CN110438000A - Double frequency standing-wave sound field separates the apparatus and method of circulating tumor cell and its micro- bolt - Google Patents
Double frequency standing-wave sound field separates the apparatus and method of circulating tumor cell and its micro- bolt Download PDFInfo
- Publication number
- CN110438000A CN110438000A CN201910668908.2A CN201910668908A CN110438000A CN 110438000 A CN110438000 A CN 110438000A CN 201910668908 A CN201910668908 A CN 201910668908A CN 110438000 A CN110438000 A CN 110438000A
- Authority
- CN
- China
- Prior art keywords
- runner
- tumor cell
- micro
- circulating tumor
- liquid outlet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Clinical Laboratory Science (AREA)
- Dispersion Chemistry (AREA)
- Mechanical Engineering (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses the apparatus and method using double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, which is set the runner pedestal there are two runner and is in contact with runner base bottom surface and is located at the first piezoelectric ceramics and the second piezoelectric ceramics below two runners for being alternately produced double frequency standing-wave sound field and formed by upper surface；The outlet end of first runner extends to form two non-wide the first liquid outlets and the second liquid outlet to two sides bifurcated from 3-point line, the outlet end of second flow channel extends to form two non-wide third liquid outlets and the 4th liquid outlet to two sides bifurcated from 3-point line, and seamlessly transits and be connected between the first liquid outlet and the 4th inlet；Due to using cascade two-stage channel, in conjunction with the alternating action of double frequency standing-wave sound field, the activity of cell has been effectively ensured in the precision and accuracy for improving sound field separation, and isolated cell flux is big, flowing is unobstructed, high sensitivity, integration is strong, good separating effect, separative efficiency are high.
Description
Technical field
The present invention relates to the separators and separation method field more particularly to one of circulating tumor cell and its micro- bolt
Kind separates the apparatus and method of circulating tumor cell and its micro- bolt using double frequency standing-wave sound field.
Background technique
Circulating tumor cell refers to the tumour cell that Peripheral Circulation is diffused by tumor focus, and under certain condition
It can develop as metastatic lesion；And the micro- bolt of circulating tumor cell (clusters) is thin by two or more circulating tumors
Born of the same parents assemble, and are as caused by the micro- bolt of circulating tumor cell greater than 50% metastases, this ratio is up in breast cancer
97%；Quick, to efficiently separate circulating tumor cell and its micro- bolt method is developed, as a result, to develop the prison with clinical meaning
Measurement equipment provides important foundation.
The diameter of the circulating tumor cell being referred to herein is general >=and 10 μm, the diameter of the micro- bolt of circulating tumor cell is tens
Microns；The method of separation circulating tumor cell and its micro- bolt is broadly divided into passive screening and actively screens two classes at present:
1) passive screening scheme: being mainly based upon the attributes such as the size, density, surface marker of cell to be separated, common
Passive screening technique have filtration method, density-gradient centrifugation method, circulating tumor cell marker screening method and based on miniflow knot
The screening technique of structure；But filtration method and the screening technique based on microfluxion would generally generate choking phenomenon and cell caused to damage
Wound, to influence to carry out subsequent culture, phenotypic evaluation and toxicity test etc. to the circulating tumor cell isolated and micro- bolt；It is close
The separative efficiency for spending gradient centrifugation is relatively low；And circulating tumor cell marker screening method is dependent on circulating tumor cell
Phenotype is normally based on EpCAM(epithelial antigen) carry out capture separation, but the circulating tumor cell in different tumours source
EpCAM expression have differences, and the albumen expression can dynamic change, thus will appear the possibility of false positive.
2) active screening scheme: it is mainly based upon the effect of applied force (such as electric field force, sound field power) to realize separation, often
Square method has dielectrophoresis method and sonic method；The advantage of dielectrophoresis method is can to separate 1 μm of cell below, but due to being situated between
Dielectric power workable for electric field field strength limits is relatively small, and separation flux is not so good as sonic method；Sonic method is produced in fluid channel
Raw standing-wave sound field acts on cell to be separated, and relative to dielectrophoresis method, sonic method is well suited to separate diameter at several microns to several
Ten microns of particle and cell；In the way of generating sound wave and the difference of Acoustic Wave Propagation mode, sonic method are subdivided into surface
Sonic method and bulk acoustic wave method: surface acoustic wave method is convenient for design sound field, but opposite sound field intensity is smaller；Bulk acoustic wave method sound field intensity
Greatly, but common bulk acoustic wave method only has single sound node, to limit the separating capacity of bulk acoustic wave method.
Either passive screening scheme and active screening scheme, the separation process of existing separator and method all do not have
It is realized in primal environment locating for circulating tumor cell, it is difficult to the activity of cell be effectively ensured, and be subsequent cell metabolism
Activity determination, multiplexed protein marker detection, culture and toxicological study etc. provide safeguard.
Therefore, the prior art still has much room for improvement and develops.
Summary of the invention
In order to solve the above technical problems, the present invention provide it is a kind of using double frequency standing-wave sound field separation circulating tumor cell and its
The device of micro- bolt can realize separation in the primal environment locating for circulating tumor cell, and the integrality of cell can be effectively ensured
And activity, and cell flux is big, flowing is unobstructed, integration is strong.
Meanwhile the present invention also provides a kind of method for separating circulating tumor cell and its micro- bolt using double frequency standing-wave sound field,
Separation can be realized in the primal environment locating for circulating tumor cell, and the integrality and activity of cell can be effectively ensured, and spirit
Sensitivity height, good separating effect, separative efficiency are high.
Technical scheme is as follows: the device of a kind of double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt,
Its core is made of runner pedestal, the first piezoelectric ceramics and the second piezoelectric ceramics；First is provided on the runner pedestal
Runner and second flow channel, first piezoelectric ceramics and the second piezoelectric ceramics are in contact with the bottom surface of runner pedestal, and described
First piezoelectric ceramics is located at the lower section of first runner, and second piezoelectric ceramics is located at the lower section of second flow channel；It is described first-class
The liquid feeding end in road extends two wide the first inlets and the second inlet from middle line to two sides bifurcated, the first runner
Outlet end extends to form two non-wide the first liquid outlets and the second liquid outlet, and described to two sides bifurcated from 3-point line
The width of one liquid outlet is twice of the width of the second liquid outlet；The liquid feeding end of the second flow channel prolongs from middle line to two sides bifurcated
It stretches to form two wide third inlets and the 4th inlet, the outlet end of the second flow channel is from 3-point line to two sides bifurcated
Two non-wide third liquid outlets and the 4th liquid outlet are extended to form, and the width of the third liquid outlet is the 4th liquid outlet
Twice of width；And it seamlessly transits and is connected between first liquid outlet and the 4th inlet.
The device of the described double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, in which: the first runner and
Second flow channel is of a straight line type, and cross section is in rectangle, and the width of the width of first runner and depth and second flow channel and
Depth is identical.
The device of the described double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, in which: the first runner with
Second flow channel is parallel and shifts to install, and the upper side of the downside of the second flow channel and first runner is located at same straight line
On.
The device of the double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, in which: first inlet
It is of a straight line type with the second inlet, and forms Y-shaped between first runner, and between the first inlet and the second inlet
Angle less than 45 °；First liquid outlet and the second liquid outlet are also of a straight line type, and Y is also formed between first runner
Font, and the angle between the central axes of the first liquid outlet and the second liquid outlet is equal between the first inlet and the second inlet
Angle.
The device of the double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, in which: the third inlet
It is of a straight line type with the 4th inlet, and forms Y-shaped between second flow channel, and between third inlet and the 4th inlet
Angle less than 45 °；The third liquid outlet and the 4th liquid outlet are also of a straight line type, and Y is also formed between second flow channel
Font, and the angle between third liquid outlet and the 4th liquid outlet is equal between third inlet and the central axes of the 4th inlet
Angle.
The device of the double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, in which: the runner pedestal is adopted
Slabbing is made with silicon substrate or silica material, and uses plasma etching industrial, produces cross in the runner pedestal upper surface
Rectangular in cross-section or trapezoidal groove are as first runner and second flow channel；The runner pedestal further includes that surface is covered on it
The glass cover-plate made of heat resistant glass, and be closely bonded by way of thermal bonding with runner pedestal；On the glass cover-plate
Position corresponding with the first inlet, the second inlet, the second liquid outlet, third inlet, third liquid outlet and the 4th liquid outlet
Place is set, processes access hole of the via hole as fluid respectively, and be connected with liquid collection test tube or syringe by miniflow hose
It connects.
The method of a kind of double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt is implemented described in any one of above-mentioned
Double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt device on, method includes the following steps:
A, will not celliferous first solution by the first syringe via corresponding miniflow hose from the first inlet injection the
One runner；By the fluid sample containing haemocyte, circulating tumor cell and its micro- bolt by the second syringe via corresponding micro-
It flows hose and injects first runner from the second inlet；
B, the injection speed or injection volume of the first syringe and the second syringe are adjusted separately, so that the first solution and fluid sample
Line of demarcation be located at first runner at the second liquid outlet side one third line；
C, the working frequency for applying 1MHz to the first piezoelectric ceramics generates standing wave sound at 1/2 standing wave node line of first runner
, so that circulating tumor cell and its micro- bolt moves to the midline of first runner and cross the line of demarcation of fluid sample into the
One solution, and haemocyte is then still in fluid sample and slowly moves；
D, the working frequency of the first piezoelectric ceramics is converted to 3MHz, is generated at the Y-direction 1/6,1/2 of first runner and 5/6 line
Standing-wave sound field, so that circulating tumor cell and its micro- bolt continue to move to the midline of first runner in the first solution, and blood
Cell is then to movement at 1/6 standing wave node line of first runner in fluid sample；
E, the working frequency of the first piezoelectric ceramics is converted repeatedly, so that the circulating tumor cell and its micro- bolt in fluid sample are always
It collects in first runner middle line nearby to move, and enters second flow channel through the 4th inlet from the first liquid outlet with the first solution,
And the 1/6 standing wave node line that haemocyte collects in first runner nearby moves, and corresponding from the second liquid outlet warp with fluid sample
Miniflow hose flow out to haemocyte liquid collect test tube.
The method of the double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, wherein the step A is also wrapped
It includes: celliferous second solution being passed through into third syringe and injects second from third inlet via corresponding miniflow hose
Road.
The method of the double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, wherein the step B is also wrapped
It includes: the injection speed or injection volume of adjustment third syringe, so that the line of demarcation of the second solution and the first solution is located at second
Road is at the 4th liquid outlet side one third line.
The method of the double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, wherein after the step E also
Include:
F, the working frequency for applying 1MHz to the second piezoelectric ceramics generates standing wave sound at 1/2 standing wave node line of second flow channel
, so that the micro- bolt of circulating tumor cell move to the midline of second flow channel and to cross the line of demarcation of the first solution molten into second
Liquid, and circulating tumor cell is then still in the first solution and slowly moves；
G, the working frequency of the second piezoelectric ceramics is converted to 3MHz, is generated at the Y-direction 1/6,1/2 of second flow channel and 5/6 line
Standing-wave sound field so that the micro- bolt of circulating tumor cell continues to move to the midline of second flow channel in the second solution, and recycles swollen
Oncocyte is in the first solution to movement at 1/6 standing wave node line of second flow channel；
H, the working frequency of the second piezoelectric ceramics is converted repeatedly, so that the micro- bolt of the circulating tumor cell in the second solution collects always
It is moved near the middle line of second flow channel, and flows out to circulation through corresponding miniflow hose from third liquid outlet with the second solution and swell
The micro- bolt liquid of oncocyte collects test tube, and the 1/6 standing wave node line that circulating tumor cell then collects in second flow channel nearby moves,
And circulating tumor cell liquid is flowed out to through corresponding miniflow hose from the 4th liquid outlet with the first solution and collects test tube.
A kind of device and side separating circulating tumor cell and its micro- bolt using double frequency standing-wave sound field provided by the present invention
Method utilizes the alternating action of two-stage flow passage characteristic combination double frequency standing-wave sound field, spirit due to using cascade two-stage channel
Sensitivity is high, improves the precision and accuracy of sound field separation, realizes circulating tumor cell and micro- bolt is integrated online
Separation, effectively prevents destruction of the multiple-step form separation method to cell, also avoids off-line type separation (such as centrifugal process
Deng) many and diverse operation sequence of the off-line monitoring of method and the thus isolated uncertainty that introduces；And standing-wave sound field generation
Active force is contactless force, effectively prevents destruction of the contact force to cell, original of the separation process locating for circulating tumor cell
It is realized in beginning environment, has been effectively ensured by point cellifugal integrality and an activity, has been detected for subsequent cell metabolic activity, is multiple
Protein markers analyte detection, culture and toxicological study etc. provide safeguard；Compared to other proactives, alternate double frequency standing wave sound
Field action power is relatively large, and isolated cell flux is big, flowing is unobstructed, avoids the cell obstructing problem of passive type, can integrate
Property it is strong, and can with other phenotypic evaluations and metabolic activity detection be integrated, standing-wave sound field active force is relatively large, separating effect
Good, separative efficiency height, the expansible online separation for other cells.
Detailed description of the invention
Fig. 1 is that the present invention utilizes double frequency standing-wave sound field separation circulating tumor cell and its work of micro- bolt embodiment core
Make schematic diagram；
Fig. 2 is Installation practice core of the present invention using double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt
Amplification stereogram.
Specific embodiment
Below with reference to attached drawing, a specific embodiment of the invention and embodiment are described in detail, described tool
Body embodiment only to explain the present invention, is not intended to limit a specific embodiment of the invention.
As shown in Figure 1, Fig. 1 is that the present invention is real using the device of double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt
Apply the working principle diagram of a core, the core of the separator is made pottery by runner pedestal (not shown go out), the first piezoelectricity
Porcelain 310 and the second piezoelectric ceramics 320 composition；
It is provided on the runner pedestal for cooperating the first piezoelectric ceramics 310 separation circulating tumor cell CTC and its micro- bolt
The first runner 100 of CTCs and for cooperate the second piezoelectric ceramics 320 separation the micro- bolt CTCs of circulating tumor cell second flow channel
200；
First piezoelectric ceramics 310 and the second piezoelectric ceramics 320 are in contact with the bottom surface of runner pedestal, and first pressure
Electroceramics 310 is located at the lower section of first runner 100, for being alternately produced double frequency standing-wave sound field in first runner 100, with separation
Circulating tumor cell CTC and its micro- bolt CTCs, second piezoelectric ceramics 320 are located at the lower section of second flow channel 200, for the
It is alternately produced double frequency standing-wave sound field in two runners 200, to separate the micro- bolt CTCs of circulating tumor cell；
The liquid feeding end of the first runner 100 extends to form two wide 101 Hes of the first inlet from middle line to two sides bifurcated
Second inlet 102, first inlet 101 are used to contain haemocyte BC, circulating tumor cell CTC with laminar flow input
And its fluid sample of micro- bolt CTCs, second inlet 102 are used to input not celliferous first solution with laminar flow
(such as phosphate buffer)；The outlet end of the first runner 100 from 3-point line to two sides bifurcated extend to form two it is non-etc.
Wide the first liquid outlet 103 and the second liquid outlet 104, and the width of first liquid outlet 103 is the width of the second liquid outlet 104
Degree twice, first liquid outlet 103 be used for by isolated circulating tumor cell CTC and its micro- bolt CTCs with the first solution with
Laminar flow outflow, the fluid sample after second liquid outlet 104 is used to be separated (contain the haemocytes such as red blood cell, leucocyte
BC it) is flowed out with laminar flow；
The liquid feeding end of the second flow channel 200 extends to form two wide 201 Hes of third inlet from middle line to two sides bifurcated
4th inlet 202, the third inlet 201 are used for laminar flow input not celliferous second solution (such as phosphoric acid
Salt buffer), the 4th inlet 202 is connected with the first liquid outlet 103, and side wall between the two and bottom wall are smooth
Transition, for being flowed by isolated circulating tumor cell CTC and its micro- bolt CTCs with the first solution with laminar flow；The second
The outlet end in road 200 extends to form two non-wide third liquid outlets 203 and the 4th to two sides bifurcated from 3-point line and goes out liquid
Mouth 204, and the width of the third liquid outlet 203 is twice of the width of the 4th liquid outlet 204, the third liquid outlet 203
For being flowed out by the isolated micro- bolt CTCs of circulating tumor cell with the second solution with laminar flow, the 4th liquid outlet 204 is used
In being flowed out by isolated circulating tumor cell CTC with the first solution with laminar flow.
So-called laminar flow refers to the liquid flowed in first runner 100 and second flow channel 200 under low reynolds number
The type of flow, and a kind of dimensionless that can be used to characterize fluid mobility status of Reynolds number Reynolds number (Reynolds number)
Number is the dust suppression by spraying of viscous effects to be characterized in hydrodynamics, and differentiate the foundation of flow behavior, when Reynolds number is smaller,
The influence of viscous force stream field is greater than inertia, and the disturbance of flow velocity can decay because of viscous force in flow field, and fluid flowing is stablized, and is formed
Laminar flow；Conversely, the influence of inertia stream field is greater than viscous force, and fluid flowing is more unstable if when Reynolds number is larger, flow velocity it is micro-
Small variation is easy development, enhancing, forms disorder, irregular turbulent flow or Turbulent Flow Field；In Guan Liuzhong, Reynolds number is less than 2300
Flowing is laminar flow, and flowing of the Reynolds number equal to 2300~4000 is transition state, and flowing of the Reynolds number greater than 4000 is turbulent flow.
Preferably, the first runner 100 and second flow channel 200 are of a straight line type, and cross section is in rectangle, and first
The width of runner 100 and depth are identical with depth with the width of second flow channel 200.
Preferably, the first runner 100 is parallel with second flow channel 200 and shifts to install, and the second flow channel 200
Downside and the upper side of first runner 100 be located along the same line.
Preferably, first inlet 101 and the second inlet 102 are of a straight line type, and between first runner 100
Y-shaped is formed, and the angle between the first inlet 101 and the second inlet 102 is less than 45 °；First liquid outlet, 103 He
Second liquid outlet 104 is also of a straight line type, and Y-shaped is also formed between first runner 100, and in the first liquid outlet 103
Angle between axis and the second liquid outlet 104 is equal to the angle between the first inlet 101 and the second inlet 102.
Preferably, the third inlet 201 and the 4th inlet 202 are of a straight line type, and between second flow channel 200
Y-shaped is formed, and the angle between third inlet 201 and the 4th inlet 202 is less than 45 °；203 He of third liquid outlet
4th liquid outlet 204 is also of a straight line type, and Y-shaped is also formed between second flow channel 200, and third liquid outlet 203 and
Angle between four liquid outlets 204 is equal to the angle between third inlet 201 and the central axes of the 4th inlet 202.
The preferred embodiment of double frequency standing-wave sound field separation circulating tumor cell and its device of micro- bolt is utilized in the present invention
In, as shown in connection with fig. 2, Fig. 2 is that the present invention is implemented using the device of double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt
The amplification stereogram of example core, specifically, that silicon substrate, silica or other hardness can be used is larger for the runner pedestal 410
Metal or nonmetal solid material be fabricated to such as 500 microns of thickness of sheet, and plasma etching industrial is used, in the stream
The groove of cross section rectangular and trapezoidal shapes is produced as first runner 100 and second flow channel 200, example in pedestal 410 upper surface in road
Such as, the rectangle groove of 750 microns of width, 50 microns of depth is etched；
The first runner 100 and the circulating tumor in second flow channel 200 are clearly recorded by microscope for the ease of video camera
The flow regime of cell CTC and its micro- bolt CTCs, the runner pedestal 410 further include the glass cover-plate that surface is covered on it
420, which can be used the sheet that heat resistant glass material is fabricated to 1 millimeter of thickness, and by way of thermal bonding with
Runner pedestal 410 is closely bonded；On the glass cover-plate 420 with the first inlet 101, the second inlet 102, the second liquid outlet
104, at third inlet 201, third liquid outlet 203 and the corresponding position of the 4th liquid outlet 204, such as diameter is processed respectively
Access hole of 700 microns of the via hole (421,422,423,424,425 and 426) as fluid, and for example, by 500 microns of internal diameter
Miniflow hose (not shown go out) and liquid collect test tube (not shown go out) or syringe (not shown out) and be connected；
First piezoelectric ceramics 310 and the second piezoelectric ceramics 320, which can be used, applies voltage in thickness direction Z and in thickness side
To Z generate vibration (i.e. thickness direction Z polarization) PNT-5 type piezoelectric ceramic piece, and the width of first piezoelectric ceramics 310 and
The length of first runner 100 is adapted, and the width of second piezoelectric ceramics 320 is adapted with the length of second flow channel 200；It will
Two planes vertical with the direction ε -33 are plated as electrode surface on first piezoelectric ceramics 310 and the second piezoelectric ceramics 320
Metal silver coating is as driving electrodes, and one of face is bonded in runner pedestal 410 with a-cyanoacrylate class glue
On bottom surface；First piezoelectric ceramics 310 and the second piezoelectric ceramics 320 difference and function signal generator (not shown go out) are electrically
Connection by the alternating voltage signal of function signal generator generation sinusoidal variations as driving signal, and can exist as required
It is converted back and forth between 1MHz and the working frequency of 3MHz, then after power amplifier device (not shown go out), drives the first piezoelectricity
Ceramics 310 and the second piezoelectric ceramics 320 work.
Based on the above-mentioned device for separating circulating tumor cell and its micro- bolt using double frequency standing-wave sound field, the invention also provides
A method of circulating tumor cell and its micro- bolt being separated using double frequency standing-wave sound field, specifically includes the following steps:
Step S510, will not celliferous first solution by the first syringe via corresponding miniflow hose from via hole 421
The first inlet 101 inject first runner 100；By the liquid containing haemocyte BC, circulating tumor cell CTC and its micro- bolt CTCs
Body sample injects first runner via second inlet 102 of the corresponding miniflow hose from via hole 422 by the second syringe
100；Not celliferous second solution is passed through into third feed liquor of the third syringe via corresponding miniflow hose from via hole 423
Mouth 201 injects second flow channels 200；
Step S520, the injection speed or injection volume of the first syringe and the second syringe are adjusted separately so that the first solution with
The line of demarcation 110 (Fig. 1) of fluid sample is located at first runner 100 at 104 side one third line of the second liquid outlet, i.e. liquid-like
Product all the second liquid outlet 104 from via hole 424 through corresponding miniflow hose flow out to haemocyte BC liquid collect test tube, and
First solution all enters second flow channel 200 from the first liquid outlet 103 through the 4th inlet 202；Specifically, for example, described
The injection speed of one syringe is 0.84mm3/ s or injection volume are 100μl/h, the injection speed of the second syringe is 0.28mm3/s
Or injection volume is 20μl/h；Meanwhile the injection speed or injection volume of third syringe are adjusted, so that the second solution and the first solution
Line of demarcation 210 (Fig. 1) be located at second flow channel 200 at the 204 side one third line of the 4th liquid outlet (because of the second solution and the
The flow velocity of one solution is different, even if both using identical liquid, can also generate line of demarcation therebetween), i.e., first is molten
Liquid all the 4th liquid outlet 204 from via hole 426 through corresponding miniflow hose flow out to circulating tumor cell CTC liquid collect
Test tube, and all through corresponding miniflow hose to flow out to circulating tumor thin for the third liquid outlet 203 from via hole 425 for the second solution
The micro- bolt CTCs liquid of born of the same parents collects test tube；Specifically, for example, the injection speed of the third syringe is 1.5mm3/ s or injection volume
It is 160μl/h；
Step S530, the working frequency for applying 1MHz to the first piezoelectric ceramics 310 using function signal generator, in Fig. 1 first
Standing-wave sound field is generated at 1/2 standing wave node line 120 (i.e. middle line) of runner 100, due to physical characteristics such as cell volume, weight
The volume of difference, circulating tumor cell CTC and its micro- bolt CTCs will be significantly greater than the body of the haemocytes such as red blood cell and leucocyte BC
Product, therefore the movement of circulating tumor cell CTC and micro- bolt CTCs under 1MHz standing-wave sound field will be significantly faster than that haemocyte BC, circulation
Tumour cell CTC and its micro- bolt CTCs can be to moving at 1/2 standing wave node line 120 (i.e. middle line) of first runner 100 and cross
The line of demarcation 110 of fluid sample enters the first solution, and haemocyte BC is then still in fluid sample and slowly moves；
Step S540, using the working frequency of function signal generator the first piezoelectric ceramics 310 of transformation to 3MHz, in first runner
Standing-wave sound field is generated at 100 Y-direction 1/6,1/2 and 5/6 line, due to the difference of the physical characteristics such as cell volume, weight, circulation
The volume of tumour cell CTC and its micro- bolt CTCs will be significantly greater than the haemocytes BC such as red blood cell and leucocyte, therefore circulating tumor
Cell CTC and its micro- bolt CTCs continues in the first solution at 1/2 standing wave node line 120 (i.e. middle line) of first runner 100
Movement, and haemocyte BC is then in fluid sample then to movement at 1/6 standing wave node line 130 of Fig. 1 first runner 100；
Step S550, the described function signal generator converts the work of the first piezoelectric ceramics 310 repeatedly between 3MHz and 1MHz
Frequency, specifically, for example, the time interval of function signal generator transformation working frequency is 5-10s；Finally, in fluid sample
Circulating tumor cell CTC and its micro- bolt CTCs collect in 1/2 standing wave node line 120 of Fig. 1 first runner 100 always (in i.e.
Line) nearby move, and enter second flow channel 200 through the 4th inlet 202 from the first liquid outlet 103 with the first solution, and blood is thin
The 1/6 standing wave node line 130 that born of the same parents BC then collects in Fig. 1 first runner 100 nearby moves, and with fluid sample from via hole 424
The second liquid outlet 104 through corresponding miniflow hose flow out to haemocyte BC liquid collect test tube, thus complete from fluid sample
In isolate circulating tumor cell CTC and its micro- bolt CTCs mixture；
Step S560, the working frequency for applying 1MHz to the second piezoelectric ceramics 320 using function signal generator, in Fig. 1 second
Standing-wave sound field is generated at 1/2 standing wave node line 220 (i.e. middle line) of runner 200, due to physical characteristics such as cell volume, weight
The volume of difference, the micro- bolt CTCs of circulating tumor cell will be significantly greater than the volume of circulating tumor cell CTC, therefore circulating tumor is thin
Movement of the micro- bolt CTCs of born of the same parents under 1MHz standing-wave sound field will be significantly faster than that circulating tumor cell CTC, the micro- bolt of circulating tumor cell
CTCs can be to the line of demarcation 210 that the first solution is moved and crossed at 1/2 standing wave node line 220 (i.e. middle line) of second flow channel 200
Into the second solution, and circulating tumor cell CTC is then still in the first solution and slowly moves；
Step S570, using the working frequency of function signal generator the second piezoelectric ceramics 320 of transformation to 3MHz, in second flow channel
Standing-wave sound field is generated at 200 Y-direction 1/6,1/2 and 5/6 line, due to the difference of the physical characteristics such as cell volume, weight, circulation
The volume of the micro- bolt CTCs of tumour cell will be significantly greater than circulating tumor cell CTC, therefore the micro- bolt CTCs of circulating tumor cell is
Continue to move at 1/2 standing wave node line 220 (i.e. middle line) of second flow channel 200 in two solution, and circulating tumor cell CTC is then
To movement at 1/6 standing wave node line 230 of Fig. 1 second flow channel 200 in the first solution；
Step S580, the described function signal generator converts the work of the second piezoelectric ceramics 320 repeatedly between 3MHz and 1MHz
Frequency, specifically, for example, the time interval of function signal generator transformation working frequency is 5-10s；Finally, in the second solution
The micro- bolt CTCs of circulating tumor cell collected near 1/2 standing wave node line 220 (i.e. middle line) of Fig. 1 second flow channel 200 always
Movement, and circulating tumor cell is flowed out to through corresponding miniflow hose with third liquid outlet 203 of second solution from via hole 425
Micro- bolt CTCs liquid collects test tube, and circulating tumor cell CTC then collects in 1/6 standing wave node line of Fig. 1 second flow channel 200
230 nearby move, and flow out to circulation through corresponding miniflow hose with fourth liquid outlet 204 of first solution from via hole 426
Tumour cell CTC liquid collects test tube, thus completes to be separately separated out circulating tumor cell CTC from fluid sample and individually divide
Separate out the micro- bolt CTCs of circulating tumor cell.
It should be understood that the foregoing is merely illustrative of the preferred embodiments of the present invention, it is not sufficient to limit of the invention
Technical solution within the spirit and principles in the present invention, can add according to the above description for those of ordinary skills
With increase and decrease, replacement, transformation or improvement, and all these increases and decreases, replacement, transformation or improved technical solution, it all should belong to this
The protection scope of invention appended claims.
Claims (10)
1. the device of a kind of double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, it is characterised in that: its core by
Runner pedestal, the first piezoelectric ceramics and the second piezoelectric ceramics composition；First runner and second are provided on the runner pedestal
Road, first piezoelectric ceramics and the second piezoelectric ceramics are in contact with the bottom surface of runner pedestal, and first piezoelectric ceramics
Positioned at the lower section of first runner, second piezoelectric ceramics is located at the lower section of second flow channel；The liquid feeding end of the first runner from
Middle line extends two wide the first inlets and the second inlet to two sides bifurcated, and the outlet end of the first runner is from three points
Two non-wide the first liquid outlets and the second liquid outlet, and the width of first liquid outlet are extended to form to two sides bifurcated at line
Degree is twice of the width of the second liquid outlet；The liquid feeding end of the second flow channel extends to form two etc. from middle line to two sides bifurcated
Wide third inlet and the 4th inlet, the outlet end of the second flow channel extend to form two to two sides bifurcated from 3-point line
Non- wide third liquid outlet and the 4th liquid outlet, and the width of the third liquid outlet is the two of the width of the 4th liquid outlet
Times；And it seamlessly transits and is connected between first liquid outlet and the 4th inlet.
2. the device of double frequency standing-wave sound field separation circulating tumor cell according to claim 1 and its micro- bolt, feature exist
In: the first runner and second flow channel are of a straight line type, and cross section is in rectangle, and the width of first runner and depth and
The width of second flow channel is identical with depth.
3. the device of double frequency standing-wave sound field separation circulating tumor cell according to claim 2 and its micro- bolt, feature exist
In: the first runner is parallel with second flow channel and shifts to install, and the downside of the second flow channel and first runner
Upper side is located along the same line.
4. the device of double frequency standing-wave sound field separation circulating tumor cell according to claim 1 and its micro- bolt, feature exist
In: first inlet and the second inlet are of a straight line type, and Y-shaped is formed between first runner, and the first feed liquor
Angle between mouth and the second inlet is less than 45 °；First liquid outlet and the second liquid outlet are also of a straight line type, and with
Also Y-shaped is formed between one runner, and the angle between the central axes of the first liquid outlet and the second liquid outlet is equal to the first feed liquor
Angle between mouth and the second inlet.
5. the device of double frequency standing-wave sound field separation circulating tumor cell according to claim 1 and its micro- bolt, feature exist
In: the third inlet and the 4th inlet are of a straight line type, and Y-shaped is formed between second flow channel, and third feed liquor
Angle between mouth and the 4th inlet is less than 45 °；The third liquid outlet and the 4th liquid outlet are also of a straight line type, and with
Also Y-shaped is formed between two runners, and the angle between third liquid outlet and the 4th liquid outlet is equal to third inlet and the 4th
Angle between the central axes of inlet.
6. the device of double frequency standing-wave sound field separation circulating tumor cell according to claim 1 and its micro- bolt, feature exist
In: the runner pedestal makes slabbing using silicon substrate or silica material, and uses plasma etching industrial, in the runner base
The groove of cross section rectangular and trapezoidal shapes is produced as first runner and second flow channel in seat upper surface；The runner pedestal also wraps
Include be covered on it surface heat resistant glass production glass cover-plate, and by way of thermal bonding with the close key of runner pedestal
It closes；On the glass cover-plate with the first inlet, the second inlet, the second liquid outlet, third inlet, third liquid outlet and
At the corresponding position of four liquid outlets, access hole of the via hole as fluid is processed respectively, and collect by miniflow hose and liquid
Test tube or syringe are connected.
7. a kind of method of double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt, implements any in claim 1 to 6
Described in double frequency standing-wave sound field separation circulating tumor cell and its micro- bolt device on, which is characterized in that this method include with
Lower step:
A, will not celliferous first solution by the first syringe via corresponding miniflow hose from the first inlet injection the
One runner；By the fluid sample containing haemocyte, circulating tumor cell and its micro- bolt by the second syringe via corresponding micro-
It flows hose and injects first runner from the second inlet；
B, the injection speed or injection volume of the first syringe and the second syringe are adjusted separately, so that the first solution and fluid sample
Line of demarcation be located at first runner at the second liquid outlet side one third line；
C, the working frequency for applying 1MHz to the first piezoelectric ceramics generates standing wave sound at 1/2 standing wave node line of first runner
, so that circulating tumor cell and its micro- bolt moves to the midline of first runner and cross the line of demarcation of fluid sample into the
One solution, and haemocyte is then still in fluid sample and slowly moves；
D, the working frequency of the first piezoelectric ceramics is converted to 3MHz, is generated at the Y-direction 1/6,1/2 of first runner and 5/6 line
Standing-wave sound field, so that circulating tumor cell and its micro- bolt continue to move to the midline of first runner in the first solution, and blood
Cell is then to movement at 1/6 standing wave node line of first runner in fluid sample；
E, the working frequency of the first piezoelectric ceramics is converted repeatedly, so that the circulating tumor cell and its micro- bolt in fluid sample are always
It collects in first runner middle line nearby to move, and enters second flow channel through the 4th inlet from the first liquid outlet with the first solution,
And the 1/6 standing wave node line that haemocyte collects in first runner nearby moves, and corresponding from the second liquid outlet warp with fluid sample
Miniflow hose flow out to haemocyte liquid collect test tube.
8. the method for double frequency standing-wave sound field separation circulating tumor cell according to claim 7 and its micro- bolt, feature exist
In the step A further include: will not celliferous second solution by third syringe via corresponding miniflow hose from third
Inlet injects second flow channel.
9. the method for double frequency standing-wave sound field separation circulating tumor cell according to claim 8 and its micro- bolt, feature exist
In the step B further include: the injection speed or injection volume of adjustment third syringe, so that the second solution and the first solution
Line of demarcation is located at second flow channel at the 4th liquid outlet side one third line.
10. the method for double frequency standing-wave sound field separation circulating tumor cell according to claim 9 and its micro- bolt, feature exist
In after the step E further include:
F, the working frequency for applying 1MHz to the second piezoelectric ceramics generates standing wave sound at 1/2 standing wave node line of second flow channel
, so that the micro- bolt of circulating tumor cell move to the midline of second flow channel and to cross the line of demarcation of the first solution molten into second
Liquid, and circulating tumor cell is then still in the first solution and slowly moves；
G, the working frequency of the second piezoelectric ceramics is converted to 3MHz, is generated at the Y-direction 1/6,1/2 of second flow channel and 5/6 line
Standing-wave sound field so that the micro- bolt of circulating tumor cell continues to move to the midline of second flow channel in the second solution, and recycles swollen
Oncocyte is in the first solution to movement at 1/6 standing wave node line of second flow channel；
H, the working frequency of the second piezoelectric ceramics is converted repeatedly, so that the micro- bolt of the circulating tumor cell in the second solution collects always
It is moved near the middle line of second flow channel, and flows out to circulation through corresponding miniflow hose from third liquid outlet with the second solution and swell
The micro- bolt liquid of oncocyte collects test tube, and the 1/6 standing wave node line that circulating tumor cell then collects in second flow channel nearby moves,
And circulating tumor cell liquid is flowed out to through corresponding miniflow hose from the 4th liquid outlet with the first solution and collects test tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910668908.2A CN110438000B (en) | 2019-07-24 | 2019-07-24 | Device and method for separating circulating tumor cells and micro-plugs thereof by using dual-frequency standing wave sound field |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910668908.2A CN110438000B (en) | 2019-07-24 | 2019-07-24 | Device and method for separating circulating tumor cells and micro-plugs thereof by using dual-frequency standing wave sound field |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110438000A true CN110438000A (en) | 2019-11-12 |
| CN110438000B CN110438000B (en) | 2021-10-12 |
Family
ID=68431432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910668908.2A Active CN110438000B (en) | 2019-07-24 | 2019-07-24 | Device and method for separating circulating tumor cells and micro-plugs thereof by using dual-frequency standing wave sound field |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110438000B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115308301A (en) * | 2022-08-16 | 2022-11-08 | 中山大学 | Measuring device capable of measuring elastic modulus of cells and cell nucleuses |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030044832A1 (en) * | 1996-09-04 | 2003-03-06 | Scandinavian Micro Biodevices A/S | Microflow system for particle separation and analysis |
| CN104195028A (en) * | 2014-08-05 | 2014-12-10 | 深圳先进技术研究院 | Microfluidic chip and cell screening method for screening specific cells |
| CN107505249A (en) * | 2017-08-23 | 2017-12-22 | 中国科学院苏州生物医学工程技术研究所 | Micro-fluidic chip system for rare cell screening |
| CN109439513A (en) * | 2018-12-11 | 2019-03-08 | 中国科学院苏州生物医学工程技术研究所 | The micro-fluidic chip screened for rare cell in whole blood |
| CN109946230A (en) * | 2019-02-26 | 2019-06-28 | 山东师范大学 | A kind of micro fluidic device for the high-throughput unicellular phenotypic analysis of CTC |
-
2019
- 2019-07-24 CN CN201910668908.2A patent/CN110438000B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030044832A1 (en) * | 1996-09-04 | 2003-03-06 | Scandinavian Micro Biodevices A/S | Microflow system for particle separation and analysis |
| CN104195028A (en) * | 2014-08-05 | 2014-12-10 | 深圳先进技术研究院 | Microfluidic chip and cell screening method for screening specific cells |
| CN107505249A (en) * | 2017-08-23 | 2017-12-22 | 中国科学院苏州生物医学工程技术研究所 | Micro-fluidic chip system for rare cell screening |
| CN109439513A (en) * | 2018-12-11 | 2019-03-08 | 中国科学院苏州生物医学工程技术研究所 | The micro-fluidic chip screened for rare cell in whole blood |
| CN109946230A (en) * | 2019-02-26 | 2019-06-28 | 山东师范大学 | A kind of micro fluidic device for the high-throughput unicellular phenotypic analysis of CTC |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115308301A (en) * | 2022-08-16 | 2022-11-08 | 中山大学 | Measuring device capable of measuring elastic modulus of cells and cell nucleuses |
| CN115308301B (en) * | 2022-08-16 | 2023-03-10 | 中山大学 | Measuring device capable of measuring elastic modulus of cells and cell nucleuses |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110438000B (en) | 2021-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9606086B2 (en) | High-efficiency separation and manipulation of particles and cells in microfluidic device using surface acoustic waves at an oblique angle | |
| Ma et al. | Mechanical properties based particle separation via traveling surface acoustic wave | |
| Shaker et al. | An impedance-based flow microcytometer for single cell morphology discrimination | |
| Salmanzadeh et al. | Isolation of prostate tumor initiating cells (TICs) through their dielectrophoretic signature | |
| Nawaz et al. | Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting” | |
| Geislinger et al. | Sorting of circulating tumor cells (MV3-melanoma) and red blood cells using non-inertial lift | |
| Mernier et al. | Characterization of a novel impedance cytometer design and its integration with lateral focusing by dielectrophoresis | |
| JP2003287519A (en) | Method for sorting microfluid component and particle in fluid | |
| US20150308971A1 (en) | Microfluidic impedance flow cytometer | |
| Huang et al. | Three-dimensional cellular focusing utilizing a combination of insulator-based and metallic dielectrophoresis | |
| CN108344678A (en) | A kind of particulate matter detection means and detection method | |
| JP4152949B2 (en) | Method for measuring impedance in a liquid microsystem | |
| Lee et al. | An integrated microfluidic platform for size-selective single-cell trapping of monocytes from blood | |
| KR20090083655A (en) | Dielectrophoretic microseparator | |
| CN110438000A (en) | Double frequency standing-wave sound field separates the apparatus and method of circulating tumor cell and its micro- bolt | |
| Iliescu et al. | Dielectrophoretic field-flow method for separating particle populations in a chip with asymmetric electrodes | |
| Geislinger et al. | Hydrodynamic and label-free sorting of circulating tumor cells from whole blood | |
| CN111154620A (en) | Micro-fluidic chip for magnetic quantity analysis carried by magnetic labeled cells | |
| Zhu et al. | An easy-fabricated and disposable polymer-film microfluidic impedance cytometer for cell sensing | |
| CN110333286A (en) | Apparatus and method based on ultrasonic standing wave acoustic field cell integral, flexible modulus | |
| Mei et al. | Counting leukocytes from whole blood using a lab-on-a-chip Coulter counter | |
| Chiavaroli et al. | An optical counting technique with vertical hydrodynamic focusing for biological cells | |
| Shen et al. | A simple 3-D microelectrode fabrication process and its application in microfluidic impedance cytometry | |
| Li et al. | Axial acoustic field barrier for fluidic particle manipulation | |
| Demircan et al. | Detection of imatinib resistance in K562 leukemia cells by 3D-electrode contactless dielectrophoresis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |