate 40% and 18%, illustrate that the nano fibrous membrane after modification has good targeting.
Cancer cell static release and dead cell stain living are tested：
In order to verify cell after the release efficiency of disulfide bond different time (10,20,30,40,50,60min) and release Activity.Still the A549 cells with the high expression of CD44 acceptors are chosen as model to examine the releasing effect of targeting material.Will Circular slide covered with HA-CBAA-CNFs nanofibersOne four parts of examination is put into 24 orifice plates is consolidated with steel loop It is fixed, the radiation sterilization 1h under the conditions of ultraviolet.The serum free medium that 500 μ L are added in the orifice plate to have sterilized is soaked fiber 30min.Then culture medium is suctioned out, the A549 cell suspending liquids contaminated with calcein (green) are added in orifice plate, A549 The concentration of cell is 105Individual/mL, 500 μ L nutrient solution is separately added into per hole.It is then placed in cell culture incubator and is incubated 1h.Take Go out orifice plate, suction out nutrient solution respectively, the glutathione that concentration is 10mM is then added into each orifice plate with PBS three times (GSH), respectively reaction 10,20,30,40,50,60min, flushed three times with PBS.(10 ×) are observed under fluorescence microscope, knot Shown in fruit such as Fig. 9 (a), recovered liquid is collected, it is counted with cell counter, shown in release efficiency such as Fig. 9 (b) of cancer cell, In preceding 40min, with the growth of GSH incubation times, the release efficiency of cancer cell is higher, and when the time is more than 40min, it discharges effect Rate tends to balance substantially.Cancer cell static release test result shows in summary：40min is that GSH is broken the optimal of disulfide bond Time.
Repeat the above steps, but the A549 cells for adding orifice plate do not have to dyeing, the same period, with GSH processing, collect Recovered liquid, the cell released is dyed with dead cell kit living, (10 ×) are observed under fluorescence microscope, with thin Born of the same parents' blood counting chamber is calculated cell, and it is discharged shown in survival rate such as Fig. 9 (c)：With the growth (10- of GSH action times 60min), the survival rate of the cancer cell released is lower (99%-80%), in optimal section release time (40min), releases The survival rate of the cancer cell put down is up to 90%, can be used for follow-up cultivation and analysis.
Embodiment 8
Cancer cell Dynamical capture is tested：
In order to verify the chitosan nano fiber after HA modifications to the capture effect of cancer cell, carried out it is different in flow rate under The measure of capture rate.Dynamical capture is tested for studying capture rate of the lower microfluidic system different in flow rate to cancer cell, profit Result is observed and counted with fluorescence microscope, calculates its capture rate.
There is HA chitosan nano fiber membrane as substrate using surface modification, by plasma treatment by substrate of glass, function The chitosan nano fiber membrane of change is bonded with microfluidic channel, obtains micro-fluidic chip, including：(1) there are 277 cylindroid battle arrays The micro-fluidic chip cell capture passage of row, one section of passage have sample injection port, and the other end has outlet sample, inlet and outlet Designed in a manner of isoceles triangle；(2) HA chitosan nano fiber membrane is modified with, nano fibrous membrane is clipped in slide and poly- two The centre of first polysiloxanes (PDMS) passage, form interlayer；(3) HA nano fibrous membrane is modified with, is had according to cancer cell surfaces Particular expression CD44 antigen, CD44 acceptors have very strong adhesion with HA, can the specific cancer for capturing surface expression CD44 Cell, so as to which on the nanofiber of functionalization, cancer cell be separated for cancer cell capture.
Model of the A549 cells as cancer cell is chosen, takes the μ L of calcein (C-AM) 20 of dilution certain multiple (60 times) It is added in A549 cell suspending liquids, 12min is incubated under the conditions of 37 DEG C.Afterwards plus medium centrifugal 5min (1000r/min), use Cell counter takes 1 × 10 respectively to cell count5Individual A549 cells, under different flow velocitys (0.5mL/h, 1.0mL/h, 2.0mL/h, 4.0mL/h and 6.0mL/h) it is passed into microfluidic channel.Because nano fibrous membrane surface modification has substantial amounts of HA, profit With the combination of acceptor and part, when cancer cell flows through in microchannel along fiber surface, cell can be targeted capture, glue Fiber surface is attached to, and non-targeted cell (leucocyte) will flow out passage, so as to realize the purpose of sorting circulating tumor cell.
The cancer cell of lower capture different in flow rate is counted respectively under fluorescence microscope, obtains the capture effect of cancer cell Rate, as a result as shown in Figure 10 (a), it is known that with the raising of flow velocity, the capture rate of cancer cell reduces, when flow velocity is 0.5mL/h When, capture rate is up to 93.93%；When flow velocity is 6.0mL/h, capture rate 60.9%.Ensureing that capture rate is higher In the case of, 1mL/h is chosen as subsequent experimental flow parameters.Under the conditions of flow velocity is 1.0mL/h, walked by above-mentioned operation Suddenly, HeLa cells, KB cells and MCF-7 cells are chosen respectively by microfluidic channel, respectively to capture under fluorescence microscope Cancer cell count, obtain shown in capture rate such as Figure 10 (b) of cancer cell, under the same conditions, targeted nano fiber pair The capture rate highest of Hela cells, capture rate 94.9%；Minimum to the capture rate of KB cells, capture rate is 80.8%.In summary, cancer cell Dynamical capture test result show HA-CBAA-CNFs nano fibrous membranes can specificity catch Obtain the circulating tumor cell of apparent height expression CD44 acceptors.
Embodiment 9
In order to verify point of the circulating tumor cell in blood of the chitosan nano fiber after HA modifications to simulating patient Effect is selected, above-mentioned syringe pump is chosen and is set under 1.0mL/h flow velocity, the cancer cell of varying number is added to strong after cracking In health human blood, capture rate of the research micro-fluidic chip to cancer cell.
The healthy human blood that 1mL is fresh is taken, centrifugation 5min (1500r/min), serum is removed, then will dilute certain multiple The DAPI (500 μ L) of (60 times) is added to dialogue cell dyeing 10min in blood, then adds PBS solution centrifugation 5min (1000r/min), to remove unnecessary uncombined dyestuff.Model of the A549 cells as lung carcinoma cell is chosen, takes dilution certain The μ L of calcein (C-AM) 20 of multiple (60 times) are added in A549 cell suspending liquids, and 12min is incubated under the conditions of 37 DEG C, it Afterwards, add nutrient solution to carry out centrifugation 5min (1000r/min), with cell counter to cell count, take 20,50,100,200 respectively It is added to 300 A549 cells in above-mentioned healthy human blood, microfluidic channel is passed through with 1.0mL/h flow velocity, shown in fluorescence The cancer cell of capture is observed and counted under micro mirror, calculates its capture rate, as a result as shown in figure 11, the microfluidic system Preferable capture rate (85%- is respectively provided with to the separative efficiency of varying number (20-300/mL) circulating tumor cell 94.9%), capture rate is substantially in the trend of first increases and then decreases, illustrates that the micro fluidic device meets to various concentrations cancer cell Capture the requirement of separation.
Embodiment 10
Immunostaining is tested：
There is the effect of separation in order to further illustrate the micro flow control chip device of the present invention to circulating tumor cell in blood Fruit, the circulating tumor cell in blood samples of patients can be sub-elected, be tested using the blood of patients with lung cancer, red blood cell will be cracked Blood samples of patients be passed through the present invention micro-fluidic chip system in, then to cell carry out immunostaining, contaminated using immunofluorescence Color is identified the circulating tumor cell of capture.
The cell captured on nanofiber is fixed with 4% paraformaldehyde first, then with 0.1% Triton-100 solution carries out penetrating 10min to cell, then closes 30min with 1% BSA solution, then passes to CK7 (dilutions 50 times, 10 μ L) and anti-CD45 (50 times of dilution, 20 μ L) dyeing 1h, last 7min be passed through DAPI (60 times of dilution, 500 μ L), Then in fluorescence microscopy Microscopic observation, according to cancer cell judging standard, DAPI dyes to all cells, Anti- CD45 only dyes to leucocyte, and CK7 can only differentiate circulating tumor cell.CK+/CD45-/DAPI+ is following in blood Ring tumour cell, CK-/CD45+/DAPI+ are leucocyte, show that the device can be realized to circulating tumor cell in blood samples of patients The purpose for differentiating and separating.

Claims (9)

1. a kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film, including：
(1) it is 9 by mass ratio：0.5~1.5 chitosan is dissolved in solvent with PEO PEO, at room temperature stirring reaction, Room temperature is cooled to, obtains chitosan/PEO spinning solutions, electrostatic spinning, vacuum drying obtains chitosan nano fiber membrane, with penta 2 Aldehyde is crosslinked, and obtains cross-linked chitosan nano fibrous membrane CNFs；
(2) by hyaluronic acid HA, 1- (3- dimethylamino-propyls) -3- ethyl diimmonium salt hydrochlorate EDCHCl, N- hydroxysuccinimidyls Acid imide NHS is dissolved in solvent, stirring reaction, the HA-COOH activated, is added dropwise to ethylcysteine hydrochloride H- Continue stirring reaction in the Cys-OEt.HCl aqueous solution, through dialysing, being freeze-dried, obtain HA-Cys；Wherein HA, EDCHCl, NHS, H-Cys-OEt.HCl mass ratio are 1：2.4~2.5：1.4~1.5：0.8~0.9；
(3) HA-Cys that step (2) obtains is dissolved in solvent, oxydol H is added dropwise2O2, mercaptosilane coupling agents MPTMS, Ice-water bath stirring reaction, through dialysing, being freeze-dried, obtain HA-Cys-MPTMS；Wherein HA-Cys, MPTMS, H2O2Mol ratio For 1：3：5~8；
(4) CNFs for obtaining step (1) is added in methanol and the mixed solution of NaCl solution, adds amphion carboxylic acid beet Alkali acrylamide CBAA solution, stirring reaction, is then washed, naturally dry at room temperature, obtains the chitosan of amphion modification Nano fibrous membrane CBAA-CNFs；Wherein CNFs, CBAA mass ratio are 4~5：1；
(5) CBAA-CNFs that step (4) obtains is dissolved in solvent, it is water-soluble adds the HA-Cys-MPTMS that step (3) obtains Liquid, stirring reaction, then washing, naturally dry, obtain the nano fibrous membrane HA-CBAA-CNFs of HA functionalization；Wherein CBAA- CNFs, HA-Cys-MPTMS mass ratio are 1：1.5~1.7；
(6) slide for the HA-CBAA-CNFs for obtaining load step (5) is micro- with polydimethylsiloxane as substrate Stream control passage cover plate, is bonded by plasma, obtains embedding the micro-fluidic chip of the nano fibrous membrane of HA functionalization.
A kind of 2. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that：Solvent in the step (1) is the acetic acid that concentration is 85%；Chitosan in chitosan/PEO spinning solutions Concentration be 2.5~3.5wt%；Stir as magnetic agitation, the time of stirring reaction is 7~9h；The technological parameter of electrostatic spinning For：Spinning voltage is 30kV, flow velocity 0.1mL/h, spinning 12~15cm of distance, 20~30 DEG C of environment temperature, humidity 20~40%； The vacuum drying time is 12~24h；The time of crosslinking is 4~8h.
A kind of 3. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that：The molecular weight of HA in the step (2) is 5830；Solvent is dimethyl sulfoxide (DMSO) DMSO；Stir as magnetic force Stirring；The time of stirring reaction is 2.5~3h；The time for continuing stirring reaction is 2~3d.
A kind of 4. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that：Solvent in the step (3) is ethanol；The concentration of hydrogen peroxide is 28~32%；Stir and stirred for magnetic force Mix, the time of stirring reaction is 3~5h.
A kind of 5. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that：Amphion CBAA in the step (4) is by the way that dimethylamino propylamine, beta-propiolactone are dissolved in In anhydrous propanone, add polymerization inhibitor 1,1- diphenyl -2- trinitrophenyl-hydrazine DPPH, under nitrogen protection ice-water bath reaction 2.5~ 3.5h, purified, vacuum drying are made；Wherein dimethylamino propylamine, beta-propiolactone, DPPH, the amount ratio of anhydrous propanone are 1.6g：1~1.1g：50mg：18mL.
A kind of 6. preparation side of micro-fluidic chip for embedding hyaluronic acid decorated nano fibrous membrane according to claim 1 Method, it is characterised in that：The volume ratio of methanol and NaCl solution is 1 in mixed solution in the step (4)：1；NaCl solution Concentration is 0.1~0.2M；The concentration of CBAA solution is 0.07~0.09mg/mL；The time of stirring reaction is 2~3d.
A kind of 7. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that：Solvent in the step (5) is isopropanol；The technological parameter of stirring reaction is：Whipping temp be 70~ 80 DEG C, the stirring reaction time is 7~9h.
A kind of 8. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that：The micro-fluidic chip for the embedding hyaluronic acid functionalized nano-fiber film that the step (5) obtains is used to follow Ring tumour cell CTCs sorting and lossless release.
A kind of 9. preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 8 Method, it is characterised in that：The sorting of the CTCs and the technological parameter of lossless release are：Received to the embedding hyaluronic acid functionalization The cell suspending liquid containing cancer cell or cancer patient's blood are passed through in the micro-fluidic chip of rice tunica fibrosa, utilizes cancer cell surfaces Particular expression CD44 antigen and HA adhesion, complete the sorting of cancer cell；To the embedding hyaluronic acid functionalized nano Glutathione GSH solution is continually fed into the micro-fluidic chip of tunica fibrosa, and collects recovered liquid, the lossless of cancer cell is completed and releases Put.
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