CN110438000B - Device and method for separating circulating tumor cells and micro-plugs thereof by using dual-frequency standing wave sound field - Google Patents
Device and method for separating circulating tumor cells and micro-plugs thereof by using dual-frequency standing wave sound field Download PDFInfo
- Publication number
- CN110438000B CN110438000B CN201910668908.2A CN201910668908A CN110438000B CN 110438000 B CN110438000 B CN 110438000B CN 201910668908 A CN201910668908 A CN 201910668908A CN 110438000 B CN110438000 B CN 110438000B
- Authority
- CN
- China
- Prior art keywords
- flow channel
- circulating tumor
- micro
- liquid outlet
- plugs
- 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.)
- Active
Links
Images
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 a device and a method for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave sound field, wherein the core part of the device consists of a flow channel base, a first piezoelectric ceramic and a second piezoelectric ceramic, wherein the upper surface of the flow channel base is provided with two flow channels, and the first piezoelectric ceramic and the second piezoelectric ceramic are in contact with the bottom surface of the flow channel base and are respectively positioned below the two flow channels and used for alternately generating the dual-frequency standing wave sound field; the liquid outlet end of the first flow passage extends from the three-branch position to two sides in a branching manner to form a first liquid outlet and a second liquid outlet which are not equal in width, the liquid outlet end of the second flow passage extends from the three-branch position to two sides in a branching manner to form a third liquid outlet and a fourth liquid outlet which are not equal in width, and the first liquid outlet and the fourth liquid inlet are communicated in a smooth transition manner; due to the adoption of two cascaded channels and the combination of the alternate action of a dual-frequency standing wave sound field, the precision and the accuracy of sound field separation are improved, the activity of cells is effectively ensured, and the separated cells have the advantages of large flux, smooth flow, high sensitivity, strong integratability, good separation effect and high separation efficiency.
Description
Technical Field
The invention relates to the field of separation devices and separation methods for circulating tumor cells and micro-plugs thereof, in particular to a device and a method for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave sound field.
Background
Circulating tumor cells refer to tumor cells which diffuse from tumor lesions into peripheral blood circulation and can develop into tumor metastatic lesions under certain conditions; circulating tumor cell micro-plugs (clusters) are formed by gathering two or more circulating tumor cells, more than 50% of tumor metastasis is caused by the circulating tumor cell micro-plugs, and the proportion in breast cancer is as high as 97%; therefore, a method for quickly and effectively separating the circulating tumor cells and the micro-thrombus thereof is developed, and an important basis is provided for developing monitoring equipment with clinical significance.
The diameter of the circulating tumor cells related to the method is generally more than or equal to 10 micrometers, and the diameter of the circulating tumor cell micro-plugs is about tens of micrometers; the current methods for separating circulating tumor cells and micro-suppositories thereof can be roughly divided into two types of passive screening and active screening:
1) passive screening protocol: the method mainly separates based on the attributes of the size, density, surface markers and the like of cells, and common passive screening methods comprise a filtration method, a density gradient centrifugation method, a circulating tumor cell marker screening method and a screening method based on a microfluidic structure; however, the filtration method and the screening method based on the microfluidic structure usually generate a blocking phenomenon to cause cell damage, thereby affecting the subsequent culture, phenotype identification, toxicological analysis and the like of the separated circulating tumor cells and the micro-plugs; the separation efficiency of the density gradient centrifugation method is relatively low; while the circulating tumor cell marker screening method relies on the phenotype of the circulating tumor cells, and usually capture and isolation are performed based on EpCAM (epithelial cell antigen), the EpCAM expression of circulating tumor cells of different tumor sources is different, and the expression of the protein is dynamically changed, so that the possibility of false positive is generated.
2) Active screening scheme: the separation is mainly realized based on the action of external force (such as electric field force, sound field force and the like), and common methods comprise a dielectrophoresis method and a sound wave method; the dielectrophoresis method has an advantage in that cells of 1 μm or less can be separated, but the separation flux is inferior to that of the sonic method because the usable dielectric force is relatively small due to the field intensity limitation of the dielectric electric field; the acoustic wave method is to generate standing wave acoustic field in the micro-channel to act on the cell to be separated, and is more suitable for separating the particle and the cell with the diameter of several micrometers to dozens of micrometers compared with the dielectrophoresis method; the acoustic wave method is further divided into a surface acoustic wave method and a bulk acoustic wave method according to the difference between the mode of generating the acoustic wave and the mode of propagating the acoustic wave: the surface acoustic wave method is convenient for designing a sound field, but has smaller intensity relative to the sound field; the sound field intensity of the bulk acoustic wave method is high, but the common bulk acoustic wave method only has a single sound node, so that the separation capability of the bulk acoustic wave method is limited.
Regardless of a passive screening scheme and an active screening scheme, the separation process of the existing separation device and method is not realized in the original environment where the circulating tumor cells are located, the activity of the cells is difficult to effectively ensure, and the subsequent detection of the metabolic activity of the cells, the detection of multiple protein markers, the culture, the toxicological research and the like are guaranteed.
Therefore, there is still a need for improvement and development of the prior art.
Disclosure of Invention
In order to solve the technical problems, the invention provides a device for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave sound field, which can realize separation in the original environment where the circulating tumor cells are located, can effectively ensure the integrity and activity of the cells, and has the advantages of large cell flux, smooth flow and strong integratability.
Meanwhile, the invention also provides a method for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field, which can realize separation in the original environment where the circulating tumor cells are located, can effectively ensure the integrity and activity of the cells, and has the advantages of high sensitivity, good separation effect and high separation efficiency.
The technical scheme of the invention is as follows: a device for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave sound field comprises a core part, a flow channel base, first piezoelectric ceramics and second piezoelectric ceramics; the flow channel base is provided with a first flow channel and a second flow channel, the first piezoelectric ceramic and the second piezoelectric ceramic are both contacted with the bottom surface of the flow channel base, the first piezoelectric ceramic is positioned below the first flow channel, and the second piezoelectric ceramic is positioned below the second flow channel; the liquid inlet end of the first flow channel branches from the middle line to two sides to form a first liquid inlet and a second liquid inlet which are equal in width, the liquid outlet end of the first flow channel branches from the three-line to two sides to form a first liquid outlet and a second liquid outlet which are not equal in width, and the width of the first liquid outlet is twice that of the second liquid outlet; the liquid inlet end of the second flow channel branches from the middle line to two sides to form a third liquid inlet and a fourth liquid inlet which are equal in width, the liquid outlet end of the second flow channel branches from the three-line to two sides to form a third liquid outlet and a fourth liquid outlet which are not equal in width, and the width of the third liquid outlet is twice that of the fourth liquid outlet; and the first liquid outlet is communicated with the fourth liquid inlet in a smooth transition mode.
The device for separating the circulating tumor cells and the micro-plugs thereof by the dual-frequency standing wave sound field comprises: the first flow channel and the second flow channel are both linear, the cross sections of the first flow channel and the second flow channel are both rectangular, and the width and the depth of the first flow channel are the same as those of the second flow channel.
The device for separating the circulating tumor cells and the micro-plugs thereof by the dual-frequency standing wave sound field comprises: the first flow channel and the second flow channel are parallel and arranged in a staggered mode, and the lower side face of the second flow channel and the upper side face of the first flow channel are located on the same straight line.
The device for separating the circulating tumor cells and the micro-plugs thereof by the dual-frequency standing wave sound field comprises: the first liquid inlet and the second liquid inlet are both linear and form a Y shape with the first flow channel, and the included angle between the first liquid inlet and the second liquid inlet is less than 45 degrees; the first liquid outlet and the second liquid outlet are also in a linear type and form a Y shape with the first flow channel, and an included angle between the central axis of the first liquid outlet and the second liquid outlet is equal to an included angle between the first liquid inlet and the second liquid inlet.
The device for separating the circulating tumor cells and the micro-plugs thereof by the dual-frequency standing wave sound field comprises: the third liquid inlet and the fourth liquid inlet are linear and form a Y shape with the second flow channel, and the included angle between the third liquid inlet and the fourth liquid inlet is less than 45 degrees; the third liquid outlet and the fourth liquid outlet are also in a linear type and form a Y shape with the second flow channel, and the included angle between the third liquid outlet and the fourth liquid outlet is equal to the included angle between the central axes of the third liquid inlet and the fourth liquid inlet.
The device for separating the circulating tumor cells and the micro-plugs thereof by the dual-frequency standing wave sound field comprises: the runner base is made of silicon-based or silicon oxide materials into a sheet shape, and a groove with a rectangular or trapezoidal cross section is made on the upper surface of the runner base as a first runner and a second runner by adopting a plasma etching process; the runner base also comprises a glass cover plate which is covered on the upper surface and made of heat-resistant glass, and the glass cover plate is tightly bonded with the runner base in a thermal bonding mode; through holes are respectively processed at positions on the glass cover plate corresponding to the first liquid inlet, the second liquid outlet, the third liquid inlet, the third liquid outlet and the fourth liquid outlet to serve as inlet and outlet holes of fluid, and the glass cover plate is connected with a liquid collecting test tube or an injector through a microflow hose.
A method for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave sound field is implemented on the device for separating circulating tumor cells and micro-plugs thereof by using the dual-frequency standing wave sound field, and comprises the following steps:
A. injecting a first solution without cells from a first liquid inlet into a first flow channel through a corresponding microfluidic hose by a first injector; injecting a liquid sample containing blood cells, circulating tumor cells and a micro-plug thereof into the first flow channel from the second liquid inlet through the corresponding micro-flow hose by a second injector;
B. adjusting the injection speed or the injection amount of the first injector and the second injector respectively so that the boundary of the first solution and the liquid sample is positioned at a third line of the first flow passage close to the second liquid outlet side;
C. applying a working frequency of 1MHz to the first piezoelectric ceramic, and generating a standing wave sound field at an 1/2 standing wave node line of the first flow channel, so that the circulating tumor cells and the micro-plugs thereof move to the central line of the first flow channel and cross a boundary line of the liquid sample to enter the first solution, while the blood cells still slowly move in the liquid sample;
D. converting the working frequency of the first piezoelectric ceramic to 3MHz, and generating a standing wave sound field at lines 1/6, 1/2 and 5/6 in the Y direction of the first flow channel, so that the circulating tumor cells and the micro-plugs thereof continue to move to the central line of the first flow channel in the first solution, and the blood cells move to the 1/6 standing wave node line of the first flow channel in the liquid sample;
E. and repeatedly changing the working frequency of the first piezoelectric ceramic, so that the circulating tumor cells and the micro-plugs thereof in the liquid sample are always gathered near the central line of the first flow channel to move, enter the second flow channel along with the first solution from the first liquid outlet through the fourth liquid inlet, and the blood cells are gathered near the 1/6 standing wave node line of the first flow channel to move, and flow out to the blood cell liquid collecting test tube along with the liquid sample from the second liquid outlet through the corresponding micro-flow hose.
The method for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field is characterized in that the step A further comprises the following steps: and injecting a second solution without cells into the second flow channel from the third liquid inlet through the corresponding microfluidic hose by a third injector.
The method for separating the circulating tumor cells and the micro-plugs thereof by the dual-frequency standing wave sound field is characterized in that the step B further comprises the following steps: the injection speed or the injection amount of the third syringe is adjusted so that the boundary between the second solution and the first solution is located at a third line on the fourth liquid outlet side of the second flow path.
The method for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field is characterized in that the method further comprises the following steps after the step E:
F. applying a working frequency of 1MHz to the second piezoelectric ceramic, and generating a standing wave sound field at an 1/2 standing wave node line of the second flow channel, so that the circulating tumor cell micro-plug moves to the central line of the second flow channel and enters the second solution by crossing the boundary line of the first solution, and the circulating tumor cell still slowly moves in the first solution;
G. converting the working frequency of the second piezoelectric ceramic to 3MHz, and generating a standing wave sound field at 1/6, 1/2 and 5/6 lines in the Y direction of the second flow channel, so that the circulating tumor cell micro-plugs continue to move to the central line of the second flow channel in the second solution, and the circulating tumor cells move to the 1/6 standing wave node line of the second flow channel in the first solution;
H. and repeatedly changing the working frequency of the second piezoelectric ceramic to ensure that the circulating tumor cell micro-plugs in the second solution always gather near the central line of the second flow channel to move and flow out to the circulating tumor cell micro-plug liquid collection test tube from the third liquid outlet through the corresponding micro-flow hose along with the second solution, and the circulating tumor cells gather near the 1/6 standing wave node line of the second flow channel to move and flow out to the circulating tumor cell liquid collection test tube from the fourth liquid outlet through the corresponding micro-flow hose along with the first solution.
The device and the method for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field have the advantages that the device and the method for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field have high sensitivity, improve the precision and the accuracy of sound field separation, realize the integrated online separation of the circulating tumor cells and the micro-plugs, effectively avoid the destructive effect of a multi-step separation method on the cells, and also avoid the complicated operation procedures of offline monitoring of an offline separation method (such as a centrifugal method and the like) and the separation uncertainty introduced by the operation procedures; the acting force generated by the standing wave sound field is a non-contact force, so that the damage of the contact force to the cells is effectively avoided, the separation process is realized in the original environment where the circulating tumor cells are located, the integrity and the activity of the separated cells are effectively ensured, and the subsequent cell metabolic activity detection, multiple protein marker detection, culture, toxicological research and the like are guaranteed; compared with other active methods, the alternating dual-frequency standing wave sound field has relatively large acting force, large separated cell flux and smooth flow, avoids the problem of passive cell blockage, has strong integratability, can be integrated with other phenotype identification and metabolic activity detection, has relatively large acting force of the standing wave sound field, good separation effect and high separation efficiency, and can be expanded to be used for the online separation of other cells.
Drawings
FIG. 1 is a schematic diagram of the operation of the core part of the embodiment of the present invention for separating circulating tumor cells and micro-plugs thereof using dual-frequency standing wave sound field;
FIG. 2 is an enlarged perspective view of the core part of the apparatus for separating circulating tumor cells and their micro-plugs by using dual-frequency standing wave sound field according to the present invention.
Detailed Description
The embodiments and examples of the present invention will be described in detail below with reference to the accompanying drawings, and the described embodiments are only for the purpose of illustrating the present invention and are not intended to limit the embodiments of the present invention.
As shown in fig. 1, fig. 1 is a schematic diagram of the operation of the core part of the device for separating circulating tumor cells and their micro-plugs by using dual-frequency standing wave acoustic field according to the present invention, wherein the core part of the separation device is composed of a flow channel base (not shown), a first piezoelectric ceramic 310 and a second piezoelectric ceramic 320;
the runner base is provided with a first runner 100 for matching with first piezoelectric ceramics 310 to separate circulating tumor cells CTC and micro-plugs CTCs thereof and a second runner 200 for matching with second piezoelectric ceramics 320 to separate circulating tumor cell micro-plugs CTCs;
the first piezoelectric ceramics 310 and the second piezoelectric ceramics 320 are both contacted with the bottom surface of the flow channel base, the first piezoelectric ceramics 310 is positioned below the first flow channel 100 and used for alternately generating a dual-frequency standing wave sound field in the first flow channel 100 to separate circulating tumor cells CTC and micro-plugs CTCs thereof, and the second piezoelectric ceramics 320 is positioned below the second flow channel 200 and used for alternately generating a dual-frequency standing wave sound field in the second flow channel 200 to separate circulating tumor cells micro-plugs CTCs;
the liquid inlet end of the first flow channel 100 is divergently extended from the central line to two sides to form a first liquid inlet 101 and a second liquid inlet 102 with equal width, the first liquid inlet 101 is used for inputting a liquid sample containing blood cells BC, circulating tumor cells CTC and micro-thrombus CTCs thereof in a laminar flow manner, and the second liquid inlet 102 is used for inputting a first solution (such as phosphate buffer solution) without cells in a laminar flow manner; the liquid outlet end of the first flow channel 100 is bifurcated from a three-line position to two sides to form a first liquid outlet 103 and a second liquid outlet 104 which are not equal in width, the width of the first liquid outlet 103 is twice that of the second liquid outlet 104, the first liquid outlet 103 is used for allowing separated circulating tumor cells CTCs and micro-plugs CTCs thereof to flow out with the first solution in a laminar flow manner, and the second liquid outlet 104 is used for allowing a separated liquid sample (blood cells BC including red blood cells, white blood cells and the like) to flow out in a laminar flow manner;
the liquid inlet end of the second flow channel 200 is branched and extended from the central line to two sides to form a third liquid inlet 201 and a fourth liquid inlet 202 with equal width, the third liquid inlet 201 is used for inputting a second solution (such as phosphate buffer solution) without cells in a laminar flow manner, the fourth liquid inlet 202 is communicated with the first liquid outlet 103, and the side wall and the bottom wall between the third liquid inlet and the fourth liquid outlet are in smooth transition, so that separated circulating tumor cells CTC and micro-plugs CTCs thereof flow in the laminar flow manner along with the first solution; the liquid outlet end of the second flow channel 200 branches from the three-branch line to two sides to form a third liquid outlet 203 and a fourth liquid outlet 204 which are not equal in width, the width of the third liquid outlet 203 is twice the width of the fourth liquid outlet 204, the third liquid outlet 203 is used for separated circulating tumor cell micro-plugs CTCs to flow out with the second solution in a laminar flow manner, and the fourth liquid outlet 204 is used for separated circulating tumor cell CTCs to flow out with the first solution in a laminar flow manner.
The laminar flow form refers to a flow mode of liquid flowing in the first flow channel 100 and the second flow channel 200 under a low Reynolds number, and the Reynolds number (Reynolds number) is a dimensionless number which can be used for representing the flow condition of the fluid, is a similarity criterion number for characterizing viscous influence in fluid mechanics, and is a basis for judging flow characteristics, when the Reynolds number is small, the influence of viscous force on a flow field is greater than inertia, disturbance of flow velocity in the flow field is attenuated due to the viscous force, and the fluid flow is stable to form a laminar flow; on the contrary, if the reynolds number is larger, the influence of inertia on the flow field is larger than the viscous force, the fluid flow is unstable, the small change of the flow velocity is easy to develop and strengthen, and a turbulent flow or turbulent flow field is formed; in the pipe flow, the flow with the Reynolds number smaller than 2300 is laminar flow, the flow with the Reynolds number equal to 2300-4000 is in a transition state, and the flow with the Reynolds number larger than 4000 is turbulent flow.
Preferably, the first flow channel 100 and the second flow channel 200 are both linear, have rectangular cross sections, and the width and depth of the first flow channel 100 are the same as the width and depth of the second flow channel 200.
Preferably, the first flow channel 100 and the second flow channel 200 are parallel and staggered, and the lower side of the second flow channel 200 is located on the same straight line with the upper side of the first flow channel 100.
Preferably, the first liquid inlet 101 and the second liquid inlet 102 are both linear, and form a Y shape with the first flow channel 100, and the included angle between the first liquid inlet 101 and the second liquid inlet 102 is less than 45 degrees; the first liquid outlet 103 and the second liquid outlet 104 are also linear, and form a Y shape with the first flow channel 100, and the included angle between the central axis of the first liquid outlet 103 and the second liquid outlet 104 is equal to the included angle between the first liquid inlet 101 and the second liquid inlet 102.
Preferably, the third liquid inlet 201 and the fourth liquid inlet 202 are both linear and form a Y shape with the second flow channel 200, and an included angle between the third liquid inlet 201 and the fourth liquid inlet 202 is less than 45 °; the third liquid outlet 203 and the fourth liquid outlet 204 are also linear, and form a Y shape with the second flow channel 200, and the included angle between the third liquid outlet 203 and the fourth liquid outlet 204 is equal to the included angle between the central axes of the third liquid inlet 201 and the fourth liquid inlet 202.
In the preferred embodiment of the apparatus for separating circulating tumor cells and micro plugs thereof using dual-frequency standing wave sound field of the present invention, referring to fig. 2, fig. 2 is an enlarged perspective view of the core part of the apparatus for separating circulating tumor cells and micro plugs thereof using dual-frequency standing wave sound field of the present invention, specifically, the flow channel base 410 may be made of silicon-based, silicon oxide or other hard metal or non-metal solid material into a sheet shape with a thickness of 500 microns, and a plasma etching process is used to form a groove with a rectangular or trapezoidal cross section on the upper surface of the flow channel base 410 as the first flow channel 100 and the second flow channel 200, for example, a rectangular groove with a width of 750 microns and a depth of 50 microns is etched;
in order to facilitate the camera to clearly record the flow state of the circulating tumor cells CTCs and the micro-plugs thereof CTCs in the first flow channel 100 and the second flow channel 200 through a microscope, the flow channel base 410 further comprises a glass cover plate 420 covering the upper surface of the flow channel base, and the glass cover plate 420 can be made of heat-resistant glass materials into a sheet shape with the thickness of 1 mm and is tightly bonded with the flow channel base 410 through thermal bonding; through holes (421, 422, 423, 424, 425 and 426) with a diameter of 700 micrometers are respectively processed on the glass cover plate 420 at positions corresponding to the first liquid inlet 101, the second liquid inlet 102, the second liquid outlet 104, the third liquid inlet 201, the third liquid outlet 203 and the fourth liquid outlet 204 as fluid inlets and outlets, and are connected with a liquid collecting test tube (not shown) or a syringe (not shown) through a micro-fluid hose (not shown) with an inner diameter of 500 micrometers;
the first piezoelectric ceramic 310 and the second piezoelectric ceramic 320 may both be PNT-5 type piezoelectric ceramic plates that apply voltage in the thickness direction Z and generate vibration in the thickness direction Z (i.e., Z polarization in the thickness direction), and the width of the first piezoelectric ceramic 310 is adapted to the length of the first flow channel 100, and the width of the second piezoelectric ceramic 320 is adapted to the length of the second flow channel 200; two planes perpendicular to the epsilon-33 direction on the first piezoelectric ceramic 310 and the second piezoelectric ceramic 320 are used as electrode surfaces, metal silver coatings are plated on the two planes to be used as driving electrodes, and one of the two planes is adhered to the bottom surface of the flow channel base 410 by alpha-cyanoacrylate glue; the first piezoelectric ceramic 310 and the second piezoelectric ceramic 320 are respectively electrically connected to a function signal generator (not shown), each function signal generator generates a sinusoidally varying alternating voltage signal as a driving signal, the alternating voltage signals can be converted back and forth between working frequencies of 1MHz and 3MHz as required, and the first piezoelectric ceramic 310 and the second piezoelectric ceramic 320 are driven to work after passing through a power amplification device (not shown).
Based on the device for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field, the invention also provides a method for separating the circulating tumor cells and the micro-plugs thereof by using the dual-frequency standing wave sound field, which specifically comprises the following steps:
step S510, injecting a first solution without cells from a first liquid inlet 101 at a via hole 421 into a first flow channel 100 through a first injector via a corresponding micro-flow hose; injecting a liquid sample containing blood cells BC, circulating tumor cells CTC and micro-plugs CTCs thereof into the first flow channel 100 from a second liquid inlet 102 at the through hole 422 through a corresponding micro-fluid hose by a second syringe; injecting a second solution without cells into the second flow channel 200 from a third liquid inlet 201 at the via hole 423 through a third syringe via a corresponding microfluidic hose;
step S520, adjusting the injection speed or the injection amount of the first syringe and the second syringe respectively, so that the boundary 110 (fig. 1) between the first solution and the liquid sample is located at a third line of the first flow channel 100 on the second liquid outlet 104 side, that is, the liquid sample completely flows out from the second liquid outlet 104 at the through hole 424 to the blood cell BC liquid collection tube through the corresponding microfluidic tube, and the first solution completely flows into the second flow channel 200 from the first liquid outlet 103 through the fourth liquid inlet 202; in particular, for example, the first injector has an injection speed of 0.84mm3/s or injection quantity of 100μl/hThe injection speed of the second injector is 0.28mm3/s or injection quantity of 20μl/h(ii) a At the same time, the injection speed or injection amount of the third syringe is adjusted so that the boundary 210 (fig. 1) between the second solution and the first solution is located in the third part of the second flow channel 200 near the fourth outlet 204At the line (because the flow rates of the second solution and the first solution are different, even if the two solutions use the same liquid, a boundary line is generated between the two solutions), that is, the first solution all flows out from the fourth liquid outlet 204 at the through hole 426 to the circulating tumor cell CTCs liquid collection tube through the corresponding microfluidic tube, and the second solution all flows out from the third liquid outlet 203 at the through hole 425 to the circulating tumor cell microembolus CTCs liquid collection tube through the corresponding microfluidic tube; in particular, for example, the injection speed of the third injector is 1.5mm3/s or injection quantity of 160μl/h；
Step S530, applying a working frequency of 1MHz to the first piezoelectric ceramic 310 by using a function signal generator, generating a standing wave sound field at 1/2 standing wave node line 120 (i.e. a central line) of the first flow channel 100 in fig. 1, wherein due to the difference of physical characteristics such as cell volume and weight, the volume of the circulating tumor cell CTC and its microembolus CTCs is significantly larger than the volume of blood cells BC such as red blood cells and white blood cells, so that the circulating tumor cell CTC and its microembolus CTCs move significantly faster than the blood cells BC under the standing wave sound field of 1MHz, the circulating tumor cell CTC and its microembolus CTCs move to 1/2 standing wave node line 120 (i.e. a central line) of the first flow channel 100 and cross the boundary line 110 of the liquid sample to enter the first solution, and the blood cells BC still move slowly in the liquid sample;
step S540, transforming the operating frequency of the first piezoelectric ceramic 310 to 3MHz by using the function signal generator, and generating a standing wave sound field at lines 1/6, 1/2, and 5/6 in the Y direction of the first flow channel 100, wherein the volume of the circulating tumor cells CTCs and their micro-plugs CTCs is significantly larger than the volume of blood cells BC such as red blood cells and white blood cells due to the difference in physical characteristics such as cell volume and weight, so that the circulating tumor cells CTCs and their micro-plugs CTCs continue to move to the 1/2 standing wave node line 120 (i.e., the central line) of the first flow channel 100 in the first solution, and the blood cells BC move to the 1/6 standing wave node line 130 of the first flow channel 100 of fig. 1 in the liquid sample;
step S550, the function signal generator repeatedly converts the working frequency of the first piezoelectric ceramic 310 between 3MHz and 1MHz, specifically, for example, the time interval for the function signal generator to convert the working frequency is 5-10S; finally, the circulating tumor cells CTC and their micro-plugs CTCs in the liquid sample always collect near the 1/2 standing wave node line 120 (i.e. the central line) of the first flow channel 100 in fig. 1 and move with the first solution from the first liquid outlet 103 through the fourth liquid inlet 202 into the second flow channel 200, while the blood cells BC collect near the 1/6 standing wave node line 130 of the first flow channel 100 in fig. 1 and move with the liquid sample from the second liquid outlet 104 at the via hole 424 out through the corresponding micro-flow hose to the blood cell BC liquid collection tube, thereby completing the separation of the circulating tumor cells CTC and their micro-plugs CTCs mixture from the liquid sample;
step S560, applying a working frequency of 1MHz to the second piezoelectric ceramic 320 by using the function signal generator, generating a standing wave sound field at 1/2 standing wave node line 220 (i.e. central line) of the second flow channel 200 in fig. 1, wherein the volume of the circulating tumor cell micro-plugs CTCs is significantly larger than the volume of the circulating tumor cell CTCs due to the difference of physical characteristics such as cell volume and weight, so that the circulating tumor cell micro-plugs CTCs move significantly faster than the circulating tumor cell CTCs under the 1MHz standing wave sound field, the circulating tumor cell micro-plugs CTCs move to 1/2 standing wave node line 220 (i.e. central line) of the second flow channel 200 and cross the boundary 210 of the first solution to enter the second solution, and the circulating tumor cell CTCs still move slowly in the first solution;
step S570, transforming the operating frequency of the second piezoelectric ceramic 320 to 3MHz by using the function signal generator, generating a standing wave sound field at lines 1/6, 1/2, and 5/6 in the Y direction of the second flow channel 200, wherein the volume of the circulating tumor cell microemboli CTCs is significantly larger than that of the circulating tumor cell CTCs due to the difference in physical properties such as cell volume, weight, and the like, so that the circulating tumor cell microemboli CTCs continue to move to the node line 1/2 (i.e., the central line) 220 of the second flow channel 200 in the second solution, and the circulating tumor cell CTCs move to the node line 1/6 of the second flow channel 200 in fig. 1 in the first solution;
step S580, the function signal generator repeatedly converts the operating frequency of the second piezoelectric ceramic 320 between 3MHz and 1MHz, specifically, for example, the time interval for the function signal generator to convert the operating frequency is 5 to 10S; finally, the circulating tumor cell micro-plugs CTCs in the second solution always gather near the 1/2 standing wave node line 220 (i.e., the center line) in the second flow channel 200 of fig. 1 and move with the second solution flowing out from the third liquid outlet 203 at the through hole 425 to the circulating tumor cell micro-plug CTCs liquid collection tube through the corresponding microfluidic tube, while the circulating tumor cell CTCs gather near the 1/6 standing wave node line 230 in the second flow channel 200 of fig. 1 and move with the first solution flowing out from the fourth liquid outlet 204 at the through hole 426 to the circulating tumor cell CTCs liquid collection tube through the corresponding microfluidic tube, thereby completing the separation of the circulating tumor cell CTCs from the liquid sample and the separation of the circulating tumor cell micro-plugs CTCs.
It should be understood that the above-mentioned embodiments are merely preferred examples of the present invention, and not restrictive, but rather, all the changes, substitutions, alterations and modifications that come within the spirit and scope of the invention as described above may be made by those skilled in the art, and all the changes, substitutions, alterations and modifications that fall within the scope of the appended claims should be construed as being included in the present invention.
Claims (10)
1. A device for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave sound field is characterized in that: the core part of the piezoelectric ceramic flow channel consists of a flow channel base, first piezoelectric ceramics and second piezoelectric ceramics; the flow channel base is provided with a first flow channel matched with a first piezoelectric ceramic to separate circulating tumor cells CTC and micro-plugs CTCs thereof and a second flow channel matched with a second piezoelectric ceramic to separate circulating tumor cells micro-plugs CTCs, the first piezoelectric ceramic and the second piezoelectric ceramic are both contacted with the bottom surface of the flow channel base, the first piezoelectric ceramic is positioned below the first flow channel and used for alternately generating a dual-frequency standing wave sound field in the first flow channel so as to separate the circulating tumor cells CTC and the micro-plugs CTCs thereof, and the second piezoelectric ceramic is positioned below the second flow channel and used for alternately generating a dual-frequency standing wave sound field in the second flow channel so as to separate the circulating tumor cells micro-plugs CTCs;
the liquid inlet end of the first flow channel is branched from the middle line to two sides to form a first liquid inlet and a second liquid inlet with equal width, the first liquid inlet is used for inputting a liquid sample containing blood cells BC, circulating tumor cells CTC and micro-plugs CTCs thereof in a laminar flow mode, the second liquid inlet is used for inputting a first solution without cells in a laminar flow mode, the liquid outlet end of the first flow channel is branched from a three-line position to two sides to form a first liquid outlet and a second liquid outlet with unequal width, the width of the first liquid outlet is twice of that of the second liquid outlet, the first liquid outlet is used for enabling the separated circulating tumor cells CTC and micro-plugs CTCs thereof to flow out with the first solution in a laminar flow mode, and the second liquid outlet is used for enabling the separated liquid sample to flow out in a laminar flow mode;
the liquid inlet end of the second flow channel branches and extends from the middle line to two sides to form a third liquid inlet and a fourth liquid inlet which are equal in width, the third liquid inlet is used for inputting a second solution without cells in a laminar flow mode, the fourth liquid inlet is used for enabling separated circulating tumor cell CTCs and micro-plugs CTCs thereof to flow in the laminar flow mode along with the first solution, the liquid outlet end of the second flow channel branches and extends from the three-line part to two sides to form a third liquid outlet and a fourth liquid outlet which are not equal in width, the width of the third liquid outlet is twice of the width of the fourth liquid outlet, the third liquid outlet is used for enabling the separated circulating tumor cell micro-plugs CTCs to flow out in the laminar flow mode along with the second solution, and the fourth liquid outlet is used for enabling the separated circulating tumor cell CTCs to flow out in the laminar flow mode along with the first solution; and the first liquid outlet is communicated with the fourth liquid inlet in a smooth transition mode.
2. The device for separating circulating tumor cells and micro-plugs thereof according to claim 1, wherein: the first flow channel and the second flow channel are both linear, the cross sections of the first flow channel and the second flow channel are both rectangular, and the width and the depth of the first flow channel are the same as those of the second flow channel.
3. The device for separating circulating tumor cells and micro-plugs thereof according to claim 2, wherein: the first flow channel and the second flow channel are parallel and arranged in a staggered mode, and the lower side face of the second flow channel and the upper side face of the first flow channel are located on the same straight line.
4. The device for separating circulating tumor cells and micro-plugs thereof according to claim 1, wherein: the first liquid inlet and the second liquid inlet are both linear and form a Y shape with the first flow channel, and the included angle between the first liquid inlet and the second liquid inlet is less than 45 degrees; the first liquid outlet and the second liquid outlet are also in a linear type and form a Y shape with the first flow channel, and an included angle between the central axis of the first liquid outlet and the second liquid outlet is equal to an included angle between the first liquid inlet and the second liquid inlet.
5. The device for separating circulating tumor cells and micro-plugs thereof according to claim 1, wherein: the third liquid inlet and the fourth liquid inlet are linear and form a Y shape with the second flow channel, and the included angle between the third liquid inlet and the fourth liquid inlet is less than 45 degrees; the third liquid outlet and the fourth liquid outlet are also in a linear type and form a Y shape with the second flow channel, and the included angle between the third liquid outlet and the fourth liquid outlet is equal to the included angle between the central axes of the third liquid inlet and the fourth liquid inlet.
6. The device for separating circulating tumor cells and micro-plugs thereof according to claim 1, wherein: the runner base is made of silicon-based or silicon oxide materials into a sheet shape, and a groove with a rectangular or trapezoidal cross section is made on the upper surface of the runner base as a first runner and a second runner by adopting a plasma etching process; the runner base also comprises a glass cover plate which is covered on the upper surface and made of heat-resistant glass, and the glass cover plate is tightly bonded with the runner base in a thermal bonding mode; through holes are respectively processed at positions on the glass cover plate corresponding to the first liquid inlet, the second liquid outlet, the third liquid inlet, the third liquid outlet and the fourth liquid outlet to serve as inlet and outlet holes of fluid, and the glass cover plate is connected with a liquid collecting test tube or an injector through a microflow hose.
7. A method for separating circulating tumor cells and micro-plugs thereof by using a dual-frequency standing wave acoustic field, which is implemented on the device for separating circulating tumor cells and micro-plugs thereof by using the dual-frequency standing wave acoustic field as claimed in any one of claims 1 to 6, and is characterized in that the method comprises the following steps:
A. injecting a first solution without cells from a first liquid inlet into a first flow channel through a corresponding microfluidic hose by a first injector; injecting a liquid sample containing blood cells, circulating tumor cells and a micro-plug thereof into the first flow channel from the second liquid inlet through the corresponding micro-flow hose by a second injector;
B. adjusting the injection speed or the injection amount of the first injector and the second injector respectively so that the boundary of the first solution and the liquid sample is positioned at a third line of the first flow passage close to the second liquid outlet side;
C. applying a working frequency of 1MHz to the first piezoelectric ceramic, and generating a standing wave sound field at an 1/2 standing wave node line of the first flow channel, so that the circulating tumor cells and the micro-plugs thereof move to the central line of the first flow channel and cross a boundary line of the liquid sample to enter the first solution, while the blood cells still slowly move in the liquid sample;
D. converting the working frequency of the first piezoelectric ceramic to 3MHz, and generating a standing wave sound field at lines 1/6, 1/2 and 5/6 in the Y direction of the first flow channel, so that the circulating tumor cells and the micro-plugs thereof continue to move to the central line of the first flow channel in the first solution, and the blood cells move to the 1/6 standing wave node line of the first flow channel in the liquid sample;
E. and repeatedly changing the working frequency of the first piezoelectric ceramic, so that the circulating tumor cells and the micro-plugs thereof in the liquid sample are always gathered near the central line of the first flow channel to move, enter the second flow channel along with the first solution from the first liquid outlet through the fourth liquid inlet, and the blood cells are gathered near the 1/6 standing wave node line of the first flow channel to move, and flow out to the blood cell liquid collecting test tube along with the liquid sample from the second liquid outlet through the corresponding micro-flow hose.
8. The method for separating circulating tumor cells and micro-plugs thereof according to the dual-frequency standing wave acoustic field of claim 7, wherein the step A further comprises: and injecting a second solution without cells into the second flow channel from the third liquid inlet through the corresponding microfluidic hose by a third injector.
9. The method for separating circulating tumor cells and micro-plugs thereof according to claim 8, wherein the step B further comprises: the injection speed or the injection amount of the third syringe is adjusted so that the boundary between the second solution and the first solution is located at a third line on the fourth liquid outlet side of the second flow path.
10. The method for separating circulating tumor cells and micro-plugs thereof by using the dual-frequency standing wave acoustic field according to claim 9, wherein the step E is further followed by:
F. applying a working frequency of 1MHz to the second piezoelectric ceramic, and generating a standing wave sound field at an 1/2 standing wave node line of the second flow channel, so that the circulating tumor cell micro-plug moves to the central line of the second flow channel and enters the second solution by crossing the boundary line of the first solution, and the circulating tumor cell still slowly moves in the first solution;
G. converting the working frequency of the second piezoelectric ceramic to 3MHz, and generating a standing wave sound field at 1/6, 1/2 and 5/6 lines in the Y direction of the second flow channel, so that the circulating tumor cell micro-plugs continue to move to the central line of the second flow channel in the second solution, and the circulating tumor cells move to the 1/6 standing wave node line of the second flow channel in the first solution;
H. and repeatedly changing the working frequency of the second piezoelectric ceramic to ensure that the circulating tumor cell micro-plugs in the second solution always gather near the central line of the second flow channel to move and flow out to the circulating tumor cell micro-plug liquid collection test tube from the third liquid outlet through the corresponding micro-flow hose along with the second solution, and the circulating tumor cells gather near the 1/6 standing wave node line of the second flow channel to move and flow out to the circulating tumor cell liquid collection test tube from the fourth liquid outlet through the corresponding micro-flow hose along with the first solution.
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 CN110438000A (en) | 2019-11-12 |
| CN110438000B true 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) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115308301B (en) * | 2022-08-16 | 2023-03-10 | 中山大学 | Measuring device capable of measuring elastic modulus of cells and cell nucleuses |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0925494T3 (en) * | 1996-09-04 | 2002-07-01 | Scandinavian Micro Biodevices | Microfluidic system for particle separation and analysis |
-
2019
- 2019-07-24 CN CN201910668908.2A patent/CN110438000B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110438000A (en) | 2019-11-12 |
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 | |
| Yan et al. | Continuous microfluidic 3D focusing enabling microflow cytometry for single-cell analysis | |
| CN107505249B (en) | Microfluidic chip system for rare cell screening | |
| US8865003B2 (en) | Apparatus and method for separation of particles suspended in a liquid from the liquid in which they are suspended | |
| JP5018879B2 (en) | Component separation device | |
| US8425749B1 (en) | Microfabricated particle focusing device | |
| US10082455B2 (en) | Apparatus and method for microparticle separation based on microfluidic chromatography using surface acoustic wave | |
| Nawaz et al. | Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting” | |
| Lin et al. | Vertical focusing device utilizing dielectrophoretic force and its application on microflow cytometer | |
| Kim et al. | Highly efficient single cell arraying by integrating acoustophoretic cell pre-concentration and dielectrophoretic cell trapping | |
| Tajik et al. | Simple, cost-effective, and continuous 3D dielectrophoretic microchip for concentration and separation of bioparticles | |
| Mernier et al. | Characterization of a novel impedance cytometer design and its integration with lateral focusing by dielectrophoresis | |
| CN110314715B (en) | Particle enrichment microfluidic chip based on focusing surface acoustic wave and micro-droplet technology | |
| Huang et al. | Three-dimensional cellular focusing utilizing a combination of insulator-based and metallic dielectrophoresis | |
| CN110438000B (en) | Device and method for separating circulating tumor cells and micro-plugs thereof by using dual-frequency standing wave sound field | |
| Wang et al. | Dielectrophoresis microsystem with integrated flow cytometers for on‐line monitoring of sorting efficiency | |
| CN111154620A (en) | Micro-fluidic chip for magnetic quantity analysis carried by magnetic labeled cells | |
| CN109647557B (en) | Direct particle separation chip based on induced charge electroosmosis micro vortex and application and separation method thereof | |
| CN209034379U (en) | A kind of high-pass cell separation device based on nano-micrometre combination channel AC dielectrophoresis | |
| CN106807459A (en) | A kind of micro-fluidic chip and preparation method thereof, application | |
| CN111735853A (en) | Integrated pre-sorting cell mechanical and electrical multi-parameter joint detection device | |
| CN114164104B (en) | Single-cell non-Newton droplet packaging device and method based on microfluidic chip | |
| Yousuff et al. | Acoustic Based Separation of Blood Cells Using Microfluidic Device | |
| Dougherty et al. | Ultrasonic particle manipulation in microchannels using phased co-planar transducers | |
| Peng et al. | Investigation on submicron particle separation and deflection using tilted-angle standing surface acoustic wave microfluidics |
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 |