CN106076444A - A kind of ultrasonic standing wave type micro-fluidic chip and preparation method thereof - Google Patents
A kind of ultrasonic standing wave type micro-fluidic chip and preparation method thereof Download PDFInfo
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- CN106076444A CN106076444A CN201610420922.7A CN201610420922A CN106076444A CN 106076444 A CN106076444 A CN 106076444A CN 201610420922 A CN201610420922 A CN 201610420922A CN 106076444 A CN106076444 A CN 106076444A
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/12—Specific details about materials
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
- B01L2300/163—Biocompatibility
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
- B01L2400/0439—Moving fluids with specific forces or mechanical means specific forces vibrational forces ultrasonic vibrations, vibrating piezo elements
Abstract
The present invention relates to a kind of ultrasonic standing wave type micro-fluidic chip and preparation method thereof, particle or cell are handled, capture and separate by the PLGA PEI microsphere in chip channel or PLGA short nanofiber lines in ultrasonic standing wave by this chip.Method includes: (1) utilizes double emulsion to synthesize PLGA microsphere, then is modified it with polymine；(2) prepare PLGA nano fibrous membrane with method of electrostatic spinning, then the process that carries out homogenizing obtains short nanofiber line；(3) ultrasonic standing wave type micro-fluidic chip is made.The present invention can efficiently control particle or cell, and preparation process is simple, with low cost, has the highest controllability and operability, has a extensive future.
Description
Technical field
The invention belongs to microfluidic art, particularly to a kind of ultrasonic standing wave type micro-fluidic chip and preparation side thereof
Method.
Background technology
Microflow control technique (Microfluidics) is one of science and technology of forefront on our times.It is one and melts
Chemistry, physics, life sciences, microelectronics, material, computer etc., in the height subject crossing technology of one, currently mainly should
It is used in chemistry and life science.It is a kind of in the passage of micro-meter scale to nanoliter or picoliters magnitude liquid handle
Or the technology controlled, from sampling, sample pretreatment, reaction, separation and concentration to the whole analysis process detected at integrated micro-core
Complete on sheet, while greatly reducing sample and reagent dosage, be more greatly enhanced analysis speed, it is achieved analyze the micro-of system
Type, integrated and automatization, and have low cost, high flux, easy to use, can the advantage such as multi-part combination.And miniflow
Control technology needs to realize by micro-fluidic chip, and micro-fluidic chip (Microfluidic chip) is also known as chip lab
(Lab-on-a-chip), it is by micro-processing technology, at silicon, quartz, glass, polymethyl methacrylate
The material such as (polymethylmethacrylate, PMMA), polydimethylsiloxane (polydimethylsiloxane, PDMS)
On, according to the actual requirements, produce various structure, size tests at the pipeline of micron dimension.Owing to it has minimum
Volume and the function handled of trace, this allows for, and it is not only few to the demand of sample size, and has the highest detection spirit
Sensitivity and analysis speed, can carry out every experiment continuously and automatically, have the extremely wide suitability and application prospect, is to work as
The emphasis of front micro-total analysis system research.
Meanwhile, owing to the line size of micro-fluidic chip is mated in micron dimension and micro-nano particle very much, the most permissible
Well handle fluid, but also can accurately control particle position in passage, have very much and be applied to the latent of particle manipulation
Power.Manipulation to particle, such as, realize the migration of particle in fluid media (medium) at present, assembles, transport, and sorting, is micro-fluidic skill
One important feature in art field.Due to the acoustic operation method non-destructive to living body biological pattern detection so that it is with micro-
Another focus being combined into research of fluidics.Acoustics microflow control technique refers to that the driving relying on piezoelectric ceramic piece excites
Ultrasonic standing wave micro fluidic device produces acoustic radiation force and realizes the manipulation to particle, and this acoustic radiation force is situated between by particle and fluid
The character difference of matter controls suspended particles and shifts to node or antinode.Owing to it is to the fine-grained suitability, therefore based on ultrasonic
It is a kind of more satisfactory mode that Principle of Standing-wave handles particle in micro-fluidic chip.CheolGi Kim et al. (CheolGi,
K.,et al.,Ultrasonic Manipulation of Magnetic Particles in a Microfluidic
Channel.International Journal of Precision Engineering and Manufacturing,
2014.15 (7): p.1411-1416) utilize ultrasonic standing wave to realize the behaviour to magnetic particle and living cells in micro-fluidic chip
Vertical.At present, in microfluidic system, typically by wet etching method, the hard materials such as silicon chip etch micronetwork knot
Structure, then by anode key and by the way of, by sheet glass key with on the silicon chip being etched with micro structure, make sound wave core with this
Sheet, the sound wave chip effect of this method processing is preferable, but, it needs to use the millions of anode key of value and equipment, from
And make the chip cost prepared the highest, it is unfavorable for the marketization.
Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid), PLGA) by two kinds of monomers-
Lactic acid and hydroxyacetic acid are polymerized at random, are a kind of degradable functional polymer organic compound, have good biology
The compatibility, nontoxic, good encystation and the performance of film forming, be widely used in pharmacy, medical engineering material and modernization industry
Field, at U.S. PLGA by FDA certification, is formally included into American Pharmacopeia as pharmaceutic adjuvant.Various PLGA medicine microspheres
The common report of application and preparation, wherein PLGA microsphere is as protein, the carrier of enzyme drug, is the focus of research.But at sound
Learn and microflow control technique does not also find handle PLGA microsphere and the report of PLGA nanofiber line based on ultrasonic standing wave principle.
Electrostatic spinning technique because its equipment is simple, processing ease and the feature such as efficiently, and by receiving that it is prepared
Rice pulp freeness is high, homogeneity is good and the adjustable advantage such as controlled of fiber morphology, and becomes and receive much concern in recent years, apply
Most methods preparing nanofiber.Use the method can prepare diameter 10nm～the superfine fibre of 10 μm, in catalysis
The aspects such as agent carrier, biomedicine, reinforcing material, filtering material, electrode material, sensor have well application.Grind in a large number
Study carefully and show that nanofiber easily carries out surface modification, and bioactive molecule can be loaded, such as protein, nucleic acid, saccharide and life
The long factor etc..Mallouk, Thomas E et al. (Mallouk, T.E., et al., Steering Acoustically
Propelled Nanowire Motors toward Cells in a Biologically Compatible
Environment Using Magnetic Fields.Langmuir, 2013.29 (52): p.16113-16118.) prepare sound
Learn nano wire targeting HeLa cell under the manipulation of externally-applied magnetic field and sound field of performance.There is not yet at ultrasonic standing wave micro-fluidic chip
In handle the report of short and scattered nanofiber line.
Retrieve domestic and international pertinent literature and patent results shows: also do not find to stay based on ultrasonic in acoustics microflow control technique
Ripple principle handles PLGA microsphere and the report of PLGA nanofiber line.There is not yet handle in ultrasonic standing wave micro-fluidic chip short and
The report of scattered nanofiber line.
Summary of the invention
The technical problem to be solved is to provide a kind of ultrasonic standing wave type micro-fluidic chip and preparation method thereof, should
Chip can efficiently control particle or cell, and preparation process is simple, with low cost, has the highest controllability and operable
Property, have a extensive future.
A kind of ultrasonic standing wave type micro-fluidic chip of the present invention, short by the PLGA-PEI microsphere in chip channel or PLGA
Particle or cell are handled, capture and are separated in ultrasonic standing wave by nanofiber line.
A length of 10～30 μm of described PLGA short nanofiber line.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip of the present invention, comprises the steps:
(1) PLGA microsphere is prepared；Then PLGA microsphere is dispersed in water, adds carbodiimide EDC and N-hydroxysuccinimidyl
The mixed aqueous solution activation of acid imide NHS, adds polymine PEI aqueous solution, shaking table oscillating reactions, centrifuge washing, divides
Dissipate, obtain PLGA-PEI microsphere；Wherein, PLGA microsphere and PEI mass ratio are 1:2-4；
(2) using oxolane and DMF as mixed solvent, prepare PLGA spinning liquid, pass through Static Spinning
Silk prepares PLGA nano fibrous membrane, is dried；Lamellar PLGA nano fibrous membrane is immersed in PVAC polyvinylalcohol solution,
Carry out the process that homogenizes under the conditions of ice-water bath, obtain the dispersion liquid of nanofiber, centrifuge washing, obtain the short nanofiber of PLGA
Line；
(3) prepare micro-fluidic chip, chip channel is injected PLGA-PEI microsphere or PLGA short nanofiber line, to obtain final product
Ultrasonic standing wave type micro-fluidic chip.
PLGA microsphere in described step (1) is 100:40:24 with the mass ratio of EDC, NHS.
The preparation method of the PLGA microsphere in described step (1) includes:
1) being dissolved in organic solvent by PLGA PLGA and obtain oil phase, ultra-pure water is as aqueous phase, so
After oil phase and aqueous phase are mixed, supersound process 20-30s in ice-water bath, obtain Water-In-Oil W/O emulsion；The wherein molecule of PLGA
Amount Mw is 25000, and the organic solvent of PLGA is dichloromethane CH2Cl2, the mass ratio of PLGA and ultra-pure water is 500:1, oil phase and
The volume ratio of aqueous phase is 5-10:1；
2) above-mentioned W/O emulsion is added in PVAC polyvinylalcohol aqueous solution, under the conditions of ice-water bath, carry out the process that homogenizes,
To W/O/W W/O/W emulsion；Wherein the molecular weight Mw of PVA is 20000-30000, and the mass percentage concentration of PVA solution is
2%-5%, homogenizes and is processed as homogenizer shearing, and rotating speed is 6000rpm, and the time is 5-10min, W/O/W emulsion three phase volume
Ratio is 25-100:5-10:1；
3) above-mentioned W/O/W emulsion is added in isopropanol water solution, about 35-40 DEG C stirring 3-5h, centrifuge washing, freezing
It is dried, obtains PLGA microsphere；Wherein the volume ratio of W/O/W emulsion and isopropanol water solution is 1:40-80.
Shaking table oscillating reactions temperature in described step (1) is 25-28 DEG C, and the response time is 2-3d.
The molecular weight of polymine PEI in described step (1) is 25000；The molecular weight of PLGA is 25000.
PLGA in described step (2) is 1g:4mL with the mass volume ratio of mixed solvent；Oxolane in mixed solvent
It is 3:1 with the volume ratio of DMF.
The molecular weight of the PLGA in described step (2) is 81000.
Electrostatic spinning process parameter in described step (2) is: voltage is 15-20kV, and receiving range is 15cm, and flow velocity is
0.6mL/h, two-tube shower nozzle, ambient temperature is 20-25 DEG C, and relative humidity is 40-50%.
Being dried as natural air drying in described step (2), drying time is 12-24h.
Lamellar PLGA nano fibrous membrane in described step (2) is 50-with the mass volume ratio of PVAC polyvinylalcohol solution
100mg:10-20mL.Lamellar PLGA nano fibrous membrane block puts into a size of 5 × 2mm2.The mass fraction of PVA solution is 2-5%.
Homogenizing and be processed as homogenizer shearing, rotating speed is 15000rpm, and the time is 15-20min.
The preparation method of the micro-fluidic chip in described step (3) is: using rectangle capillary tube as chip channel, use ring
Epoxy resins glue is fixed on microscope slide；By epoxide-resin glue, piezoelectric ceramic piece is bonded in rectangle capillary tube side；Growing
Two side outlet bonded polyethylene pipes of square capillary tube are as the suspension of PLGA-PEI microsphere or PLGA short nanofiber line
Input port and delivery outlet.
The size of described rectangle capillary tube is 50mm × 0.2mm × 2mm.
The size of described piezoelectric ceramic piece is 15mm × 2mm × 0.2mm, drives the waveform generator of piezoelectric ceramic piece
Voltage-regulation is 20Vpp, and frequency is adjusted to 4.6MHz.
Described polyethylene tube internal diameter is 1mm, and external diameter is 1.4mm.
The present invention uses the easy double emulsion of operation to prepare PLGA microsphere, demonstrates the acoustics contrast of this material of PLGA
The factor, and PLGA microsphere is carried out functional modification, utilize electrostatic adsorption to combine cell, it was demonstrated that through Electrostatic Absorption
Processing with ultrasonic standing wave and not injure cytoactive, therefore handling particle in micro-fluidic chip based on ultrasonic standing wave principle is
A kind of more satisfactory mode.
In the present invention, nanofiber by film forming homogenizes and is processed into short and scattered nanofiber line, it is achieved that its
Manipulation in ultrasonic standing wave micro-fluidic chip, owing to ultrasonic standing wave can handle short and mono-dispersed nano single, future is again
It is carried out functional modification, is applied to bio-sensing and biomedicine.
The ultrasonic standing wave chip of present invention design, manufacturing process is simple, with low cost, has the highest controllability and can grasp
The property made, integration degree is high, it is easy to realize particle and biological living sample manipulations.
In the present invention, PLGA is chain macromolecule, it is more difficult to carry out modification, and PEI molecule have more amino and its
The carboxyl reaction on surface, the electropositive of recycling PEI, by Electrostatic Absorption, cell is adsorbed at PLGA microsphere surface, successfully grasp
Vertical cell is arranged in the middle of passage with the complex of microsphere.
The present invention is successfully prepared short and scattered nanofiber line, it is achieved its row in ultrasonic standing wave micro-fluidic chip
Row, are expected to be applied to bio-sensing and biomedical aspect.
Beneficial effect
(1) the PLGA microsphere that prepared by the present invention and PLGA nanofiber, material has good pattern, Stability Analysis of Structures；
(2) PLGA microsphere adherent cell modified for the PEI that prepared by this method is effective；And PLGA microsphere and PLGA nanometer
Fibrous material has sensitive ultrasonic standing wave response characteristic, presents more neat arrangement in micro-fluidic chip；
(3) ultrasonic standing wave type micro-fluidic chip of the present invention, processing is simple, with low cost, is not required to charge in manufacturing process
The anode linkage equipment that lattice are expensive；The generation of ultrasonic standing wave field and regulate controllable；Chip easy cleaning, Reusability does not affect effect
Rate；Utilize the present invention can be easily carried out the manipulation to the biological living sample such as particle and cell, capture and separate；This
Bright can be widely applied to the fields such as life sciences, pharmaceutical science and medical science.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1, the SEM of the PLGA microsphere of preparation schemes (left) and diameter distribution profile (right)；
Fig. 2 is the uv-visible absorption spectra figure of PEI and PEI-FI solution in embodiment 1；
Fig. 3 is the fluorescence microscope picture of the functionalization PLGA microsphere after the PEI-FI in embodiment 1 modifies；Wherein,
A (), (b), (c) are respectively the picture after the fluorescence picture of fluorescence PLGA microsphere, difference picture and fluorescence and difference superposition；
Fig. 4 be in embodiment 2 PLGA microsphere ultrasonic standing wave stimulate before random distribution in the channel (a), ultrasonic standing wave thorn
It is arranged in after Jiing in the middle of passage (b)；
Fig. 5 is that in embodiment 3, the cell of Electrostatic Absorption PLGA-PEI microsphere is randomly dispersed in logical before ultrasonic standing wave stimulates
In road (a), ultrasonic standing wave is arranged in the middle of passage (b) after stimulating；
Fig. 6 is in embodiment 4, through the HeLa cell of ethanol postincubation shows fluorescent microscopy images (on), through ultrasonic standing wave
Before process, the shows fluorescent microscopy images of the conjugate of HeLa cell and PLGA-PEI Electrostatic Absorption (in), process through ultrasonic standing wave
After, the shows fluorescent microscopy images of HeLa cell and PLGA-PEI Electrostatic Absorption conjugate (under)；
Fig. 7 is FCM test result in embodiment 4, is through ethanol postincubation respectively, processes and ultrasonic standing wave without ultrasonic standing wave
The fluorescence intensity figure of the HeLa cell processed；
Fig. 8 is that in embodiment 5, the SEM of the PLGA electrostatic spinning nano fiber of preparation schemes (left) and diameter distribution profile (right)；
Fig. 9 is PLGA nano fibrous membrane block hydrophilic in different solvents in embodiment 6；
Figure 10 is the phase contrast microscope figure (left) of the PLGA short nanofiber line of preparation in embodiment 6 and short nanofiber line
Staple diagram (right)；
Figure 11 is that in embodiment 7, PLGA nanofiber line random distribution before ultrasonic standing wave stimulates is in the channel (a), ultrasonic
Standing wave is arranged in the middle of passage (b) after stimulating；
Figure 12 is the schematic diagram of ultrasonic standing wave micro-fluidic chip in embodiment 2.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, people in the art
The present invention can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims equally and limited
Scope.
Embodiment 1
The preparation of PLGA microsphere and the detailed process of functional modification thereof are as follows:
(1) weigh 100mg PLGA and be dissolved in 2mLCH2Cl2In, 200 μ L ultra-pure waters are as interior aqueous phase, by organic facies and Nei Shui
Phase two-phase mixtures, supersound process 30s under ice-water bath state, form W/O emulsion；Above-mentioned W/O emulsion is proceeded to 50mL 5%PVA
In aqueous solution, process (6000rpm, 5min) with homogenizer under ice-water bath state, make the W/O/W that droplet formation size is the most homogeneous
Emulsion；The W/O/W emulsion obtained is instilled in 80mL 2% isopropanol water solution, under 39 DEG C of water bath condition, after magnetic agitation 4h,
Centrifugal (4000rpm, 3min), abandoning supernatant, centrifuged residue 30mL ultra-pure water is washed, and repeats above-mentioned centrifugal water washing operations
So that supernatant clear, colorless, after being centrifuged, centrifuged residue is scattered in 30ml ultra-pure water again ,-80 DEG C of refrigerator freezings for three times
After 3h, it is placed in freezer dryer lyophilization and processes 3d, i.e. obtain PLGA microsphere.As shown in Figure 1, it can be clearly seen that
The smooth surface of PLGA, balling-up is preferable, and distribution of sizes is narrower, has good monodispersity, and average diameter is 3.46 μm.
(2) the PLGA microsphere 100mg weighing step (1) lyophilizing is dissolved in 5mL ultra-pure water prepared microspheres solution, adds wherein
Enter carbodiimide EDC (40mg, 0.2mmol) and N-hydroxy-succinamide NHS (24mg, 0.2mmol) mixed aqueous solution, activation
Reaction 2h, be eventually adding polymine PEI aqueous solutions of polymers (200mg, 8 × 10-3Mmol), under the conditions of 25 DEG C, shaking table
Upper oscillating reactions 3d, obtains PLGA microsphere based on polyethyleneimine-modified, then with 30mL ultra-pure water centrifugal (4000rpm,
3min) washing 3 times, abandoning supernatant, centrifuged residue is dispersed in 5mL water standby.
(3) weigh polymine PEI (200mg, 8.0 × 10-3Mmol), it is dissolved in 5mL dimethyl sulfoxide DMSO, adds
Fluorescein isothiocyanate (FITC) (6.20mg, 1.6 × 10-2Mmol) lucifuge reaction 1d.Then completely reacted mixed liquor transfer
To the bag filter that molecular cut off is 8000-14000, first with 0.02M PBS that pH value is 7.4 dialysis 24h (3 times,
2L/ time), then the 24-48h (6 times, 2L/ time) that dialyses in distilled water.Then carry out lyophilization process, i.e. obtain product P EI-
FI.Being required for lucifuge in dialysis and freeze-drying process to process, by uv-visible absorption spectra figure, FI successfully modifies
PEI surface (Fig. 2).
(4) the PLGA microsphere 100mg weighing step (1) lyophilizing is dissolved in 5mL ultra-pure water in prepared microspheres solution, adds carbon
Diimine EDC (40mg, 0.2mmol) and N-hydroxy-succinamide NHS (24mg, 0.2mmol) mixed aqueous solution, react 2h,
Add and be dissolved in the product 200mg that in 5mL water, step (3) obtains, under room temperature condition, lucifuge, shaking table oscillating reactions 72h.Reaction
After end, being centrifuged gained solution washing (4000rpm, 3min) 3 times, abandoning supernatant, centrifuged residue is dispersed in 5mL
In water standby, prove that PEI successfully modifies at PLGA microsphere surface (Fig. 3) by the fluorescence microscope picture of PLGA-PEI-FI.And
And the PLGA microsphere that dynamic light scattering test result display PLGA microsphere surface electromotive force-22.97 ± 1.03mV, PEI is modified
The surface potential of PLGA-PEI is 48.40 ± 0.35mV, further demonstrate that PEI successfully modifies at PLGA microsphere surface.
Embodiment 2
Ultrasonic standing wave type micro-fluidic chip as shown in figure 12, specifically comprises the following steps that
(1) at 76 × 26mm2Microscope slide on, fix rectangle capillary tube (a length of 50mm, width with epoxide-resin glue
For 0.2mm, height is 2mm), this rectangle capillary tube is as the passage of micro-fluidic chip；
(2) at two side outlets of rectangle capillary tube, bonding internal diameter is 1mm, external diameter is that the polyethylene tube of 1.4mm is as outstanding
The input port of supernatant liquid and delivery outlet；
(3) last in the rectangle capillary tube side fixed, fix piezoelectric ceramic piece by epoxide-resin glue is bonding
(15mm × 2mm × 0.2mm), then the positive and negative welding lead at piezoelectric ceramic piece connects wire as circuit.
In Example 1, aqueous solution (0.2mg/mL) 2mL of (1) products obtained therefrom is placed in 5mLEP pipe as liquid storage pipe, enters
Before row test, micro-fluidic chip 2%BSA soaks 2h, puts up ultrasonic standing wave micro fluidic device platform, first test piezoelectricity pottery
Whether porcelain two ends add voltage, the output signal of regulation signal generator be voltage be 20Vpp, frequency is 10KHz, and standard is
Plus hearing buzzer after this signal, otherwise piezoelectric ceramics two ends are not the most plus signal, are at this moment accomplished by inspection and connect
Whether head is correct, and signal generator output channel chooses whether consistent etc. with input channel.After debugging is good, signal generator
The output signal of telecommunication to be transferred to voltage be 20Vpp, frequency is 4.6MHz.Polyethylene tube as suspension inlet is inserted liquid storage pipe
In, syringe pump uses the pattern taken out, and flow rate set is 30 μ L/min, when microsphere flowing velocity is stablized, opens on computer
MetaVue image capture software, recalls live real time imaging and observes interface, opens the outfan button of waveform generator, permissible
Observe (Fig. 4) between arrangement of microspheres is in the channel.
Embodiment 3
In Example 1, the PLGA-PEI aqueous solution 500 μ L (1mg) of (2) products obtained therefrom, is diluted to serum-free medium
1mL (concentration is 0.5mg/mL), is placed in 5mLEP pipe, and the HeLa that addition digestion is centrifuged the serum-free medium suspension got off is thin
Born of the same parents (1,000,000), cumulative volume is 2mL, and 37 DEG C co-culture 30min, obtain the solution of cell and microsphere conjugate.Before testing,
Micro-fluidic chip 2%BSA soaks 2h, puts up ultrasonic standing wave micro fluidic device platform, and first test piezoelectric ceramics two ends are
The no voltage that adds, regulation signal generator output signal be voltage be 20Vpp, frequency is 10KHz, and standard is plus this
Can hear buzzer after signal, otherwise piezoelectric ceramics two ends are not the most plus signal, are at this moment accomplished by the most just checking joint
Really, signal generator output channel chooses whether consistent etc. with input channel.After debugging is good, the output electricity of signal generator
It is 20Vpp that signal is transferred to voltage, and frequency is 4.6MHz.Polyethylene tube insertion cell as suspension inlet is combined with microsphere
In the solution liquid storage pipe of thing, syringe pump uses the pattern taken out, and flow rate set is 30 μ L/min, when microsphere flowing velocity is stablized,
Open the MetaVue image capture software on computer, recall live real time imaging and observe interface, open the defeated of waveform generator
Go out end button, it can be observed that combine the cell arrangement of microsphere in the channel between (Fig. 5).
Embodiment 4
(1) digestion is in the HeLa cell of logarithmic (log) phase, collects centrifugal, counts with cell counter, take 900,000, be divided into 3 parts,
Every part of 300,000 cells.Wherein adding 0.5mg PLGA-PEI microspheres solution in two parts of cell suspending liquids, serum-free medium is made
For cell and microsphere culture fluid, cumulative volume 1mL, 37 DEG C co-culture 30min.
(2) mixed liquor that in step (1), a cell and PLGA-PEI microsphere co-culture is selected to be passed through ultrasonic standing wave miniflow
In control passage, building ultrasonic standing wave device according to above-mentioned steps, omnidistance unlatching ultrasonic standing wave signal (continuing 2min), until all
Solution passes through this device, it was observed that combine the cell arrangement of microsphere in the channel between, finally collect through super in channel outlet
The cell that sound standing wave processes.
(3) then with a cell remaining in the ethanol postincubation step (1) of 70%, the process time is 25min.Last same
One time dyeed these three parts of cells respectively with Calcein-AM (live cell dye), and dye strength is 5 μMs, and dyeing time is
10min.After dyeing, add 10mLPBS dilution, the dyestuff (1000rpm, 3min) that centrifuge washing is unnecessary, be more again dispersed in
In 500 μ L PBS.
(4) the product 10 μ L taking out step (3) gained respectively drops on microscope slide, observation of cell under fluorescence microscope
Fluorescent effect (Fig. 6).Result shows that the dead cell with ethanol postincubation dyeing is compared, and does not process staining cell through ultrasonic standing wave
Good activity is presented with the cell processing dyeing through ultrasonic standing wave.
(5) in order to further determine that the cytoactive through Different treatments, step (3) through three kinds of different modes
The cell ice bath being dissolved in 500 μ L PBS processed makes cell reduce vigor, strong with flow cytometer (FCM) detection cell fluorescence
Degree.Interpretation of result, the most weak through the cell fluorescence intensity of ethanol postincubation, not through the cell of ultrasonic standing wave process with through super
The cell that sound standing wave processes has the strongest fluorescence intensity (Fig. 7), contrasts the dead cell through ethanol postincubation, and its fluorescence intensity is very
It is weak, and the cell fluorescence intensity that experimental group processes through ultrasonic standing wave is the strongest, hence it is demonstrated that ultrasonic standing wave processes and quiet
The mode of electro-adsorption does not results in injury to cell.
Embodiment 5
The preparation of PLGA nanofiber comprises the steps: to weigh the PLGA of 1.0g, be dissolved in oxolane (THF) and
In N-N-dimethylformamide (DMF) mixed liquor (volume ratio is THF:DMF=3:1), under magnetic agitation effect, until PLGA
All dissolving complete, obtaining concentration of dope is 25% (1g/4mL).Spinning liquid is drawn in syringe, utilizes high-pressure electrostatic
Spinning-drawing machine carries out electrostatic spinning, and spinning condition is: temperature 25 DEG C, humidity 40-50%, voltage 20Kv；Receiving range 15cm；Liquid
Flow 0.8mL/h.Wherein receiving plate is clean masking foil.Natural air drying 12h.SEM result shows, it is thus achieved that Nanowire dimension table
Face is smooth, uniform diameter, and average fibre diameter is 724nm (Fig. 8).
Embodiment 6
In Example 5, the PLGA nano fibrous membrane of gained is cut into the nano fibrous membrane block (a size of 5 × 2mm of lamellar2),
Weighing 100mg to be immersed in 12.5mL5%PVA aqueous solution, contrast swims in the nano fibrous membrane block on ultra-pure water surface, can see
Nanofibers film block is completely submerged in PVA aqueous solution preferable hydrophilic (Fig. 9), so selecting PVA as emulsifying agent.
Carry out, with homogenizer (15000rpm, 15min), the process that homogenizes under ice-water bath state, obtain the dispersion liquid of fiber.Use 30mL again
Ultra-pure water centrifuge washing (5000rpm, 3min) 3 times, abandoning supernatant, centrifuged residue is dispersed in 5mL ultra-pure water, the most again
1000rpm is centrifugal once takes supernatant, and 60 DEG C of oven dryings are standby.Dried nano wire is weighed 5mg, and to be dissolved in 5mL ultrapure
In water (1mg/mL), taking 10 μ L and drop on microscope slide, observe under phase contrast microscope, result display single dispersibility is preferable,
Within length is mainly distributed on 30 μm, it is concentrated mainly on (Figure 10) about 17.73 μm.
Embodiment 7
In Example 6, nanofiber line dispersion liquid (1mg/mL) 2mL of gained is placed in 5mLEP pipe as liquid storage pipe,
Before testing, micro-fluidic chip (with embodiment 2) soaks 2h with 2%BSA, puts up ultrasonic standing wave micro fluidic device platform,
Whether first test piezoelectric ceramics two ends add signal, the output signal of regulation signal generator be voltage be 20Vpp, frequency
For 10KHz, standard is plus hearing buzzer after this signal, and otherwise piezoelectric ceramics two ends are not the most plus signal, this
Time be accomplished by checking that joint is whether correct, signal generator output channel chooses whether consistent etc. with input channel.Debugging gets well it
After, it is 20Vpp that the output signal of telecommunication of signal generator is transferred to voltage, and frequency is 4.6MHz.Using gathering as suspension inlet
Ethylene tube inserts in liquid storage pipe, and syringe pump uses the pattern taken out, and flow rate set is 30 μ L/min, when microsphere flowing velocity is stable
Time, open the MetaVue image capture software on computer, recall live real time imaging and observe interface, open waveform generator
Outfan button, it can be observed that nanofiber line is arranged in (Figure 11) in the middle of passage.
Claims (10)
1. a ultrasonic standing wave type micro-fluidic chip, it is characterised in that: by the PLGA-PEI microsphere in chip channel or PLGA
Particle or cell are handled, capture and are separated in ultrasonic standing wave by short nanofiber line.
A kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 1, it is characterised in that: the short Nanowire of described PLGA
A length of 10～30 μm of dimension line.
3. a preparation method for ultrasonic standing wave type micro-fluidic chip, comprises the steps:
(1) PLGA microsphere is prepared；Then PLGA microsphere is dispersed in water, adds carbodiimide EDC and N-hydroxysuccinimidyl acyl is sub-
The mixed aqueous solution activation of amine NHS, adds polymine PEI aqueous solution, shaking table oscillating reactions, centrifuge washing, dispersion, obtains
To PLGA-PEI microsphere；Wherein, PLGA microsphere and PEI mass ratio are 1:2-4；
(2) using oxolane and DMF as mixed solvent, PLGA spinning liquid is prepared, by electrostatic spinning system
For obtaining PLGA nano fibrous membrane, it is dried；Lamellar PLGA nano fibrous membrane is immersed in PVAC polyvinylalcohol solution, at frozen water
Carry out the process that homogenizes under the conditions of bath, obtain the dispersion liquid of nanofiber, centrifuge washing, obtain PLGA short nanofiber line；
(3) prepare micro-fluidic chip, chip channel is injected PLGA-PEI microsphere or PLGA short nanofiber line, obtains ultrasonic
Standing wave declines fluidic chip.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described step
Suddenly the PLGA microsphere in (1) is 100:40:24 with the mass ratio of EDC, NHS.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described step
Suddenly the shaking table oscillating reactions temperature in (1) is 25-28 DEG C, and the response time is 2-3d.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described step
Suddenly the molecular weight of the PLGA in (1) is 25000；The molecular weight of polymine PEI is 25000.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described step
Suddenly the PLGA in (2) is 1g:4mL with the mass volume ratio of mixed solvent；Oxolane and N in mixed solvent, N-dimethyl methyl
The volume ratio of amide is 3:1.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described step
Suddenly the electrostatic spinning process parameter in (2) is: voltage is 15-20kV, and receiving range is 15cm, and flow velocity is 0.6mL/h, two-tube spray
Head, ambient temperature is 20-25 DEG C, and relative humidity is 40-50%.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described step
Suddenly the lamellar PLGA nano fibrous membrane in (2) is 50-100mg:10-20mL with the mass volume ratio of PVAC polyvinylalcohol solution.
The preparation method of a kind of ultrasonic standing wave type micro-fluidic chip the most according to claim 3, it is characterised in that: described
The preparation method of the micro-fluidic chip in step (3) is: using rectangle capillary tube as chip channel, fix with epoxide-resin glue
On microscope slide；By epoxide-resin glue, piezoelectric ceramic piece is bonded in rectangle capillary tube side；At rectangle capillary tube
Two side outlet bonded polyethylene pipes are as the input port of the suspension of PLGA-PEI microsphere or PLGA short nanofiber line and output
Mouthful.
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| CN110643486A (en) * | 2019-09-25 | 2020-01-03 | 东南大学 | Ultrasonic sound field device and digital PCR liquid drop array chip manufacturing method |
| CN112574877A (en) * | 2020-12-02 | 2021-03-30 | 武汉大学 | Cell assembling method and device based on medical B-ultrasonic machine |
| CN114522649A (en) * | 2022-02-15 | 2022-05-24 | 浙江大学 | Acoustic particle capturing and track control method based on magnetofluid reconstruction |
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