WO2019165770A1 - 颗粒物检测装置及检测方法 - Google Patents

颗粒物检测装置及检测方法 Download PDF

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WO2019165770A1
WO2019165770A1 PCT/CN2018/103171 CN2018103171W WO2019165770A1 WO 2019165770 A1 WO2019165770 A1 WO 2019165770A1 CN 2018103171 W CN2018103171 W CN 2018103171W WO 2019165770 A1 WO2019165770 A1 WO 2019165770A1
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Prior art keywords
electrical impedance
dielectrophoretic
main channel
microfluidic chip
electrode
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PCT/CN2018/103171
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English (en)
French (fr)
Inventor
李珍仪
陈昭宏
王竣宏
陆祎
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南京怡天生物科技有限公司
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Priority to US16/617,634 priority Critical patent/US20200086320A1/en
Publication of WO2019165770A1 publication Critical patent/WO2019165770A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0656—Investigating concentration of particle suspensions using electric, e.g. electrostatic methods or magnetic methods
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502776—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for focusing or laminating flows
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1023—Microstructural devices for non-optical measurement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the invention relates to a device and a method for detecting particulate matter by using dielectrophoresis and electrical impedance measurement, in particular to an electrical impedance measurement microfluidic chip enhanced by dielectrophoresis and a detection method using the same.
  • the inventors have unexpectedly discovered that if a dielectrophoretic generating device is added to an electrical impedance measuring device, the dielectric field exerts different effects on different cells at a specific frequency window, for example, for a target cell.
  • a dielectrophoretic generating device such as CTC
  • CTC produces positive dielectric power to move to the surface of the DEP electrode; at the same time, it produces negative dielectric power to other blood cells, so that it is repelled by the DEP electrode away from the electrode surface, so that two or more of the flow channels can be originally mixed together.
  • Cells are arranged at different vertical heights depending on the cell type and/or characteristics.
  • the inventors completed the present invention.
  • the present invention provides a microfluidic chip for separating and detecting particulate matter, comprising an injection portion, a main channel (3), a dielectrophoretic electric field generating portion, and an electrical impedance detecting portion.
  • the injection portion comprises one or more sample channels (1), and optionally one or more sheath fluid channels (2), such as 1, 2 or 3 sample channels And 1, 2, 3, 4, 5 or 6 sheath fluid channels, more for example 1 sample channel and 2 sheath fluid channels, said 1 or more sample channels (1) and optionally said 1 One or more sheath liquid channels (2) meet at one end and are connected to the main channel (3), so that the sample stream and optionally the sheath liquid flow are concentrated and injected into the main channel (3);
  • sheath fluid channels (2) such as 1, 2 or 3 sample channels And 1, 2, 3, 4, 5 or 6 sheath fluid channels, more for example 1 sample channel and 2 sheath fluid channels, said 1 or more sample channels (1) and optionally said 1
  • sheath liquid channels (2) meet at one end and are connected to the main channel (3), so that the sample stream and optionally the sheath liquid flow are concentrated and injected into the main channel (3);
  • the main channel (3) is sequentially provided with a dielectrophoretic electric field generating portion and an electrical impedance detecting portion;
  • the dielectrophoretic electric field generating portion includes a dielectrophoretic generating electrode (4) disposed at an upper portion or a lower portion of the main channel (3), and the dielectrophoretic generating electrode is configured to generate an electric field that is uneven in a direction perpendicular to the main channel, thereby Performing dielectrophoresis of particles in the flow of the main channel (3) in a direction perpendicular to the main channel;
  • the electrical impedance detecting portion includes an electrical impedance detecting electrode (5) provided at an upper portion or a lower portion of the main passage (3), capable of generating an electrical impedance detecting signal in response to particles flowing through the main passage (3).
  • the microfluidic chip comprises one or more of the dielectrophoretic electric field generating portions, for example 1, 2, 3, 4 or 5; in particular when comprising 2 or more
  • the dielectrophoretic electric field generating portion it is preferable that the dielectrophoretic generating electrodes are respectively provided at an upper portion and a lower portion of the main channel.
  • At least one of said dielectrophoretic generating electrodes (4) is disposed in a lower portion of the main channel (3), such as on a substrate of the chip.
  • At least one of said dielectrophoretic generating electrodes (4) is disposed on an upper portion of the main channel (3), for example, on a cover sheet overlying a substrate of the microfluidic chip.
  • the electrical impedance detecting electrode (5) is disposed at a lower portion of the main channel (3), preferably on a substrate of the chip.
  • the electrical impedance detecting electrode (5) is disposed on the upper portion of the main channel (3), preferably on a cover sheet overlying the substrate of the microfluidic chip.
  • the dielectrophoretic generating electrode (4) is a conductive metal electrode or a photovoltaic material having a photoconductive effect, such as amorphous silicon or zinc oxide (ZnO), etc., which may be locally Illumination produces a non-uniform electric field which in turn produces dielectrophoretic forces.
  • a photoconductive effect such as amorphous silicon or zinc oxide (ZnO), etc.
  • the particulate matter is a cell of interest, such as a tumor cell, especially a circulating tumor cell.
  • the invention provides an electrical impedance detecting device for separating and detecting particulate matter, comprising the microfluidic chip of any of the foregoing first aspects.
  • a dielectrophoretic electric field signal generator coupled to the dielectrophoretic generating electrode (4), and/or an electrical impedance signal receiver coupled to the electrical impedance detecting portion, preferably further comprising an injection portion Connected applicator and/or electrical impedance signal analyzer connected to the electrical impedance signal receiver.
  • the present invention also provides a method of separating and detecting particulate matter, which uses the microfluidic chip described in the first aspect and/or the electrical impedance detecting device described in the second aspect.
  • the method includes
  • the sample is derived from whole blood, plasma, urine, tissue fluid a cerebrospinal fluid, a cell culture fluid or a mixture of cells; specifically, the sheath fluid is a PBS solution of sucrose, preferably, the sheath fluid comprises 280 mM sucrose, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 and 1.47 mM KH 2 PO 4 , pH 7.4; preferably, the sheath liquid has an electric conductivity of 10 to 500 mS/m, 30 to 300 mS/m or 50 to 100 mS/m, and more preferably, the conductivity of the sheath liquid It is 50mS/m.
  • the target particulate matter is a tumor cell, preferably a circulating tumor cell;
  • the non-target cell is a white blood cell, preferably a peripheral blood mononuclear cell (PBMC) or a macrophage, and/or a red blood cell; with
  • PBMC peripheral blood mononuclear cell
  • the target particulate matter and the non-target particulate matter are subjected to an opposite dielectric power in a particular separation frequency window.
  • the present invention provides the use of the microfluidic chip of the first aspect and the electrical impedance detecting device of the second aspect, in particular for detecting tumor cells, in particular said tumor
  • the cells are circulating tumor cells.
  • the present invention provides the use of the microfluidic chip of the first aspect and the electrical impedance detecting apparatus of the second aspect for preparing a medical device for detecting tumor cells, in particular the tumor Cells are circulating tumor cells.
  • the present invention can at least provide advantages and benefits of at least one of the following aspects.
  • the present invention selectively treats a target cell by a dielectrophoretic generating electrode (4), and sensitively and accurately targets a target cell in a sample (especially an isolated blood cell sample or a PBMC sample) without using a label and an antibody (for example) Tumor cells, circulating tumor cells) are detected or counted.
  • a sample especially an isolated blood cell sample or a PBMC sample
  • an antibody for example
  • FIG. 1 is a schematic view showing the structure of a microfluidic chip according to an embodiment of the present invention, wherein Fig. 1(a) is a plan view and Fig. 1(b) is a side view.
  • Figure 2 is a motion trajectory diagram (side view) of PBMC and A549 cells in the main channel (3).
  • the left picture is an electron micrograph, and the right picture is a schematic diagram.
  • the left circle shows the PBMC cells. It is away from the lower electrode under the action of negative dielectric power (nDEP), and the circle with a triangular arrow indicates A549 cells, which are close to the lower electrode under the action of positive dielectric power (pDEP).
  • nDEP negative dielectric power
  • pDEP positive dielectric power
  • Figure 3 is a diagram showing the electrical impedance signals of A549 cell samples, wherein Figure 3(a) shows the electrical impedance signal without focusing by dielectrophoresis cells, while Figure 3(b) shows the focus after dielectrophoresis. Electrical impedance signal diagram.
  • Figure 4 (a) shows the results of electrical impedance measurements obtained when stratified with or without dielectrophoresis using 1.5 mL of PBMC samples.
  • 4(b) is a result of counting A549 cells after stratification by dielectrophoresis in a method in which a sample of PBMC mixed with a different amount of A549 cells is detected according to the method of Example 5 of the present disclosure.
  • Example 4(c) is a result of counting A549 cells when the sample mixed with different numbers of A549 cells is detected according to the method of Example 5 of the present disclosure, without stratification by dielectrophoresis.
  • Dielectrophoresis also known as two-dimensional electrophoresis, refers to the phenomenon of uncharged particles or particles with a low dielectric constant (both called dielectrics) that are forced and oriented in a non-uniform electric field.
  • dielectrics dielectric constant
  • electrode design depending on the dielectric properties, size and shape of the biological particles themselves, the particles can be manipulated or separated.
  • a positive dielectrophoretic force (pDEP) is generated, which causes the particle to be attracted to the strong electric field by the electric field;
  • nDEP negative dielectrophoretic force
  • ⁇ m is the dielectric constant of the solution
  • a is the particle radius
  • E is the electric field strength
  • Re ⁇ f CM ( ⁇ ) ⁇ is the real part of the Clausius Mossotti factor, which can be expressed as:
  • ⁇ p * and ⁇ m * are the plural forms of the dielectric constant of particles and medium (which can be regarded as the ability to induce charge generation), ⁇ is the angular frequency of the applied electric field signal, and ⁇ is the conductivity of the substance. ( ⁇ m is the conductivity of the electrolyte, ⁇ p is the conductivity of the particles), ⁇ 0 is the vacuum dielectric constant, and ⁇ r is the dielectric constant of the substance relative to the vacuum. It can be seen from the above equation that the positive and negative values of f CM ( ⁇ ) determine the direction of the induced electric dipole, and by selecting the frequency of the applied electric field, the positive and negative values can be changed, and then the same particle can be generated under different conditions.
  • Dielectrophoretic force or negative dielectrophoretic force two modes, or different dielectrophoretic forces are generated at different times for different particles to separate them. It should be noted that the above theory is well known to those skilled in the art, and that the required electric field frequency can be obtained by conventional experiments to achieve manipulation and/or separation of any target particles using any applied electric field.
  • the dielectrophoretic generating electrode refers to an electrode device integrated in a microfluidic chip for generating a non-uniform electric field to initiate dielectrophoresis of particles, which can be carried out by methods known in the art, such as photolithography stripping, or magnetron sputtering.
  • the method is prepared on a substrate or a cover sheet of the chip, and the electrode material may be any material selected, such as Pt or Au.
  • the dielectrophoretic generating electrode (4) located at the lower part of the main channel (3) means: the main channel (3)
  • the upper surface of the lower chip substrate i.e., the inner surface of the lower wall of the main channel
  • the inner portion or the lower outer surface.
  • the upper portion of the main channel (3) of the dielectrophoretic generating electrode (4) means the lower surface, the inner surface or the upper surface of the cover sheet at the upper portion of the main channel (3).
  • the electrode generates an electric field by an external signal generator, and an appropriate electric field size can be selected according to the nature of the particle. Illustratively, for cell separation, the electric field strength E can be selected to be greater than 10 5 V/m.
  • the microfluidic chip comprises a dielectrophoretic generating electrode.
  • two or more dielectrophoretic generating electrodes are sequentially disposed in the extending direction of the main channel of the microfluidic chip, wherein a part of the electrodes are located at an upper portion of the main channel, and another part of the electrodes are located at a lower portion of the main channel, preferably, more
  • the dielectrophoresis generating electrodes may be alternately disposed on the upper and lower portions of the main channel, respectively.
  • the dielectrophoretic generating electrode (4) is an electrode prepared from a conductive metal; in another embodiment, the dielectrophoretic generating electrode (4) is prepared from an optoelectronic material having a photoconductive effect.
  • the obtained electrode can be prepared, for example, from amorphous silicon or zinc oxide (ZnO) or the like, which can generate a dielectrophoretic force by generating a non-uniform electric field by local illumination.
  • the separation frequency window means that a dielectrophoretic force of different polarity can be generated for the target cell and the non-target cell under the action of applying an alternating current non-uniform electric field (that is, an electric field applied by the dielectrophoresis generating electrode (4)).
  • an alternating current non-uniform electric field that is, an electric field applied by the dielectrophoresis generating electrode (4).
  • a specific frequency or frequency band that separates the target cell stream from the non-target cell stream.
  • the separation frequency window is sometimes a specific frequency value, but it can also be a specific frequency range or "band”, so in the present invention, "separation frequency window”, “separation frequency” Or “separated frequency bands” are used interchangeably when referring to the meanings referred to by the separation frequency window, which have substantially the same meaning.
  • the microfluidic chip comprises a dielectrophoretic generating electrode, and by selecting a specific separation frequency window, the attraction or repulsion of the target particulate matter can be achieved, and the opposite impurity is generated, thereby targeting the target particulate matter and the impurity. Phase separation.
  • a suitable separation frequency window can be selected to utilize the positive dielectrophoretic force to move the circulating tumor cells (CTC) in the whole blood sample to the surface of the dielectrophoretic generating electrode (4) while generating a negative dielectrophoretic force to repel the blood cells away from the electrode surface.
  • An exemplary separation frequency is, for example, 150 kHz used in the present invention.
  • the negative electrophoresis force is used to make the circulating tumor cells (CTC) in the whole blood sample away from the surface of the electrophoresis generating electrode (4), and the positive dielectrophoretic force is used to attract the blood cells to the surface of the electrode, and the electrical impedance detecting electrode can be Dielectrophoretic generating electrodes are designed on opposite surfaces in the microchannel. At this time, when CTC and blood cells are arranged at different heights due to dielectrophoresis, and then flow through the rear electrical impedance detecting electrode (5), since the CTC is closer to the electrode surface, significant electrical impedance change can be measured, and blood cells can be measured.
  • the number of CTCs in the blood sample can be accurately and accurately measured by the number of impedance changes of the chip of the present invention, without any interference of the blood cell signal.
  • the microfluidic chip comprises two or more dielectrophoretic generating electrodes, and different dielectrophoretic generating electrodes can select different separation frequency windows to apply opposite dielectric power to the target particles, but Through the adjustment of the installation position of the dielectrophoretic electrode, it is ensured that the target particles can maintain the deviation in the same direction.
  • the microfluidic chip comprises two dielectrophoretic generating electrodes, and the first dielectrophoretic generating electrode is disposed on the upper part of the main channel, and a separation frequency window can be selected to cause the electrode to repel the target particles and attract the impurities, thereby The target particles are placed closer to the lower side; the second dielectrophoretic generating electrode is disposed at the lower portion of the main channel, and a separation frequency window can be selected to attract the target particles and repel the impurities, so that the target particles are further closer to the lower side.
  • the separation frequency window varies due to the distribution of the uneven electric field, the difference in the type, size, and dielectric properties of the target particle and the non-target particle, so there is no universally fixed frequency or frequency band, but after knowing the present invention It is within the ability of those skilled in the art to adjust the applied electric field frequency for the target particle to obtain the desired separation frequency window.
  • the present invention does not require detailed explanation of how to obtain an appropriate separation frequency window for the target particulate matter for a particular target particulate matter and conditions.
  • Microfluidics also known as lab on chips, microfluidics or microfluidic chips, integrate the basic operating units of a particular operational flow into a micron-sized channel structure of several square centimeters On the chip.
  • microfluidic chip also known as lab on chips, microfluidics or microfluidic chips, integrate the basic operating units of a particular operational flow into a micron-sized channel structure of several square centimeters On the chip.
  • microfluidic chip may include only a substrate in which channels and/or electrodes are integrated, or may include both a substrate and a cover sheet, wherein the cover sheet may be integrated with electrodes or may form a channel with the substrate.
  • a sample channel (1) On the substrate and/or the cover sheet, components such as a sample channel (1), a sheath liquid channel (2), a main channel (3), a dielectrophoretic generating electrode (4), and an electrical impedance detecting electrode (5) are integrated, and the above components are integrated. It can be prepared by a conventional process of the prior art, such as photolithography, etching, and the like. The dimensions and relative proportions of the various elements are also custom made by those skilled in the art depending on the size of the substrate and/or cover sheet, as well as the size of the target particles.
  • the sample channel (1) and the sheath fluid channel (2) prepared by the present invention each have a width of 60 ⁇ m, and the main channel has a width of 125 ⁇ m and a height of 120 ⁇ m.
  • the material of the microfluidic chip can be selected from any conventional materials, such as various suitable rigid materials and/or elastic materials, and the rigid material can be an inorganic rigid material such as single crystal silicon, amorphous silicon, glass, quartz. Alternatively, it may be a rigid organic polymer material such as an epoxy resin, a polyurea, a polyurethane, a polystyrene, a polymethyl methacrylate or the like; a suitable elastic material mainly includes polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the microfluidic chip can also be directly connected to an external device through a medium such as a catheter or a wire to achieve a predetermined function, such as connecting the injection channel to a device such as a sample pump or an autosampler, for Continuous or automated injection of the sample and / or sheath fluid; connecting the dielectrophoretic generating electrode (4) to an electrical signal generator (such as a DC power source, an AC power source, a transformer, etc.) to generate an inhomogeneous electric field, thereby introducing particles The function of electric power; after the electrical impedance detecting device is connected with the electric signal generator and the electric signal receiver and the analyzer (for example, a computer), the detection and analysis processing of the target particle electrical impedance signal can be realized.
  • a medium such as a catheter or a wire
  • an electrical signal generator such as a DC power source, an AC power source, a transformer, etc.
  • the principle of detecting and counting particles using electrical impedance techniques is well known in the art, that is, when particles suspended in an electrolyte pass through a direct current or alternating electric field with an electrolyte, they replace or partially replace the same volume of electrolyte in the circuit. This causes a transient change in the resistance between the two electrodes to generate a potential pulse.
  • the size and number of pulse signals are proportional to the size and number of particles. This can be used for counting a variety of particles including blood cells.
  • the electrical impedance detecting electrode (5) may be prepared on a substrate surface or a cover sheet of a chip by a conventional method known in the art, such as a photolithography stripping method or a magnetron sputtering method, and the electrode material may be any suitable material, for example.
  • a metal material such as Pt or Au which has a low resistance and is not susceptible to oxidation.
  • the electrical impedance detecting electrode (5) may be located at different positions as long as it matches the position of the dielectrophoretic generating electrode (4) and the dielectric power generated by the target particle. .
  • the dielectrophoretic generating electrode (4) is integrated on the substrate, it is located at the lower portion of the main channel (3) and attracts the target particles.
  • the dielectrophoretic generating electrode (4) is integrated on the cover sheet, is located at the upper portion of the main channel (3), and repels the target particles, and the target particles are brought close to the lower side of the main channel (3).
  • the electrical impedance detecting electrode (5) may be integrated under the main channel (3), that is, on the substrate.
  • the dielectrophoretic generating electrode (4) is integrated on the substrate, it is located below the main channel (3) and repels the target particles.
  • the dielectrophoretic generating electrode (4) is integrated on the cover sheet, located above the main channel (3), and attracts the target particles, and the target particles are brought close to the upper portion of the main channel (3).
  • the electrical impedance detecting electrode (5) can be integrated over the main channel (3), such as a cover sheet.
  • the electrical impedance detecting electrode (5) located below the main channel (3) means: the upper surface of the lower chip substrate of the main channel (3) (ie, the inner surface of the lower wall of the main channel), the inner portion, or the lower outer surface.
  • the electrical impedance detecting electrode (5) located above the main channel (3) means the lower surface, the inner surface or the upper surface of the upper cover sheet of the main channel (3).
  • a more common embodiment of the present invention is to prepare a specific chip in advance, at which time the dielectrophoretic generating electrode (4) and the electrical impedance detecting electrode (5) have a predetermined arrangement, for example, both are located in the main channel ( 3)
  • the target particle can be detected and/or counted by selecting a specific separation frequency window to achieve the target particle close to the electrical impedance detecting electrode (5).
  • a microfluidic chip as shown in Figure 1 is prepared by conventional methods in the art, including a sample channel (1) in the middle and a sheath fluid channel (2) on both sides, and the three channels merge at the outlet to form a main channel (3)
  • the dielectrophoretic generating electrode (4) and the electrical impedance detecting electrode (5) are both located below the main channel (3).
  • the dielectrophoretic generating electrode (4) is externally connected to the electrical signal generating device, and the electrical impedance detecting electrode (5) is connected to the electrical signal generating device and the electrical signal receiving and analyzing device.
  • Example 2 Dielectrophoresis Cell Dividing Effectively Differentiating A549 Lung Adenocarcinoma Cells and PBMC Cells to Construct a GFP-Labeled A549 Cell Line
  • A549 human non-small cell lung cancer cell line was purchased from ATCC. The cell line was originally derived from a 58-year-old white male lung cancer patient, established by D.J. Gard et al. in 1972.
  • DNA expression vector the pLEIN vector, which contains a bicistronic that simultaneously expresses the EGFP and neomycin resistance genes, and also contains an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • NIH3T3-derived packaging cell line PT67 expressing the 10AI virus envelope was purchased from Clontech Laboratories, Inc.
  • PT67 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-products, Calabasas, CA).
  • a 70% confluent packaging cell line (PT67) was incubated with a mixture of DOTAP reagent and a saturating amount of pLEIN plasmid for 18 hours, at which time fresh medium was replenished. The cells were examined by fluorescence microscopy after 48 hours. The cells were cultured for 7 days in 500-2000 ⁇ g/ml G418 (Life Technologies, Inc., Grand Island, NY) to select GFP-positive cells.
  • Retroviral transfected cells a 20% confluent A549 cell line and a retroviral precipitation mixture obtained by culturing PT67 cells, and RPMI1640 medium (Life Technologies) containing 10% fetal bovine serum (the ratio of the two is 1:1) Incubate for 72 hours while replenishing fresh medium.
  • A549 cells were harvested by trypsin/EDTA 72 hours after transfection and passaged to a selection medium containing 200 ⁇ g/ml G418 at a ratio of 1:15. G418 was gradually increased to 800 ⁇ g/ml.
  • Cell clones expressing GFP were isolated by trypsin/EDTA using a cloning column (Bel-Art Products. Pekounock. NJ) and amplified and transferred by conventional culture methods.
  • the conventional culture method is as follows: in MEM- ⁇ medium supplemented with 10% fetal bovine serum, cultured in an incubator at a carbon dioxide concentration of 5% and a temperature of 37 ° C for 48 hours. After taking out the culture dish, the old culture solution is taken out, and the trypsin-EDTA solution is added, and the solution is applied at 37 ° C for 1 minute. After the cells are detached from the bottle wall, an appropriate amount of fresh medium containing serum is added to terminate the trypsin action, and the cells are removed by centrifugation. The supernatant can be used as experimental cells after adding fresh medium.
  • Experimental group 1 A549 cells (with GFP label) at an initial culture concentration of 4*10 6 /mL were taken, serially diluted to about 4000 cells/ml in PBS, centrifuged at 800 rpm (1100 g) for 5 min, and PBS was removed. And resuspended using an isotonic electrolyte: 280 mM sucrose, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 and 1.47 mM KH 2 PO 4 , pH 7.4, conductivity It is 50mS/m.
  • A549 cells are injected into the microfluidic chip through the sample channel (1), and the sheath fluid is added through the sheath fluid channel (2) on both sides of the sample channel (1).
  • the composition of the sheath fluid is the same as the isotonic electrolyte component.
  • the input voltage of the dielectrophoretic generating electrode (4) was 6 V, the frequency was 150 kHz, the detection frequency of the impedance detecting electrode was set to 1 kHz - 5 MHz, and the electric resistance cut-off value was set to 0.015 mV, followed by electrical impedance cell counting.
  • Experimental group 2 First, the cells were mixed, and 3 mL of whole blood samples were taken. After standard Ficoll centrifugation, PBMC products (about 1.5 mL, containing 10 6 cells) were obtained, centrifuged and resuspended using an isotonic electrolyte.
  • the composition of the electrolyte was: 280 mM sucrose, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 and 1.47 mM KH 2 PO 4 , pH 7.4, and the conductivity was 50 mS/m; a certain amount (3-300, It can be a number gradient of A549 cells (the initial addition amount is calculated by the conventional chamber counter).
  • the mixed cells are injected into the microfluidic chip through the sample channel (1), and the sheath liquid is added through the sheath liquid channel (2) on both sides of the sample channel (1), and the composition of the sheath liquid is the same as the isotonic electrolyte component described above.
  • the input voltage of the dielectrophoretic generating electrode (4) was 6 V, the frequency was 150 kHz, the detection frequency of the impedance detecting electrode was set to 1 kHz - 5 MHz, and the electric resistance cut-off value was set to 0.015 mV, followed by electrical impedance cell counting.
  • Control group 1 The cells added in the sample channel (1) were 1.5 mL PBMC, and the conditions on the machine were the same as those in the experimental groups 1 and 2.
  • Control group 2 The cells added in the sample channel (1) were 1.5 mL of A549 cells dissolved in an isotonic electrolyte, and the dielectrophoresis generating electrode (4) was not turned on. The conditions of the machine were the same as those of the experimental groups 1 and 2.
  • Control group 3 1.5 mL of PBMC was added to the sample channel (1), and the dielectrophoretic electrode (4) was not turned on. The conditions of the machine were the same as those of the experimental groups 1 and 2.
  • Control group 4 The cells added in the sample channel (1) were mixed cells of 1.5 mL of PMBC and 3-300 A549 cells, and the dielectrophoresis generating electrode (4) was not turned on. The conditions of the machine were the same as those of the experimental groups 1 and 2.
  • PBMC cells were gradually pushed away from the bottom electrode by continuous photographing, while A549 cells gradually approached the electrode (Fig. 2).
  • Example 2 The test method is shown in Example 2, and the results of the experimental group 1 and the control group 2 were compared.
  • Figure 3 shows the electrical impedance peaks of A549 cells in experimental group 1 ( Figure 3b) and control group 2 ( Figure 3a). It can be seen from the figure that after applying the electric field by the dielectrophoresis generating electrode (4), the A549 cells are closer to the electrical impedance measuring electrode, so that a stronger signal can be generated than the control group which does not use dielectrophoresis focusing, and Because A549 cells are focused once in the vertical direction, A549 cells are also relatively constant in the direction perpendicular to the surface of the chip (Fig.
  • Example 2 The test method is shown in Example 2, and the results of Control Group 1 and Control Group 3 were compared.
  • Example 2 The test method is shown in Example 2, and the results of Experimental Group 2 and Control Group 4 were compared.
  • the accuracy of the electrical impedance measurement technique can only reach 60-72%. It can be seen that the accuracy of electrical impedance cell counting is greatly improved by the method of the present invention.

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Abstract

一种颗粒物检测装置及检测方法，具体提供了一种基于介电泳和电阻抗测量技术的检测装置及检测方法，更具体的涉及一种使用微流控芯片的电阻抗检测装置，以及其检测目标粒子的应用，该装置包括进样部、主通道(3)、介电泳电场发生部和电阻抗检测部，通过介电泳发生部(3)对目标细胞的选择性操控，在不使用标记和抗体的情况下灵敏且精确的对样本进行检测或者计数。

Description

颗粒物检测装置及检测方法
交叉引用
本申请要求发明名称为“颗粒物检测装置及检测方法”于2018年2月28日提交到中国专利局的中国专利申请201810169100.5的优先权，其内容通过引用以整体并入本文。
技术领域
本发明涉及一种利用介电泳和电阻抗测量检测颗粒物的装置和方法，特别涉及一种利用介电泳增强的电阻抗测量微流控芯片和利用该芯片的检测方法。
背景技术
电阻抗测量技术，大多经由微流控芯片或电场聚焦使颗粒物呈现单一粒子流并通过测量电极，随后基于颗粒物对测量电压或电流产生的阻碍和抵抗作用进行颗粒物检测及计数。但是在实际检测样本(比如血液)时，由于目标颗粒物(如循环肿瘤细胞，CTC)与非目标颗粒物如血细胞(特别是巨噬细胞、单核细胞等白细胞)的尺寸相当，所产生的电阻抗信号相当接近，仅依据电阻抗信号很难将不同的颗粒物进行区分。因此通常需要对目标颗粒物如细胞进行荧光染色或采用检测抗体等额外的细胞标记手段，才能够对目标颗粒物加以区分，但同时也增加了额外的步骤和成本，因此本领域迫切需要一种无需添加标记的能够区分不同种类颗粒物的电阻抗检测技术。
发明内容
本发明人出乎意料地发现，如果在电阻抗测量设备中，加入介电泳发生装置，介电场在特定频率(separation frequency window)下会对不同的细胞施加不同的影响，例如，对目标细胞(如CTC)产生正介电力，从而向DEP电极表面移动；同时对其他血细胞产生负介电力，使之被DEP 电极排斥远离电极表面，就可以使流道中原本混合在一起的两种或多种细胞，依据细胞种类和/或特性的不同，被排列在不同的垂直高度。由于目标细胞更接近电极表面，因而可测量到更加明显的阻抗信号；相反，杂质细胞离电极表面较远，无法测量到明显的阻抗信号，从而提高了信号的分辨力。在上述发现的基础上，发明人完成了本发明。
在第一个方面中，本发明提供了一种分离和检测颗粒物的微流控芯片，其包括进样部、主通道(3)、介电泳电场发生部和电阻抗检测部。
在一个实施方案中，所述进样部包括1个或更多个样品通道(1)，以及任选地1个或更多个鞘液通道(2)，例如1、2或3个样品通道和1、2、3、4、5或6个鞘液通道，更例如1个样品通道和2个鞘液通道，所述1个或更多个样品通道(1)与任选地所述1个或更多个鞘液通道(2)在一端汇合，并与主通道(3)连接，从而使样品流与任选地鞘液流汇聚后注入主通道(3)；
所述主通道(3)上依次设置有介电泳电场发生部与电阻抗检测部；
所述介电泳电场发生部包括在主通道(3)上部或下部设置的介电泳发生电极(4)，所述介电泳发生电极用于产生在垂直于主通道的方向上不均匀的电场，从而使主通道(3)液流中的颗粒物在垂直于主通道的方向发生介电泳；
所述电阻抗检测部包括在主通道(3)上部或下部设置的电阻抗检测电极(5)，能够响应于流经主通道(3)的颗粒物并产生电阻抗检测信号。
在另一个实施方案中，所述微流控芯片包括一个或更多个所述介电泳电场发生部，例如1、2、3、4或5个；特别地当包括2个或更多个所述介电泳电场发生部时，优选地所述介电泳发生电极分别设置在主通道上部和下部。
在另一个实施方案中，其中至少一个所述介电泳发生电极(4)设置在主通道(3)下部，例如设置于芯片的基片上。
在另一个实施方案中，其中至少一个所述介电泳发生电极(4)设置在主通道(3)上部，例如设置于覆盖在微流控芯片之基片上方的盖片上。
在另一个实施方案中，其中所述电阻抗检测电极(5)设置在主通道(3)下部，优选设置于芯片的基片上。
在另一个实施方案中，其中所述电阻抗检测电极(5)设置在主通道 (3)上部，优选设置于覆盖于微流控芯片之基片上方的盖片上。
在另一个实施方案中，其中所述介电泳发生电极(4)是导电金属电极或具有光导电效应的光电材料，例如非晶硅(Amorphous silicon)或氧化锌(ZnO)等，其可经由局部光照产生非均匀电场进而产生介电泳力。
在另一个实施方案中，其中所述颗粒物是感兴趣的细胞，例如肿瘤细胞，尤其是循环肿瘤细胞。
在第二个方面中，本发明提供一种分离和检测颗粒物的电阻抗检测装置，包括前述第一方面中任选实施方案的微流控芯片。
在一个实施方案中，其中还包括与介电泳发生电极(4)连接的介电泳电场信号发生器，和/或与电阻抗检测部连接的电阻抗信号接收器，优选地还包括与进样部连接的加样器和/或与电阻抗信号接收器连接的电阻抗信号分析器。
在第三个方面中，本发明还提供一种分离和检测颗粒物的方法，其使用第一方面中所述的微流控芯片和/或第二个方面中所述的电阻抗检测装置。
在一个实施方式中，所述方法包括
a)将待测样品和任选地鞘液分别通过样品通道(1)和任选地鞘液通道(2)注入进样部，例如，所述样品源自全血、血浆、尿液、组织液、脑脊液、细胞培养液或细胞混合液；具体的，所述鞘液是蔗糖的PBS溶液，优选的，所述鞘液包含280mM蔗糖、137mM NaCl、2.7mM KCl、4.3mM Na 2HPO 4和1.47mM KH 2PO 4，pH7.4；优选的，所述鞘液的电导率为10-500mS/m、30-300mS/m或50-100mS/m，更优选的，所述鞘液的电导率为50mS/m。
b)使介电泳发生电极(4)产生在垂直于主通道的方向上不均匀的电场，使样品中的目标颗粒物发生介电泳，从而使主通道(3)内的目标颗粒物与非目标颗粒物在垂直于芯片表面的方向发生分层，且目标颗粒物形成的液流靠近主通道下游的电阻抗检测电极(5)侧，而非目标颗粒物形成的液流远离主通道下游的电阻抗检测电极(5)侧；具体的，所述目标颗粒物是肿瘤细胞，优选循环肿瘤细胞；具体的，所述非目标细胞是白细胞，优选为外周血单个核细胞(PBMC)或巨噬细胞，和/或红细胞；和
c)利用电阻抗检测部对流经颗粒物进行电阻抗信号检测。
在另一个实施方案中，其中通过选择所述不均匀电场的频率，使得在特定分离频率窗口中，目标颗粒物与非目标颗粒物受到相反的介电力作用。
在第四个方面中，本发明提供了第一个方面中的微流控芯片和第二个方面中的电阻抗检测装置的用途，具体地，其用于检测肿瘤细胞，特别地所述肿瘤细胞是循环肿瘤细胞。
在第五个方面中，本发明还提供了第一个方面中的微流控芯片和第二个方面中的电阻抗检测装置在制备检测肿瘤细胞的医疗器械中的应用，特别地所述肿瘤细胞是循环中肿瘤细胞。
本发明内容仅仅举例说明了要求保护的一些具体实施方案，其中一个或更多个技术方案中所记载的技术特征可以与任意的一个或更多个技术方案相组合，这些经组合而得到的技术方案也在本申请保护范围内，就像这些经组合而得到的技术方案已经在本发明公开内容中具体记载一样。
通过以上技术方案，本发明至少可以提供以下至少一个方面的优势和利益。本发明通过介电泳发生电极(4)对目标细胞的选择性操控，在不使用标记和抗体的情况下灵敏且精确的对样本(特别是分离的血细胞样本或PBMC样本)中的目标细胞(例如肿瘤细胞，循环肿瘤细胞)进行检测或者计数，相比于传统的细胞电阻抗检测装置，显著的提高了信噪比，也大大降低了对上机样本中目标细胞的浓度要求(非目标细胞/目标细胞数量比例大于10 6仍然能够用本芯片检测)。而且本发明操作简便，相比于采用荧光标记或抗体标记的方法，具有更低的检测成本，具有很好的产业应用前景。
附图说明
下面通过对本发明的详细描述以及附图来清楚地说明本发明前面叙述的方面以及其他方面。为了举例说明本发明，在附图中的实施方案是目前优选的，然而，可以理解，本发明并不限于所公开的特定实施方案。
图1是本发明一个实施方案的微流控芯片的结构示意图，其中图1(a)是俯视图，图1(b)是侧视图。
图2是主通道(3)中PBMC和A549细胞发生介电泳时的运动轨迹图(侧视)，左图为电镜照片图，右图为示意图，其中左图中圆圈内表示的是PBMC细胞，其在负介电力(nDEP)的作用下远离下部电极，带三 角箭头的圆圈内表示的是A549细胞，其在正介电力(pDEP)的作用下靠近下部电极。
图3是A549细胞样品的电阻抗信号图，其中图3(a)显示的是并未经过介电泳细胞聚焦的电阻抗信号图，而图3(b)则显示的是经过介电泳细胞聚焦后的电阻抗信号图。
图4(a)是使用1.5mL PBMC样品时，经过或不经过介电泳细胞分层时，所得到的电阻抗计数结果。
图4(b)是根据本公开内容实施例5的方法检测PBMC与不同数量A549细胞混合的样品时，经过介电泳细胞分层后，A549细胞的计数结果。
图4(c)是根据本公开内容实施例5的方法检测PBMC与不同数量A549细胞混合的样品时，不经过介电泳细胞分层时，A549细胞的计数结果。
具体实施方式
除非另外说明，本公开中使用的术语具有所属领域普通技术人员理解的一般含义。下面是一些术语在本公开内容中的含义，如果跟其他定义有不一致，以以下定义为准。
定义
介电泳(dielectrophoresis,DEP)
介电泳也称双向电泳，是指非带电粒子或者介电常数较低的粒子(均称之为电介质)在非匀强电场中受力并进行定向运动的现象。通过适当的电极设计，依据生物性粒子本身的介电特性、大小与形状的不同，即可对粒子进行操控或进行不同粒子的分离。由于粒子与其周围溶液的极化程度有所差异，因此当粒子的极化能力高于液体时，产生正介电泳力(pDEP)，将使粒子受电场影响而往强电场区吸引；反之，当粒子的极化能力弱于液体时，此时将会产生负介电泳力(nDEP)，将使粒子被强电场排斥而远离强电场区。作用于均匀的圆形粒子的介电泳力，可通过下式表示：
Figure PCTCN2018103171-appb-000001
其中ε m为溶液的介电常数、a为粒子半径、E为电场强度、Re{f CM(ω)} 为实部部分(real part)的Clausius Mossotti factor，其又可表示成：
Figure PCTCN2018103171-appb-000002
ε p *和ε m *为粒子(particle)与溶液(medium)的介电常数的复数形式(可视为诱导产生电荷的能力)，ω为外加电场信号的角频率，σ为物质的导电系数(σ m为电解液的导电系数，σ p微粒子的导电系数)，ε 0为真空介电常数，ε r为物质相对于真空的介电常数。由上式可知，f CM(ω)值的正负决定了诱导电偶极的方向，而通过选择外加电场的频率，就可以改变其正负值，进而可以针对同一粒子在不同条件下产生正介电泳力或者负介电泳力两种模式，或者针对不同的颗粒在同一时刻产生不同极性的介电泳力从而将其分离。应当指出的是，上述理论是本领域技术人员所熟知的，并且可以通过常规的实验获得所需要的电场频率，从而利用任何外加电场实现任何目标粒子的操控和/或分离。
此外，在本发明中，还会使用“介电泳聚焦”、“介电泳细胞分层”、“DEP聚焦”、“介电泳吸引”或“介电泳牵引”的表述，有时省略称之为“聚焦”或“细胞分层”，上述表述具有相同的含义，可以互换使用，其具体代表的含义均是指通过介电泳发生电极(4)的作用，使得目标粒子或细胞在垂直于芯片的方向上靠近或远离介电泳发生电极(4)。
介电泳发生电极
介电泳发生电极是指集成于微流体芯片中用于产生不均匀电场从而引发粒子的介电泳的电极设备，可采用现有技术中已知的方法，例如光刻剥离法、或磁控溅射法等制备于芯片的基片或者盖片上，电极材料可选任意的材料，例如Pt或Au等，在本发明中，介电泳发生电极(4)位于主通道(3)下部是指：主通道(3)下部芯片基材的上表面(即主通道下壁内表面)、内部，或者下部外表面。介电泳发生电极(4)位于主通道(3)上部是指：主通道(3)上部的盖片下表面、内部或者上表面。电极通过外接信号发生器而产生电场，可以根据粒子的性质选择适当的电场大小，示例性的，对细胞分离而言，电场强度E可以选择大于10 5V/m即可。
在本发明的一个实施方式中，微流体芯片中包含一个介电泳发生电极。
在另一个实施方式中，在微流体芯片主通道的延伸方向依次布置两个或更多个介电泳发生电极，其中部分电极位于主通道的上部，另外一部分电极位于主通道下部，优选的，多个介电泳发生电极可分别交替设置于主通道的上部和下部。
在本发明的又一个实施方式中，介电泳发生电极(4)是由导电金属制备得到的电极；在另一个实施方式中，介电泳发生电极(4)是由具有光导电效应的光电材料制备得到的电极，例如可以由非晶硅(Amorphous silicon)或氧化锌(ZnO)等制备，这些光电材料可经由局部光照产生非均匀电场进而产生介电泳力。
分离频率窗口(separation frequency window)
在本发明中，分离频率窗口是指在外加交流不均匀电场(即通过介电泳发生电极(4)施加的电场)的作用下，能够对目标细胞与非目标细胞产生不同极性的介电泳力，从而将目标细胞流与非目标细胞流分离的特定频率或频段。从上述定义可以看出，分离频率窗口有时候是一个特定的频率值，但也可以是一个特定的频率范围或者叫做“频段”，因此在本发明中，“分离频率窗口”，“分离频率”或者“分离频段”在指代分离频率窗口所指代的含义时可互换使用，其具有实质性相同的含义。
在一个实施方式中，微流控芯片包含一个介电泳发生电极，通过选择特定的分离频率窗口，可以实现对目标颗粒物的吸引或排斥，而对标杂质产生相反的作用，从而将目标颗粒物与杂质相分离。
例如，可选择适当的分离频率窗口利用正介电泳力将全血样本中的循环肿瘤细胞(CTC)向介电泳发生电极(4)表面移动，同时产生负介电泳力将血细胞排斥远离电极表面，示例性的分离频率例如本发明所使用的150kHz，当电阻抗检测电极与介电泳发生电极设计于同一表面时，CTC更接近于电阻抗检测电极表面，从而可测量到明显的电阻抗变化。或者，利用负介电泳力使全血样本中的循环肿瘤细胞(CTC)远离介电泳发生电极(4)表面，同时利用正介电泳力吸引血细胞靠近电极表面，此时可将电阻抗检测电极与介电泳发生电极设计于微流道中之相对的表面。此时，CTC与血细胞因介电泳现象被排列于不同高度，再流经后方的电阻抗检 测电极(5)时，由于CTC更接近于电极表面，因而可测量到明显的电阻抗变化，而血细胞由于被排列于离检测电极较远的高度，因而几乎测量不到明显的由血细胞通过而产生的信号变化。如此，经由本发明芯片的阻抗变化次数，可精准明确的测量到纯CTC的信号而计算血液样本中的CTC数量，而无任何血球信号的干扰。
在另一个实施方式中，微流控芯片包含两个或更多个介电泳发生电极，此时不同的介电泳发生电极可以选择不同的分离频率窗口，对目标颗粒物施加相反的介电力作用，但通过介电泳电极安装位置的调整，保证目标颗粒物能够保持向同一个方向的偏离。例如，微流控芯片包含两个介电泳发生电极，第一个介电泳发生电极设置于主通道上部，可选择一个分离频率窗口使电极对目标颗粒物产生排斥作用，并对杂质产生吸引作用，从而使目标颗粒物更靠近下侧；第二个介电泳发生电极设置于主通道下部，可选择一个分离频率窗口对目标颗粒物产生吸引作用，并对杂质产生排斥作用，从而使目标颗粒物进一步靠近下侧。
分离频率窗口因外加不均匀电场的分布、目标粒子和非目标粒子的种类、大小、介电性等性质的差异而变化，因此并不存在普适固定的频率或频段，但在知晓本发明之后，针对目标粒子通过调整外加电场频率从而获得所需的分离频率窗口属于本领域技术人员的能力范围。因此，对于特定的目标颗粒物以及所处条件，本发明并不需要对如何针对目标颗粒物得到适当的分离频率窗口进行详细阐述。
微流控芯片
微流控芯片(microfluidics)，又称芯片实验室(lab on chip)、微流体或微流体芯片，可将一个特定操作流程的各个基本操作单元集成到几个平方厘米大小的具有微米级通道结构的芯片上。本发明中，“微流控芯片”，“微流体芯片”，“微流体”，“微流体芯片”和“芯片”在指代微流控芯片时具有相同的含义，可以互换使用。微流控芯片可以仅包括集成了通道和/或电极的基片，也可以同时包括基片和盖片，其中盖片上可以集成电极，也可以和基片形成通道。在基片和/或盖片上，集成了样品通道(1)、鞘液通道(2)、主通道(3)、介电泳发生电极(4)以及电阻抗检测电极(5)等元件，上述元件均可通过现有技术的常规工艺进行制备，例如光刻法、刻蚀法等。各元件的尺寸以及相对比例也是本领域技术人员根据基片和/或盖片的大小，以及目标粒子的尺寸而自行定制的。示例性的，本发明制 备的样品通道(1)和鞘液通道(2)的宽度均为60μm，主流道的宽度为125μm，高度为120μm。
本发明中，微流控芯片的材料可以选择任何常规的材料，例如各类适合的刚性材料和/或弹性材料，刚性材料可以是无机刚性材料，如单晶硅、无定形硅、玻璃、石英等，也可以是刚性有机聚合物材料如环氧树脂、聚脲、聚氨酯、聚苯乙烯和聚甲基丙烯酸甲酯等；适合的弹性材料主要包括聚二甲基硅氧烷(PDMS)。
在本发明中，微流控芯片还可以与外接的设备直接通过导管或者导线等介质连接，以实现既定的功能，例如将进样通道与加样泵或自动加样器等设备连接，用于样品和/或鞘液的连续或者自动化注入；将介电泳发生电极(4)与电信号发生器(例如直流电源、交流电源、变压器等)连接后，以产生不均匀电场，从而对粒子产生介电力作用；将电阻抗检测装置与电信号发生器和电信号接受器和分析器(例如计算机)连接后，可以实现目标粒子电阻抗信号的检测和分析处理。
电阻抗检测电极
利用电阻抗技术对粒子进行检测和计数的原理为本领域所公知，即当悬浮在电解液中的颗粒随电解液通过直流或交流电场时，取代或部分取代相同体积的电解液，在电路中导致两电极间电阻发生瞬时变化，产生电位脉冲。脉冲信号的大小和次数与颗粒的大小和数目成正比。由此可以用于包括血细胞在内的多种粒子的计数。
电阻抗检测电极(5)可以通过本领域公知的常规方法，如光刻剥离法、或磁控溅射法等制备于芯片的基片表面或者盖片上，电极材料可选任意的适用材料，例如Pt或Au等低阻值且不易起氧化反应的金属材料。
需要注意的是，为了实现本发明的目的，电阻抗检测电极(5)可以位于不同的位置，只要与介电泳发生电极(4)的位置，以及对目标粒子产生的介电力作用相匹配即可。例如，如果介电泳发生电极(4)集成于基片上，位于主通道(3)下部，并且对目标粒子产生吸引作用。或者，介电泳发生电极(4)集成于盖片上，位于主通道(3)上部，并且对目标粒子产生排斥作用，此时均会使目标粒子靠近主通道(3)的下侧。此时，为了获得区分的信号，电阻抗检测电极(5)可以集成于主通道(3)下方，即位于基片上。
反之，如果介电泳发生电极(4)集成于基片上，位于主通道(3)下方，并且对目标粒子产生排斥作用。或者，介电泳发生电极(4)集成于盖片上，位于主通道(3)上方，并且对目标粒子产生吸引作用，此时均会使目标粒子靠近主通道(3)的上部。此时为了获得区分的信号，电阻抗检测电极(5)可以集成于主通道(3)上方，例如盖片上。
在本发明中，电阻抗检测电极(5)位于主通道(3)下方是指：主通道(3)下部芯片基材的上表面(即主通道下壁内表面)、内部、或者下部外表面。电阻抗检测电极(5)位于主通道(3)上方是指：主通道(3)上部盖片的下表面、内部或者上表面。
不过，本发明更常见的实施方式是预先制备好特定的芯片，此时介电泳发生电极(4)和电阻抗检测电极(5)已经具有预设的排布，例如二者均位于主通道(3)下方，随后通过选择特定的分离频率窗口实现目标粒子靠近电阻抗检测电极(5)即可实现检测和/或计数的目的。
还可进一步通过实施例来理解本发明，然而，要理解的是，这些实施例不限制本发明。现在已知的或进一步开发的本发明的变化被认为落入本文中描述的和以下要求保护的本发明范围之内。
实施例
实施例1 微流体芯片的制备
利用本领域的常规方法制备如图1所示的微流体芯片，其中包括位于中间的样品通道(1)和两侧的鞘液通道(2)，三条通道在出口处合并形成主通道(3)，介电泳发生电极(4)和电阻抗检测电极(5)均位于主通道(3)的下方。介电泳发生电极(4)与电信号发生装置外接，电阻抗检测电极(5)与电信号发生装置与电信号接收和分析装置连接。
实施例2 介电泳细胞分层有效区别A549肺腺癌细胞和PBMC细胞构建带有GFP标记的A549细胞系
初始细胞系：从ATCC购买A549人非小细胞肺癌细胞株，该细胞系 原始来源为58岁白人男性之肺癌患者，由D.J.Gard等人于1972年建立。
DNA表达载体：pLEIN载体，包含同时表达EGFP和新霉素抗性基因的双顺反子，同时含有内部核醣体进入位点(IRES)，
细胞培养，载体生产，转染及亚克隆：表达10AI病毒包膜的NIH3T3衍生的包装细胞株PT67购自Clontech Laboratories，Inc。在补充有10％热灭活的胎牛血清(Gemini Bio-products，Calabasas，CA)的DMEM中培养PT67细胞。对于载体生产，将70％铺满的包装细胞株(PT67)与DOTAP试剂和饱和量的pLEIN质粒的沉淀混合物培养18小时，此时补充新鲜培养基。48小时后通过荧光显微镜检查细胞。将细胞于500-2000μg/ml G418(Life Technologies，Inc.，Grand Island，NY)中培养7天，以选择GFP阳性细胞。
逆转录病毒转染细胞：将20％铺满的A549细胞株与培养PT67细胞得到的反转录病毒沉淀混合物、和包含10％胎牛血清的RPMI1640培养基(Life Technologies公司)(二者比例为1:1)培养72小时，同时补充新鲜培养基。转染72小时后利用胰蛋白酶/EDTA收获A549细胞，并以1:15的比例传代至含有200μg/ml G418的选择培养基中。G418逐渐增加到800μg/ml。利用克隆柱(Bel-Art Products.Pequannock.NJ)透过胰蛋白酶/EDTA分离表达GFP的细胞克隆，并用常规培养方法进行扩增和转移。
培养：采用常规的培养方法，具体如下：于加入10％胎牛血清的MEM-α培养基中，在二氧化碳浓度为5％，温度为37摄氏度的条件下，置于培养箱培养，经过48小时后，取出培养皿吸掉旧培养液，加入胰酶-EDTA溶液，37℃作用1分钟，待细胞自瓶壁脱落后，加入适量含血清之新鲜培养基，以终止胰酶作用，离心后去除上清液，添加新鲜培养基后即可作为实验细胞使用。
上机试验，设置两个实验组和四个对照组，具体如下：
实验组1：取初始培养浓度为4*10 6个/mL的A549细胞(带有GFP标记)，以PBS连续稀释至约4000cells/ml，在800rpm(1100g)条件下离心5min，移除PBS，并使用等渗电解液进行重悬，所述等渗电解液的组成为：280mM蔗糖、137mM NaCl、2.7mM KCl、4.3mM Na 2HPO 4和1.47mM KH 2PO 4，pH7.4，电导率为50mS/m。
上机：将A549细胞通过样品通道(1)注入微流控芯片，同时通过样品通道(1)两侧的鞘液通道(2)加入鞘液，鞘液的成分与前述等渗电解液成分相同，即：280mM蔗糖、137mM NaCl、2.7mM KCl、4.3mM Na 2HPO 4和1.47mM KH 2PO 4，pH7.4，电导率为50mS/m；其中微流控芯片的通道尺寸为125μm，样品流速为0.5mL/hr，鞘液流速为2mL/hr，流速比为1:4，从而在鞘液流的作用下，样本流被压缩为约30μm的单颗粒流。介电泳发生电极(4)的输入电压为6V，频率为150kHz，阻抗检测电极的检测频率设置为1kHz-5MHz，电阻抗cut-off值设定为0.015mV，随后进行电阻抗细胞计数。
实验组2：首先进行细胞混合，取3mL全血样品，经过标准Ficoll离心后，获得PBMC产物(约1.5mL，含10 6个细胞)，离心并使用等渗电解液重悬，所述等渗电解液的组成为：280mM蔗糖、137mM NaCl、2.7mM KCl、4.3mM Na 2HPO 4和1.47mM KH 2PO 4，pH7.4，电导率为50mS/m；添加一定数量(3-300个，可以是数量梯度)的A549细胞(以传统chamber counter计算初始加入量)。
上机：将混合细胞通过样品通道(1)注入微流控芯片，同时通过样品通道(1)两侧的鞘液通道(2)加入鞘液，鞘液的成分与前述等渗电解液成分相同：280mM蔗糖、137mM NaCl、2.7mM KCl、4.3mM Na 2HPO 4和1.47mM KH 2PO 4，pH7.4，电导率为50mS/m；其中芯片的通道尺寸为125μm，样品流速为0.5mL/hr，鞘液流速为2mL/hr，流速比为1:4，从而在鞘液流的作用下，样本流被压缩为约30μm的单颗粒流。介电泳发生电极(4)的输入电压为6V，频率为150kHz，阻抗检测电极的检测频率设置为1kHz-5MHz，电阻抗cut-off值设定为0.015mV，随后进行电阻抗细胞计数。
对照组1：在样品通道(1)中加入的细胞为1.5mL PBMC，上机条件同实验组1和2。
对照组2：在样品通道(1)加入的细胞为溶解于等渗电解液中的1.5mL A549细胞，介电泳发生电极(4)不予接通。上机条件同实验组1和2。
对照组3：在样品通道(1)加入的细胞为1.5mL PBMC，介电泳发 生电极(4)不予接通。上机条件同实验组1和2。
对照组4：在样品通道(1)加入的细胞为1.5mL PMBC与3-300颗A549细胞的混合细胞，介电泳发生电极(4)不予接通。上机条件同实验组1和2。
结果：在实验组中，通过连续拍照可以看到PBMC细胞被逐渐推离底部电极，而A549细胞则逐渐靠近电极(图2)。
实施例3 介电泳聚焦提高目标信号的稳定性
试验方法见实施例2，比较实验组1和对照组2的结果。
结果：图3显示实验组1(图3b)和对照组2(图3a)中A549细胞的电阻抗峰值图。从图中可以看出，利用介电泳发生电极(4)施加电场后，A549细胞与电阻抗测量电极更加接近，因此相比较不使用介电泳聚焦的对照组，能够产生更强的信号，并且正是由于A549细胞在竖直方向发生了一次聚焦，A549细胞在垂直于芯片表面的方向位置也较为恒定(图3b)，因此信号强度也非常稳定，而不像对照组不用介电泳聚焦时，由于A549细胞在垂直方向没有约束或者聚焦，因而细胞更加分散，导致产生的电阻抗信号大小不够稳定，同时也容易出现计算的遗漏(图3a)。
实施例4 介电泳细胞分层提高检测的特异性
试验方法见实施例2，比较对照组1和对照组3的结果。
结果：经预实验(n＝20)，经介电泳细胞分层的PBMC与血球样本，有13次PBMC样本检测值皆小于0.015mV，仅7次PBMC样本结果中各自出现小于5个大于0.015mV之结果，故cut-off值设为0.015mV。经过介电泳细胞分层的对照组1在多次重复试验下平均仅能统计到了2-5个细胞，但是没有经过介电泳聚焦的对照组3却平均统计到了120个细胞(图4a)。也就是说，如果不经过介电泳细胞分层，会有更多的PBMC细胞会被误认为是目标细胞(A549)而被计算进去。
实施例5 介电泳聚焦提高计数的准确性
试验方法见实施例2，比较实验组2和对照组4的结果。
结果：经预实验(n>20)，A549之信号最大为0.039mV，最小为0.018mV，故可设定信号的cut-off值为0.015mV，该值以上是A549细胞，0.015mV以下的信号被滤除。图4b和图4c的结果显示，经过介电泳聚焦后，由于细胞被吸引靠近电阻抗检测电极表面，实验组中电阻抗测量技术得到的细胞数量与初始的加入量非常接近，准确率达到了将近93％；而在不经过介电泳聚焦和分层的对照组4中，电阻抗测量技术的准确率仅能达到60-72％之间。可见通过本发明的方法大大提高了电阻抗细胞计数的准确性。
以上描述地仅是优选实施方案，其只作为示例而不限制实施本发明所必需特征的组合。所提供的标题并不意指限制本发明的多种实施方案。
本申请中提及的所有公开物和专利通过引用方式并入本文。不脱离本发明的范围和精神，本发明的所描述的方法和组合物的多种修饰和变体对于本领域技术人员是显而易见的。虽然通过具体的优选实施方式描述了本发明，但是应该理解所要求保护的本发明不应该被不适当地局限于这些具体实施方式。事实上，那些对于相关领域技术人员而言显而易见的用于实施本发明的所描述的模式的多种变体意在包括在随附的权利要求的范围内。

Claims (16)

  1. 一种分离和检测颗粒物的微流控芯片，其包括进样部、主通道(3)、介电泳电场发生部和电阻抗检测部。
  2. 根据权利要求1所述的微流控芯片，其中
    所述进样部包括1个或更多个样品通道(1)，以及任选地1个或更多个鞘液通道(2)，例如1、2或3个样品通道和1、2、3、4、5或6个鞘液通道，更例如1个样品通道和2个鞘液通道，所述1个或更多个样品通道(1)与任选地所述1个或更多个鞘液通道(2)在一端汇合，并与主通道(3)连接，从而使样品流与任选地鞘液流汇聚后注入主通道(3)；
    所述主通道(3)上依次设置有介电泳电场发生部与电阻抗检测部；
    所述介电泳电场发生部包括在主通道(3)上部或下部设置的介电泳发生电极(4)，所述介电泳发生电极用于产生在垂直于主通道的方向上不均匀的电场，从而使主通道(3)液流中的颗粒物在垂直于主通道的方向发生介电泳；
    所述电阻抗检测部包括在主通道(3)上部或下部设置的电阻抗检测电极(5)，能够响应于流经主通道(3)的颗粒物并产生电阻抗检测信号。
  3. 根据权利要求1或2所述的微流控芯片，其包括一个或更多个所述介电泳电场发生部，例如1、2、3、4或5个；特别地当包括2个或更多个所述介电泳电场发生部时，优选地所述介电泳发生电极分别设置在主通道上部和下部。
  4. 根据权利要求1至3任一项所述的微流控芯片，其中至少一个所述介电泳发生电极(4)设置在主通道(3)下部，例如设置于芯片的基片上。
  5. 根据权利要求1至4任一项所述的微流控芯片，其中至少一个所述介电泳发生电极(4)设置在主通道(3)上部，例如设置于覆盖在微流控芯片之基片上方的盖片上。
  6. 根据权利要求1至5任一项所述的微流控芯片，其中所述电阻抗检测电极(5)设置在主通道(3)下部，优选设置于芯片的基片上。
  7. 根据权利要求1至5任一项所述的微流控芯片，其中所述电阻抗检测电极(5)设置在主通道(3)上部，优选设置于覆盖于微流控芯片之基片上方的盖片上。
  8. 根据权利要求1至7任一项所述的微流控芯片，其中所述介电泳发生电极(4)是导电金属电极或具有光导电效应的光电材料，例如非晶硅(Amorphous silicon)或氧化锌(ZnO)等，其可经由局部光照产生非均匀电场进而产生介电泳力。
  9. 根据权利要求1至8任一项所述的微流控芯片，其中所述颗粒物是感兴趣的细胞，例如肿瘤细胞，尤其是循环肿瘤细胞。
  10. 一种分离和检测颗粒物的电阻抗检测装置，包括权利要求1至9任一项所述的微流控芯片。
  11. 根据权利要求10所述的电阻抗检测装置，还包括与介电泳发生电极(4)连接的介电泳电场信号发生器，和/或与电阻抗检测部连接的电阻抗信号接收器，优选地还包括与进样部连接的加样器和/或与电阻抗信号接收器连接的电阻抗信号分析器。
  12. 一种分离和检测颗粒物的方法，其使用权利要求1至9任一项所述微流控芯片和/或权利要求10或11所述电阻抗检测装置。
  13. 根据权利要求12所述的方法，其包括
    a)将待测样品和任选地鞘液分别通过样品通道(1)和任选地鞘液通道(2)注入进样部，例如，所述样品源自全血、血浆、尿液、组织液、脑脊液、细胞培养液或细胞混合液；具体的，所述鞘液是蔗糖的PBS溶液，优选的，所述鞘液包含280mM蔗糖、137mM NaCl、2.7mM KCl、4.3mM Na 2HPO 4和1.47mM KH 2PO 4，pH7.4；优选的，所述鞘液的电导率为10-500mS/m、30-300mS/m或50-100mS/m，更优选的，所述鞘液的电导率为50mS/m；
    b)使介电泳发生电极(4)产生在垂直于主通道的方向上不均匀的电场，使样品中的目标颗粒物发生介电泳，从而使主通道(3)内的目标颗粒物与非目标颗粒物在垂直于芯片表面的方向发生分层，且目标颗粒物形成的液流靠近主通道下游的电阻抗检测电极(5)侧，而非目标颗粒物形成的液流远离主通道下游的电阻抗检测电极(5)侧；具体的，所述目标颗粒物是肿瘤细胞，优选循环肿瘤细胞；具体的，所述非目标细胞是白细胞，优选为外周血单个核细胞(PBMC)或巨噬细胞，和/或红细胞；和
    c)利用电阻抗检测部对流经颗粒物进行电阻抗信号检测。
  14. 根据权利要求12或13所述的方法，其中通过选择所述不均匀电场的频率，使得在特定分离频率窗口中，目标颗粒物与非目标颗粒物受 到相反方向的介电力作用。
  15. 权利要求1至9任一项所述的微流控芯片、权利要求10或11所述的电阻抗检测装置，其用于检测肿瘤细胞，特别地所述肿瘤细胞是循环肿瘤细胞。
  16. 权利要求1至9任一项所述的微流控芯片、权利要求10或11所述的电阻抗检测装置在制备检测肿瘤细胞的医疗器械中的应用，特别地所述肿瘤细胞是循环肿瘤细胞。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265327A (zh) * 2021-04-26 2021-08-17 大连海事大学 一种基于藻类脂质含量的交流-介电泳微藻多级分选装置及方法
CN115193358A (zh) * 2022-07-21 2022-10-18 扬州大学 一种试管形多功能微流控反应装置

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108387488B (zh) * 2018-02-28 2023-08-01 上海欣戈赛生物科技有限公司 颗粒物检测装置及检测方法
CN108344678B (zh) * 2018-04-25 2021-03-26 北京怡天佳瑞科技有限公司 一种颗粒物检测装置及检测方法
CN110918139B (zh) * 2018-09-20 2023-09-29 上海欣戈赛生物科技有限公司 微流控芯片、含有该微流控芯片的装置及样本浓缩的方法
CN110923112A (zh) * 2018-09-20 2020-03-27 北京怡天佳瑞科技有限公司 微流控芯片、含有该微流控芯片的装置及其分选和/或粒子的方法
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CN112268934B (zh) * 2020-09-16 2022-06-28 东南大学 一种针对循环肿瘤细胞的检测芯片及其检测方法
CN112903793B (zh) * 2021-01-21 2022-04-19 中国科学技术大学 一种用于水体多参数快速检测装置及检测方法
CN112834588A (zh) * 2021-02-24 2021-05-25 江苏济纶医工智能科技有限公司 一种用于病毒电阻抗实时监测的微流控芯片
CN113049475B (zh) * 2021-02-25 2023-03-17 首都医科大学宣武医院 一种m1型巨噬细胞的鉴定方法与应用
CN113418857B (zh) * 2021-06-01 2023-01-31 大连海事大学 一种结合交流-介电泳与库尔特计数的海洋微生物识别装置及方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101046458A (zh) * 2006-03-30 2007-10-03 中国科学院电子学研究所 三维纳隙网格阵列微电极生物传感芯片
CN102099666A (zh) * 2008-06-06 2011-06-15 华盛顿大学 用于从溶液浓缩颗粒的方法和系统
CN102866193A (zh) * 2012-09-04 2013-01-09 吴传勇 基于介电泳来操控液体中的粒子的器件及方法
WO2014122873A1 (ja) * 2013-02-08 2014-08-14 ソニー株式会社 微小粒子分析装置及び微小粒子分析システム
CN104195028A (zh) * 2014-08-05 2014-12-10 深圳先进技术研究院 用于对特异性细胞进行筛选的微流控芯片及细胞筛选方法
WO2015164847A1 (en) * 2014-04-25 2015-10-29 Berkeley Lights, Inc. Providing dep manipulation devices and controllable electrowetting devices in the same microfluidic apparatus
CN107189929A (zh) * 2017-05-25 2017-09-22 重庆大学 一种用于脑脊液中细胞分选富集的微流控芯片、系统和方法
CN108387488A (zh) * 2018-02-28 2018-08-10 北京怡天佳瑞科技有限公司 颗粒物检测装置及检测方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW577855B (en) * 2003-05-21 2004-03-01 Univ Nat Cheng Kung Chip-type micro-fluid particle 3-D focusing and detection device
DE102006023238A1 (de) * 2006-05-18 2007-11-22 Universität Tübingen Vorrichtung und Verfahren zur kontaktlosen Fixierung, Positionierung, Freigabe und Entnahme von Partikeln
TW200826055A (en) * 2006-12-06 2008-06-16 Gigno Technology Co Ltd Display apparatus and manufacturing method thereof
WO2010104856A2 (en) * 2009-03-09 2010-09-16 Virginia Tech Intellectual Properties, Inc. Devices and methods for contactless dielectrophoresis for cell or particle manipulation
CN106215984B (zh) * 2016-05-04 2018-07-13 大连理工大学 基于介电泳作用的微流控芯片
EP3418717A1 (en) * 2017-06-23 2018-12-26 Cellix Limited A microfluidic apparatus for separation of particulates in a fluid

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101046458A (zh) * 2006-03-30 2007-10-03 中国科学院电子学研究所 三维纳隙网格阵列微电极生物传感芯片
CN102099666A (zh) * 2008-06-06 2011-06-15 华盛顿大学 用于从溶液浓缩颗粒的方法和系统
CN102866193A (zh) * 2012-09-04 2013-01-09 吴传勇 基于介电泳来操控液体中的粒子的器件及方法
WO2014122873A1 (ja) * 2013-02-08 2014-08-14 ソニー株式会社 微小粒子分析装置及び微小粒子分析システム
WO2015164847A1 (en) * 2014-04-25 2015-10-29 Berkeley Lights, Inc. Providing dep manipulation devices and controllable electrowetting devices in the same microfluidic apparatus
CN104195028A (zh) * 2014-08-05 2014-12-10 深圳先进技术研究院 用于对特异性细胞进行筛选的微流控芯片及细胞筛选方法
CN107189929A (zh) * 2017-05-25 2017-09-22 重庆大学 一种用于脑脊液中细胞分选富集的微流控芯片、系统和方法
CN108387488A (zh) * 2018-02-28 2018-08-10 北京怡天佳瑞科技有限公司 颗粒物检测装置及检测方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265327A (zh) * 2021-04-26 2021-08-17 大连海事大学 一种基于藻类脂质含量的交流-介电泳微藻多级分选装置及方法
CN113265327B (zh) * 2021-04-26 2022-06-14 大连海事大学 一种基于藻类脂质含量的交流-介电泳微藻多级分选装置及方法
CN115193358A (zh) * 2022-07-21 2022-10-18 扬州大学 一种试管形多功能微流控反应装置
CN115193358B (zh) * 2022-07-21 2024-05-28 扬州大学 一种试管形多功能微流控反应装置

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