WO2019128841A1 - Spiral microchannel, use method thereof, and series/parallel-connected installation structure - Google Patents

Spiral microchannel, use method thereof, and series/parallel-connected installation structure Download PDF

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WO2019128841A1
WO2019128841A1 PCT/CN2018/122352 CN2018122352W WO2019128841A1 WO 2019128841 A1 WO2019128841 A1 WO 2019128841A1 CN 2018122352 W CN2018122352 W CN 2018122352W WO 2019128841 A1 WO2019128841 A1 WO 2019128841A1
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microchannel
spiral
microchannels
cell
outlet
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PCT/CN2018/122352
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French (fr)
Chinese (zh)
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黄庆
魏昆
王柳
苏宁
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黄庆
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • 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
    • 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
    • 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
    • G01N2015/1028—Sorting particles

Definitions

  • the invention belongs to the field of rare cell or particle enrichment screening, and relates to a spiral microchannel and a method for using the same, and a series and parallel installation structure.
  • Circulating tumor cells refer to tumor cells that are detached from the primary and metastatic tumor lesions, invade and enter the circulatory system such as lymphatic vessels and blood. Circulating tumor cells detected in the circulatory system suggest the presence of tumor micrometastasis and the possibility of further development into distant metastases. In recent years, many research reports have confirmed that circulating tumor cells have potential value in early diagnosis, individualized treatment, and exploration of tumor metastasis mechanisms. Compared with tumor tissue cells of solid tumors, the detection of circulating blood cells of peripheral blood has the advantages of reproducible detection, simple sampling, and no trauma. It is called liquid biopsy and has become a hot spot for accurate diagnosis of tumors in recent years.
  • Circulating tumor cells in the circulatory system are not only morphologically and genetically heterogeneous, but also very rare. Even in patients with advanced tumors, there are only 1-10 circulating tumor cells per milliliter of whole blood. Therefore, it is necessary to achieve circulation.
  • the detection of tumor cells is an indispensable step for sorting and enriching, and the advantages and disadvantages of circulating tumor cell sorting and enrichment will directly affect its subsequent detection effects (eg counting, gene amplification, genes). Sequencing, cell culture).
  • Microfluidic chip based on inertial microfluidic principle has become one of the commonly used methods for label-free sorting and enrichment of circulating tumor cells in recent years. Its main feature is that it can be implemented in microchannels without applying any external force, such as electric power and magnetic force. The focused flow of certain size particles, including cells.
  • the microfluidic chip based on the inertial microfluidic principle can be mainly divided into three types: a linear microchannel, a curved microchannel, and a spiral microchannel. Among them, the spiral microchannel is a curved channel. a special form.
  • the flow velocity of the fluid exhibits a parabolic distribution: the velocity of the fluid layer in the middle of the channel is the largest, the closer the fluid layer is to the channel wall, the lower the flow rate, and as the fluid layer approaches The distance of the channel wall is reduced, the flow rate is also reduced by a certain ratio, and the fluid velocity closest to the channel wall is the smallest.
  • This parabolic flow velocity distribution produces a shear force gradient that induces lift that pushes particles suspended in the fluid toward the channel walls. When the particles migrate to a position sufficiently close to the channel wall, the lift induced by the channel walls pushes the particles away from the channel walls.
  • the resultant force of the above two opposite lift forces is referred to as the inertia lift F L .
  • the inertial lift acts on the particles, causing the particles to move relative to each other in the cross section of the channel.
  • the inertial lift is zero, and the particle is stabilized at the cross-section, so that the particle is focused in a stable position in the cross-section. Focus on the flow and flow downstream.
  • the position at which the particles are balanced in the cross section of the linear microchannel is related to the shape of the cross section of the microchannel: in the microchannel of the circular cross section, the particles are focused to form a ring; in the microchannel of the square cross section, the particles
  • the position of the focus is reduced to four, located in the middle of the four faces; in the microchannel of the rectangular cross section, when the vertical and horizontal of the microchannel is relatively large, that is, when the microchannel is particularly wide or particularly high, the position of the particle focus will be Reduce to two, located in the middle of the two longer faces.
  • the fluid flowing in a parabola has the highest velocity in the middle of the channel.
  • the fluid in the middle of the microchannel is subjected to the largest centrifugal force due to its maximum flow rate, thereby flowing to the outer side wall of the curved microchannel.
  • the fluid flow near the walls of the microchannel is minimal and the centrifugal force is minimal, which is squeezed by the intermediate high flow rate fluid.
  • a pair of counter-rotating and symmetrical vortices are formed in a direction perpendicular to the flow of the fluid, respectively located at the upper and lower portions of the cross-section of the microchannel, thereby creating a type of Dean The secondary flow of the eddy current.
  • the Dean vortex acts as a drag force on particles in the fluid, known as Dean drag F D . Therefore, in a curved arc-shaped microchannel, the flowing particles are simultaneously subjected to inertial lift and Dean drag, and the relative magnitudes of the two forces determine the focused flow of the particles in the curved passage.
  • the microchannel spiral microchannel is a special curved microchannel which has the above-mentioned characteristic characteristics of the curved flow of the curved microchannel and also has its own unique characteristics, and therefore, is relatively straight and curved with respect to the above.
  • Curved microchannels, spiral microchannels have unique application advantages in inertial microfluidic chips. In combination with the inertial migration force of the laminar fluid in the spiral microchannel and the Dean drag, the particles move back and forth between the inner and outer sidewalls of the pipe and form a Dean vortex.
  • the above lateral migration is called the Dean cycle: when a particle initially on the outer sidewall moves laterally to the inner sidewall through a length of microchannel, it is said to complete 0.5 Dean cycles; when the same particle passes through When a certain length of downstream microchannel returns to the original outer sidewall, it is said to complete a complete Dean cycle, or complete one Dean cycle.
  • larger particles especially a diameter of a/h ⁇ 0.07) are also subjected to inertial lift, so that the focus position of the particles is extremely dependent on inertial lift and Dean.
  • the ratio of the drag force (F L /F D ), the ratio is exponentially related to the size of the particle, and the focus of the particle is mainly dependent on the smallest microchannel scale parameter (such as the height h of the spiral microchannel).
  • Larger particles ie: a/h ⁇ 0.07), Dean drag and inertial lift are in an equilibrium state, after a certain length of microchannels, they are focused on the inner side wall of the spiral channel and in the downstream microchannel It is always kept on the inner side wall of the microchannel; while the smaller particles (ie: a/h ⁇ 0.07), the Dean drag force predominates, and the particles are continuously in the form of Dean vortex in the outer and inner sidewalls of the microchannel. Move back and forth.
  • a sheath inlet can be designed in the inner sidewall portion of the microchannel by designing a particle inlet in the outer sidewall portion of the microchannel.
  • the sheath fluid with higher velocity will squeeze the particle flow to the outer sidewall of the microchannel; when the particles flow in the microchannel, all the initial state (ie, the entrance) of the particles at Dean drag
  • the microchannel length of 0.5 Dein cycle from the outer sidewall to the inner sidewall when reaching the inner sidewall, the larger particles (ie: a/h ⁇ 0.07) are strongly resisted by strong inertial lift.
  • Dean vortices in the form of smaller particles migrate back and forth laterally outer and inner sidewalls of the microchannel wall. It can be seen that the larger particles always have an inertial focusing stream on the inner side wall of the spiral microchannel, while the smaller particles migrate laterally back and forth in the outer and inner sidewalls of the microchannel in the form of a Dean vortex.
  • a spiral microchannel of a particular length is designed with its exit just at or near the outer sidewall, larger particles can be separated from the smaller particles by appropriate outlets to achieve enrichment of particles of a particular size.
  • the above characteristics of the spiral microchannel are widely used for the sorting and enrichment of peripheral blood circulating tumor cells in patients with malignant tumors.
  • the main principle is that the size of circulating tumor cells (about 20-30 ⁇ m in diameter) is larger than that of red blood cells (about 6-8 ⁇ m in diameter) and white blood cells (about 8-12 ⁇ m in diameter) in peripheral blood. Ratio of rectangular cross-section, at a specific fluid flow rate, peripheral blood circulating tumor cells will flow along the inner sidewall of the spiral microchannel to the cell outlet, and the remaining cells in the sample will flow along the outer wall of the channel. To the waste liquid outlet, thereby achieving the separation of circulating tumor cells. At the same time, cells with different sizes can be shunted from each particular outlet by further designing multiple outlets.
  • spiral inertial microfluidic chips Compared with linear and curved inertial microfluidic chips, spiral inertial microfluidic chips have unique advantages in the field of sorting and enriching circulating tumor cells, and are mainly manifested in the ability to rapidly process whole blood samples to 3 mL/min. speed.
  • the existing spiral inertial microfluidic chip is mainly a single-channel single-spiral microchannel inertial microfluidic chip.
  • the main drawback is that the obtained circulating tumor cells have low purity, which leads to subsequent identification and clinical practice of circulating tumor cells. There are major difficulties in application, and its actual clinical application value is low.
  • spiral microchannels still have large deficiencies and related technical bottlenecks in sorting and enriching circulating tumor cells, the existing research results suggest that if you can subtly "cascade" multiple individual applications
  • the spiral microchannel can greatly improve the purity of circulating tumor cells in the sorted and enriched cell mixture under the premise of ensuring good capture efficiency and processing flux, so as to meet the actual clinical needs.
  • an object of the present invention is to provide a spiral microchannel, a method of using the same, and a series and parallel mounting structure.
  • the present invention provides the following technical solutions:
  • a spiral microchannel for sorting and enriching rare cells or particles comprising N single spiral microchannels, the single single spiral microchannel including an inlet, a microchannel sealingly connected to the inlet, and a channel away from the inlet An outlet that is sealed at one end, the inlet is formed by a cell inlet and a sheath inlet, and the outlet is formed by a waste liquid outlet and a cell outlet; the (M-1) single spiral microchannel cells The outlet is sealingly connected to the cell inlet of the Mth single spiral microchannel into the (M-1)th graded circulation point, the waste liquid outlet of the (M-1) single spiral microchannel, the Mth single helix
  • the sheath liquid inlet of the microchannel and the (M-1)th graded circulation point constitute the (M-1)th classification cycle;
  • a liquid pressure flow rate is provided at a waste liquid outlet of the (M-1) single spiral microchannel in the (M-1)th classification cycle, and a liquid flow rate of the cell inlet of the Mth single spiral microchannel is V1, the liquid flow rate of the cell inlet of the (M-1) single spiral microchannel is v2; the liquid flow rate of the cell outlet of the Mth single spiral microchannel is v3, the (M-1) single spiral
  • the liquid flow rate of the cell outlet of the microchannel is v4; the liquid flow rate of the waste liquid outlet of the Mth single spiral microchannel is v5, and the liquid flow rate of the waste liquid outlet of the (M-1) single spiral microchannel is v6 ;
  • the N single spiral microchannels form spiral microchannels that do not intersect each other;
  • the cell inlet of the first single spiral microchannel is used to enter a sample liquid containing rare cells or particles; the cell outlet of the Nth single spiral microchannel is used to collect rare cells or particles after sorting and enrichment;
  • the sheath fluid inlet is used to add a diluent with osmotic pressure to ensure the integrity of rare cells or particles; 1 ⁇ M ⁇ N, M, N are positive integers.
  • the pressure balance module is disposed at a waste liquid outlet of the (M-1) single spiral microchannel disposed in the (M-1)th classification cycle and is sealingly connected to the waste liquid outlet.
  • the pressure equalization microchannel is disposed in the same plane as the N single spiral microchannels.
  • the pressure balance module is a pressure pump disposed at a waste liquid outlet of the (M-1) single spiral microchannel in the (M-1)th classification cycle.
  • the inlet of the first single helical microchannel and the outlet of the Nth single helical microchannel are located outside of the helical microchannel geometry.
  • the spiral microchannels are formed by N single spiral microchannels forming mutually parallel loops.
  • the horizontal distance between the single spiral microchannel between the spiral microchannels and the single spiral microchannel adjacent thereto and parallel is 0.1-1000 ⁇ m.
  • the single single spiral microchannel has a rectangular or trapezoidal cross section.
  • the single spiral microchannel has a trapezoidal cross section, and the height of one side of the trapezoid near the geometric center of the spiral microchannel is lower than the height of the trapezoid away from the geometric center of the spiral microchannel.
  • the microchannel has a width w of 0.1-1000 ⁇ m and a height of 0.1-800 ⁇ m.
  • the spiral microchannel consists of a first single spiral microchannel and a second single spiral microchannel.
  • the distance between the entrance of the single single helical microchannel and the exit of the single helical microchannel is 1.7-2.0 Dean cycles of non-target or non-target particle hydrodynamic characteristics, or, 2.7- 3.0 Dean cycles, or 3.7-4.0 Dean cycles, or 4.7-5.0 Dean cycles, or 5.7-6.0 Dean cycles, or 6.7-7.0 Dean cycles, or 7.7- 8.0 Dean cycles, or 8.7-9.0 Dean cycles, or 9.7-10.0 Dean cycles.
  • the single-spiral microchannel has a channel cross-sectional width at a maximum width of l1, and the single-spiral microchannel has a cell inlet and/or a cell outlet having a width of l2, 0.1*l1 ⁇ l2 ⁇ 0.4*l1;
  • the width of the sheath liquid inlet and/or the waste liquid outlet of the single spiral microchannel is l3, 0.1*l1 ⁇ l3 ⁇ 0.9*l1.
  • the spiral microchannel comprises a first single spiral microchannel, a second single spiral microchannel, a third single spiral microchannel, and a fourth single spiral Microchannel composition.
  • the rare cells or particles have a diameter of a, and the height of the microchannels is h, 0.07 ⁇ a/h ⁇ 0.5.
  • the rare cells or particles are circulating tumor cells, circulating progenitor cells, circulating fetal cells, cell vesicles, exosomes or pathogens.
  • a detection unit for identifying and/or counting rare cells or particles sorted and enriched for the spiral channel; the detection unit being disposed in the cell of the Nth single helical microchannel exit.
  • the detecting unit performs the identification of the characteristics of the rare cells or particles by a specific technical means;
  • the specific technical means is selected from the group consisting of bio-optics, bioelectrics, bioacoustics, biomagnetism and any combination thereof.
  • the detecting unit performs identification of a rare cell or particle trait by detecting a specific feature selected from a terahertz spectrum, a Raman spectrum, an impedance, a differential impedance, and any of the rare cells or particles.
  • a specific feature selected from a terahertz spectrum, a Raman spectrum, an impedance, a differential impedance, and any of the rare cells or particles.
  • the pressure-balanced microchannels and the N single-spiral microchannels form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed in a spiral micro-channel Outside the geometry of the channel, the flow rate through the contents of the (M-1) cell outlet is adjusted by adjusting the length and inner diameter of the (M-1)th pressure-balanced microchannel.
  • outlets of the (N-1) pressure equalization microchannels are each disposed outside of the geometry of the spiral microchannel.
  • one or more of the N of said cell outlets are disposed outside of the geometry of the helical microchannel.
  • all cell outlets are disposed outside of the geometry of the helical microchannel.
  • one or more of the N inlets are disposed outside of the geometry of the spiral microchannel.
  • all of the cell inlets are disposed outside of the geometry of the spiral microchannel.
  • all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
  • a method of using a spiral microchannel comprising the steps of:
  • S2 collecting a fluid rich in rare cells or particles at the cell outlet of the Nth single spiral microchannel; discharging the waste liquid in the first single spiral microchannel to the waste liquid outlet of the Nth single spiral microchannel Drain the waste liquid.
  • the sample liquid used in step S1 is a blood sample having a certain ratio of red blood cells, or a blood sample in which a large amount of red blood cells is removed by hemolysis; the ratio of red blood cells in the blood sample is 0% to 70%.
  • the diluent used in step S1 has a certain osmotic pressure for protecting blood cell integrity, and is phosphate buffer, culture serum or physiological saline.
  • step S1.1 further comprising step S1.1;
  • a series installation structure of spiral microchannels characterized in that, by means of P spiral microchannels arranged in series, the cell outlet of the Nth single spiral microchannel of the (Q-1) spiral microchannel Connected to the first single spiral microchannel inlet of the Qth spiral microchannel to form a spiral microchannel series installation, 1 ⁇ Q ⁇ P.
  • a pressure equalization module is also provided at the waste liquid outlet of the Nth single spiral microchannel of the (Q-1)th spiral microchannel at the spiral microchannel series installation.
  • the pressure balance module is a pressure balanced microchannel sealingly connected to a waste liquid outlet of the Nth single spiral microchannel of the (Q-1)th spiral microchannel in a series installation of the spiral microchannel.
  • the pressure-balanced microchannel and the matching helical microchannel form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed outside the geometry of the spiral microchannel By adjusting the length and inner diameter of the pressure-balanced microchannel to adjust the flow rate through the contents in the cell outlet that matches it.
  • outlets of all of the pressure balanced microchannels are disposed outside of the geometry of the helical microchannels.
  • one or more of the cell outlets are disposed outside of the geometry of the helical microchannel.
  • all cell outlets are disposed outside of the geometry of the helical microchannel.
  • one or more of the inlets are disposed outside of the geometry of the spiral microchannel.
  • all of the cell inlets are disposed outside of the geometry of the spiral microchannel.
  • all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
  • the pressure equalization module is a pressure pump disposed at the waste liquid outlet of the Nth single spiral microchannel of the (Q-1)th spiral microchannel at the spiral microchannel series installation.
  • the spiral microchannel is a double helix microchannel.
  • a parallel installation structure of spiral microchannels adopts a method in which X spiral microchannels are arranged in parallel, and sample solution enters from the cell inlet of the first single spiral microchannel of the X spiral microchannels, and rare cells or particles Flowing out of the cell outlet of the Nth single helical microchannel of the X helical microchannels; X>1.
  • the cell inlets of the first single helical microchannel of the X helical microchannels are interconnected.
  • the cell outlets of the Nth single helical microchannels of the X helical microchannels are interconnected.
  • the X spiral microchannels are disposed in the same plane.
  • the X spiral microchannels are concentrically disposed in the same plane.
  • the X spiral microchannels are concentric and arranged in a direction perpendicular to the plane of the single spiral microchannel.
  • the sample solution enters from the cell inlet of the first single spiral microchannel of the two helical microchannels, and the rare cells or particles are from two The cell outlet of the Nth single helical microchannel of the helical microchannel flows out.
  • the sample solution enters from the cell inlet of the first single spiral microchannel of the X double helix microchannels, and the rare cells or particles are The cell outlets of the second single helical microchannel of the X double helix microchannels flow out.
  • a pressure equalization module is provided at the waste liquid outlet of the Nth single spiral microchannel of the Xth spiral microchannel in the spiral microchannel.
  • the pressure balance module is a pressure pump.
  • the pressure balance module is a pressure balanced microchannel.
  • the pressure-balanced microchannel and the matching helical microchannel form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed outside the geometry of the spiral microchannel By adjusting the length and inner diameter of the pressure-balanced microchannel to adjust the flow rate through the contents in the cell outlet that matches it.
  • outlets of all of the pressure balanced microchannels are disposed outside of the geometry of the helical microchannels.
  • one or more of the cell outlets are disposed outside of the geometry of the helical microchannel.
  • all cell outlets are disposed outside of the geometry of the helical microchannel.
  • one or more of the inlets are disposed outside of the geometry of the spiral microchannel.
  • all of the cell inlets are disposed outside of the geometry of the spiral microchannel.
  • all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
  • a combined connection mounting structure of a spiral microchannel comprising K microchannel monomers, the microchannel monomer being the spiral microchannel according to any one of claims 1 to 27;
  • the body is combined with each other in series, parallel, multiple series, multiple parallel, or series-parallel combination, wherein K is a positive integer and K ⁇ 2;
  • the connection form of the series is the (K-1) microchannel single
  • the Nth cell outlet of the body is sealingly connected with the first cell inlet of the Kth microchannel monomer to form a series spiral microchannel;
  • the parallel connection form is the (K-1) microchannel monomer
  • the first cell inlet is combined with the first cell inlet of the Kth microchannel monomer to form a uniform cell inlet;
  • the first sheath inlet of the (K-1) microchannel monomer is The first sheath fluid inlet sealing junction of the K microchannel monomers is combined into a uniform sheath fluid inlet to form a parallel spiral microchannel.
  • the plurality of series connection forms are a combination of a plurality of series spiral microchannels connected in series in a series connection; or a series spiral microchannel and a microchannel monomer are combined with each other in a series connection.
  • the parallel connection form further comprises sealingly combining the Nth cell outlet of the (K-1) microchannel monomer with the Nth cell outlet of the Kth microchannel monomer to form a unified cell.
  • the outlet; the Nth waste liquid outlet of the (K-1) microchannel monomer and the Nth waste liquid outlet of the Kth microchannel monomer are sealed and combined to form a unified waste liquid outlet.
  • the multiple parallel connection forms are a combination of a plurality of parallel spiral microchannels in parallel connection; or a parallel spiral microchannel and a microchannel unit are combined in parallel.
  • the series-parallel combination is in the form of a series connection of one or more parallel spiral microchannels and/or one or more series spiral microchannels and/or one or more microchannel monomers.
  • Formal or parallel connection forms are combined with each other to form a hybrid spiral flow path.
  • a single spiral microchannel comprising an inlet, a microchannel sealingly connected to the inlet, and an outlet sealingly connected to an end of the channel remote from the inlet, the inlet being formed by a cell inlet and a sheath fluid inlet, the outlet being The waste liquid outlet and the cell outlet are aggregated, wherein the cell inlet is used to enter a sample liquid containing rare cells or particles; the cell outlet is used to collect the rare cells or particles after sorting and enrichment; the sheath liquid inlet is used for adding Osmotic pressure ensures a complete dilution of rare cells or particles.
  • the cell inlet and/or sheath fluid inlet and/or waste outlet and/or cell outlet are disposed outside of the geometry of the microchannel.
  • the cell inlets are connected, 1 ⁇ Q ⁇ P.
  • a pressure equalization module is disposed at the waste liquid outlet of the (Q-1) single spiral microchannel.
  • the pressure balance module is a pressure pump or a pressure balanced microchannel.
  • the pressure equalization module is a pressure balanced microchannel that is sealingly coupled to the waste liquid outlet of the (Q-1) single spiral microchannel.
  • the pressure-balanced microchannel and all of the single-helical microchannels matched thereto form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed in the geometry of the spiral microchannel Outside the graph, the flow rate through the contents of the cell outlet is matched by adjusting the length and inner diameter of the pressure-balanced microchannel.
  • outlets of all of the pressure balanced microchannels are disposed outside of the geometry of the helical microchannels.
  • the cell inlet and/or sheath fluid inlet and/or waste outlet and/or cell outlet are disposed outside of the geometry of the helical microchannel.
  • a parallel installation structure of single spiral microchannels adopts X single spiral microchannels in parallel, the sample solution enters from the cell inlet of X single spiral microchannels, and rare cells or particles from X single spirals The cell outlet of the microchannel flows out; X>1.
  • the cell inlets and/or cell outlets of the X single helical microchannels are interconnected.
  • a pressure equalization module is disposed at the waste liquid outlet of the Xth single spiral microchannel; the pressure balance module is a pressure pump or a pressure balanced microchannel.
  • the pressure-balanced microchannel and the matched single-helical microchannel form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed on the geometry of the spiral microchannel Externally, the flow rate through the contents of the cell outlet that matches it is adjusted by adjusting the length and inner diameter of the pressure-balanced microchannel.
  • all cell inlets and/or sheath fluid inlets and/or waste outlets and/or cell outlets are disposed outside of the geometry of the microchannels.
  • all cell inlets and/or sheath fluid inlets are disposed outside of the geometry of the helical microchannels.
  • the invention aims at the technical problem of the spiral microchannel in the field of sorting and enriching the peripheral blood circulation tumor cells of malignant tumor patients, develops a simpler and more effective technology, and skillfully designs a series connection of a plurality of spiral microchannels by rational cascade.
  • the parallel spiral microchannel inertial separation structure realizes cascading superposition hierarchical enrichment and/or purification of circulating tumor cells on a single spiral inertial microfluidic chip.
  • the present invention not only has a good collection efficiency and processing flux of circulating tumor cells, but also maximizes the purity of circulating tumor cells, for sorting enrichment and/or peripheral blood circulating tumor cells.
  • Embodiment 1 is a schematic structural view of Embodiment 1 of the present invention.
  • Embodiment 2 is a schematic structural view of Embodiment 2 of the present invention.
  • Embodiment 3 is a schematic structural view of Embodiment 3 of the present invention.
  • Figure 4 is a schematic structural view of Embodiment 4 of the present invention.
  • Figure 5 is a schematic structural view of Embodiment 5 of the present invention.
  • Figure 6 is a schematic structural view of Embodiment 6 of the present invention.
  • Figure 7 is a schematic structural view of Embodiment 7 of the present invention.
  • Figure 8 is a motion trajectory diagram of particles of different sizes at different flow rates in the first embodiment of the present invention.
  • Figure 8a is a motion trajectory of the particles of different sizes at a flow rate of 150 ⁇ l/min in the first embodiment
  • Figure 8b is a motion trajectory of the particles of different sizes at a flow rate of 300 ⁇ l/min in the first embodiment
  • Figure 8c is a motion trajectory of the particles of different sizes at a flow rate of 450 ⁇ l/min in the first embodiment
  • Figure 8d is a motion trajectory of the particles of different sizes at a flow rate of 600 ⁇ l/min in the first embodiment
  • Figure 8e is a motion trajectory of the particles of different sizes at a flow rate of 750 ⁇ l/min in the first embodiment
  • Figure 8f is a motion trajectory of the particles of different sizes at a flow rate of 900 ⁇ l/min in the first embodiment
  • Figure 9 is a micrograph of the exit of different spiral microchannels in the first embodiment of the present invention.
  • Figure 9a is a photomicrograph at the exit of a conventional single-spiral microchannel cell
  • Figure 9b is a micrograph of the outlet of the double helix microchannel cell of the present invention.
  • Figure 9c is a photomicrograph of the double spiral microchannel waste liquid outlet 7 of the present invention.
  • Figure 10 is a diagram showing the fluid flow velocity in the spiral microchannel in the first embodiment of the present invention.
  • Figure 11 is a conventional single spiral microchannel
  • Figure 12 is a schematic structural view of Embodiment 8 of the present invention.
  • Figure 13 is a schematic structural view of Embodiment 9 of the present invention.
  • Figure 14 is a schematic structural view of Embodiment 10 of the present invention.
  • Figure 15 is a schematic structural view of Embodiment 11 of the present invention.
  • Figure 16 is a schematic structural view of Embodiment 12 of the present invention.
  • Figure 17 is a schematic structural view of Embodiment 13 of the present invention.
  • Figure 18 is a schematic structural view of Embodiment 14 of the present invention.
  • Figure 19 is a schematic structural view of Embodiment 15 of the present invention.
  • Figure 20 is a schematic structural view of Embodiment 16 of the present invention.
  • Figure 21 is a schematic structural view of Embodiment 17 of the present invention.
  • Figure 22 is a schematic structural view of Embodiment 18 of the present invention.
  • Figure 23 is a simulation result of the eighth embodiment of the present invention, showing the trajectories of the polystyrene microspheres at different flow rates;
  • the flow rate in Figure 23a is 200 ul / min
  • the flow rate in Figure 23b is 350 ul / min
  • the flow rate in Figure 23c is 500 ul / min
  • the flow rate in Figure 23d is 650 ul / min
  • the flow rate in Figure 23e is 800 ul / min
  • the flow rate in Figure 23f is 950 ul / min
  • Figure 24 is a comparison diagram of simulation results of Embodiment 8 and Embodiment 14 of the present invention.
  • Figure 24a shows the enrichment at the cell exit of the second single helical microchannel in Example VIII
  • Figure 24b shows the enrichment at the cell exit of the conventional single-spiral microchannel of Figure 11;
  • Figure 24c shows the enrichment at the cell outlet of the first single helical microchannel in Example VIII
  • Figure 24d shows the enrichment of the waste liquid outlet at the conventional single spiral microchannel shown in Figure 11;
  • Figure 24e shows the enrichment at the waste liquid outlet of the second single spiral microchannel in the eighth embodiment
  • Figure 25 is a view showing the results of immunofluorescence staining of the waste liquid outlet of the second single-spiral microchannel in the eighth embodiment and the cells collected at the cell outlet;
  • Figure 25a shows the cell morphology of the cells collected at the waste liquid outlet of the second single-spiral microchannel at 40 times the light microscope
  • Figure 25b shows the results of DAPI fluorescence staining of leukocytes enriched by double-helical chip sorting
  • Figure 25c is a result of fluorescent staining of leukocyte CD45 antibody enriched by double-helical chip sorting
  • Figure 25d shows the result of fluorescent staining of leukocytes by double-stranded chip sorting
  • Figure 25e is the cell morphology of the cells collected at the cell exit of the second single-spiral microchannel at 40 times the light microscope;
  • Figure 25f shows the SW480DAPI fluorescence staining results of the double-helical chip sorting and enrichment
  • Figure 25g shows the results of fluorescent staining of SW480EPCAM enriched by double-helical chip sorting.
  • Figure 25h is a result of SW480 fluorescence staining Merge obtained by double-helical chip sorting and enrichment
  • Figure 26 is a result of analysis of the capture efficiency of tumor cells by a double helix chip
  • Figure 26a shows the recovery of different concentrations of tumor cells in a whole blood sample by a double helix chip
  • Figure 26b is a histogram of the capture efficiency of different tumor cells by a double helix chip.
  • the cell inlet 1 of the first single spiral microchannel, the sheath fluid inlet 2 of the first single spiral microchannel, and the first single spiral micro The inlet A of the channel, the pressure-balanced microchannel 3, the cell outlet 4 of the first single-spiral microchannel, the sheath inlet 5 of the second single-spiral microchannel, and the waste outlet of the first single-spiral microchannel 6.
  • the outlet B of the first single spiral microchannel, the waste outlet 7 of the second single spiral microchannel, the cell outlet 8 of the second single spiral microchannel, and the second single spiral microchannel The outlet liquid C, the sheath liquid inlet 9 of the first single spiral microchannel in the second spiral microchannel, the waste liquid outlet 10 of the first single spiral microchannel of the second spiral microchannel, and the second The sheath flow inlet 11 of the second single spiral microchannel of the spiral microchannel, the cell outlet 12 of the second single spiral microchannel of the second spiral microchannel, and the second spiral microchannel
  • the waste liquid outlet 13 of the two single spiral microchannels, the first single of the second spiral microchannel The cell inlet 14 of the spiral microchannel, the sheath inlet 15 of the third single helical microchannel, the waste outlet 16 of the third single helical microchannel, and the sheath inlet 17 of the fourth single helical microchannel
  • the present invention relates to a spiral microchannel for sorting and enriching rare cells or particles, in this embodiment comprising two single spiral microchannels, the two single spiral microchannels being disposed in the same plane and mutually It is wound into a ring without crossing, as shown in Figure 1.
  • the single single spiral microchannel includes an inlet A, a microchannel sealingly connected to the inlet, and an outlet B sealingly connected to an end of the channel remote from the inlet, the inlet being aggregated by the cell inlet 1 and the sheath inlet 2,
  • the outlet B is formed by the waste liquid outlet 6 and the cell outlet 4;
  • the cell outlet 4 of the first single-spiral microchannel is sealingly connected to the cell inlet of the second single-spiral microchannel to form a first staged circulation point, and the waste liquid outlet of the first single-spiral microchannel is 6,
  • the sheath liquid inlet 5 of the two single spiral microchannels and the first staged circulation point constitute the first classification cycle;
  • the waste liquid outlet 6 of the first single spiral microchannel in the first classification cycle is provided with a pressure equalization microchannel 3, and the liquid flow rate of the cell inlet of the second single spiral microchannel is v1, the first The liquid flow rate of the cell inlet 1 of the single spiral microchannel is v2; the liquid flow rate of the cell outlet 8 of the second single spiral microchannel is v3, and the liquid flow rate of the cell outlet 4 of the first single spiral microchannel is v4
  • the liquid flow rate of the waste liquid outlet 7 of the second single spiral microchannel is v5, and the liquid flow rate of the waste liquid outlet 6 of the first single spiral microchannel is v6;
  • the cell inlet 1 of the first single-spiral microchannel is used to enter a sample liquid containing rare cells or particles;
  • the cell outlet 8 of the second single-spiral microchannel is used to collect rare cells after sorting and enrichment or Particles;
  • the sheath fluid inlet 2 of the first single spiral microchannel and the sheath fluid inlet 5 of the second single spiral microchannel are used to add osmotic pressure to ensure rare cell or particle integrity and biological activity Diluent.
  • the second single-helical microchannel can directly sort and enrich the rare cells or particles enriched by the first single-spiral microchannel, thereby A single single-helical microchannel captures rare cells or particles more efficiently, which is equivalent to two sorts of enrichment and purification of rare cells or particles, which capture the target cells, throughput, cell purity, and cells.
  • the four parameters of activity are superior to single spiral microchannels.
  • the double helix microchannel of the present invention comprises a first single spiral microchannel and a second single spiral microchannel formed by a direct connection between the first spiral and the microchannel, and a double spiral microchannel having a parallel appearance and the same center point.
  • the geometric structure is characterized in that the inlet A of the first single spiral microchannel of the double helix microchannel and the outlet of the second single spiral microchannel are located outside the geometry of the microchannel body.
  • Each of the single spiral microchannels of the double helix microchannel of the present invention has a cell inlet 1, a sheath inlet 2, a cell outlet 4, and a waste liquid outlet 6, wherein the cell inlet 4 and the sheath fluid inlet 2 converge into each The inlet A of the single spiral microchannel, the cell outlet 4 and the waste outlet 6 converge into the outlet B of each single spiral microchannel.
  • the cell inlet 1 is typically located at the outer sidewall portion of the inlet of the intended single helical microchannel
  • the sheath fluid inlet 2 is typically located at the inner sidewall portion of the inlet of the intended single helical microchannel
  • the cell outlet 4 is typically located at the inner sidewall of the exit of the intended single helical microchannel.
  • the waste liquid outlet 6 is typically located at the outer sidewall portion of the outlet of the intended single spiral microchannel.
  • the two single spiral microchannels of the double spiral microchannel of the present invention have the same or similar rectangular cross-sectional planes defining the aspect ratio, and may also be a trapezoidal cross-section with an inner sidewall height lower than the outer sidewall height for the target.
  • the difference in size between the cells and the non-target cells allows only the target cells to form a focused flow in the inner walls of the microchannels, while the non-target cells migrate laterally back and forth between the inner and outer sidewalls of the microchannels in a Dean vortex.
  • the width of the microchannel is 0.1-1000 ⁇ m
  • the depth is 0.1-800 ⁇ m, or further, the width of the microchannel is 100-800 ⁇ m, the depth is 50-500 ⁇ m, or further, the width of the microchannel is 600 ⁇ m, and the depth is 140-180 ⁇ m.
  • the two single helical microchannels of the double helix microchannel of the present invention have the same or similar length and are characterized by the length from the start point to the end point of the spiral microchannel of the first and second single spiral microchannels 1.7-2.0 Dean cycles for non-target cell hydrodynamics, or 2.7-3.0 Dean cycles, or 3.7-4.0 Dean cycles, or 4.7-5.0 Dean cycles, or 5.7-6.0 A Dean cycle, or 6.7-7.0 Dean cycles, or 7.7-8.0 Dean cycles, or 8.7-9.0 Dean cycles, or 9.7-10.0 Dean cycles. Under this condition, at the end of the first and second single-spiral microchannels, the non-target cells migrate along the Dean vortex to the outer sidewall portion of the helical microchannel.
  • the waste liquid outlet of the first single spiral microchannel of the double spiral microchannel of the present invention has a pressure balance microchannel 3.
  • the size and shape of the pressure balance microchannel 3 of the present invention have no special requirements, and There is no special requirement for the ratio, radius of curvature, width, height and length.
  • the function is to balance the pressure of the cell outlet 1 of the first single-spiral microchannel, so that the cell inlet of the second single-spiral microchannel or the first one
  • the flow rate and direction of the cell outlet 4 of the single-spiral microchannel are the same or very similar to the flow rate and direction of the cell inlet 1 of the first single-spiral microchannel and the cell outlet 4 of the second single-spiral microchannel, while The flow rate and direction of the waste liquid outlet 6 of the first single spiral microchannel are kept the same or very close to the flow rate and direction of the waste liquid outlet 7 of the second single spiral microchannel.
  • the horizontal parallel spacing of the first and second helical microchannels of the double helix microchannel of the present invention is from 0.1 to 1000 ⁇ m.
  • the width of the cell inlet 1 microchannel of the first and/or second helical microchannel of the double helix microchannel of the present invention is 10-40% of the overall width of the microchannel, the first and/or the second
  • the width of the microchannel of the waste liquid outlet 6 of the spiral microchannel is 40-90% of the overall width of the microchannel.
  • the blood sample according to the present invention refers to a blood sample having a certain ratio of red blood cells, or a blood sample for removing a large amount of red blood cells by hemolysis, or a red blood cell or only a small amount or a pole obtained by other methods or devices known in the art.
  • a small blood sample of red blood cells refers to a blood sample having a certain ratio of red blood cells, or a blood sample for removing a large amount of red blood cells by hemolysis, or a red blood cell or only a small amount or a pole obtained by other methods or devices known in the art.
  • the sheath fluid of the present invention refers to any of various solutions known in the art having a certain osmotic pressure and not destroying the integrity of blood cells, such as phosphate buffer, culture serum, physiological saline and the like.
  • Whether the particle can form an inertial focusing flow in a curved arc microchannel is related to the size and radius of curvature of the microchannel, the size and flow velocity of the particle.
  • Previous studies have shown that in a rectangular cross-section channel with a channel width greater than the height, a certain size of particles forming an inertial focusing flow usually needs to satisfy the condition a/h ⁇ 0.07, a is the diameter of the particle, and h is the minimum size of the microchannel.
  • the height of the microchannels in an inertial microfluidic chip is typically the smallest size. If the particle size is too small, it may result in the particle not being able to focus inertially within a finite length microchannel. Studies have also shown that a / h ⁇ 0.5 is also a limiting condition, too large particle size may cause the channel to be easily blocked. Therefore, in design preparation, the size of the microchannels is determined according to the size of the separated particles.
  • the invention provides a method for enriching peripheral blood circulating tumor cells from peripheral blood cascade or tandem superposition, specifically comprising: the sample solution to be enriched is injected from the cell inlet 1 of the first single spiral microchannel, The sheath fluid is injected from the sheath fluid inlet 2 of the first single spiral microchannel and the sheath fluid inlet 5 of the second single spiral microchannel, at a certain fluid flow rate, in the second single spiral microchannel.
  • the cell outlet 8 collects the fluid enriched in the target cells, while the non-target cells are collected from the waste liquid outlet 7 of the second single spiral microchannel.
  • the working principle of the double helix microchannel of the present invention is as follows: at the inlet A of the first single spiral microchannel, the blood sample enters the microchannel from the cell inlet 1, and the sheath fluid enters the microchannel from the sheath inlet 2, Since the fluid flow rate of the sheath liquid inlet 2 is higher than the fluid flow rate of the cell inlet 1, all cells of the cell inlet 1 are squeezed to the outer side wall of the first single spiral microchannel, and thereby enter the subsequent microchannel, thus, It can be said that at 0 Dean cycles, the starting position of all cells is the outer side wall of the first single spiral microchannel.
  • the target cells Under the combined action of inertial lift and Dean drag, the target cells form a focused stream on the inner side wall of the first single spiral microchannel due to its large size at 0.5 Dein cycles, and due to its inertia With the effect of lift and Dean's drag, the target cells always maintain a focused flow on their inner sidewalls in the subsequent microchannels of the first single-helical microchannel; non-target cells are mainly subjected to Dean drag due to their small size.
  • the target cells flow out from the cell outlet 4 located on the inner side wall of the first single-spiral microchannel and directly enter the second
  • the cell inlet of the single-spiral microchannel, but not the target cell fluid flows out of the waste liquid outlet 5 located on the outer side wall of the first single-spiral microchannel and is collected via the pressure-balanced microchannel 3; due to the first single-spiral micro
  • the cell outlet 4 of the channel is the cell inlet of the second single-spiral microchannel, so in the second single-spiral microchannel, the first single is operated according to the same principle as the first single-spiral microchannel
  • Spiral microchannel sorting enriched target cells are enriched by the second sorting in the second single spiral microchannel, or cascaded enrichment, or tandem sorting enrichment, or superposition Sorting and enriching.
  • the target cells in the blood sample are subjected to two-stage superposition sorting enrichment in cascade or tandem, and at the same time, since most non-target cells have been Removed by the first helical microchannel, the total number of cells in the cell suspension entering the second single helical microchannel is generally much lower than the total number of cells in the cell suspension of the second single helical microchannel, Cells have less interference due to their density, and their sorting enrichment effect is better than using a single spiral microchannel alone, and finally high-purity target cells can be obtained.
  • the following part of this embodiment is actually comparing the sorting enrichment effect of the double helix channel of the present invention with a conventional single spiral microchannel (as shown in FIG. 11).
  • the spiral microchannel has a width l of 600 ⁇ m, a radius of curvature of 1.15 cm, and a microchannel height h of 130 ⁇ m.
  • the conventional single-helical microchannel (shown in Figure 11) is designed to be designed inside the spiral planar structure, and the outer surface of the planar structure is constructed.
  • the width of the cell inlet and the cell outlet is 0.15 mm, respectively located in the spiral microchannel.
  • the outer and inner side walls, the sheath liquid inlet and the waste liquid outlet have a width of 0.45 mm, respectively located on the inner side wall and the outer side wall of the spiral microchannel, and the cell inlet and the waste liquid inlet are both located inside the spiral microchannel planar geometry.
  • the cell outlet and the sheath fluid outlet are both outside the planar geometry of the spiral microchannel.
  • the width of the cell inlet 1 and the cell outlet 4 of the double helix microchannel of the present invention is 0.15 mm, and the width of the sheath liquid inlet 2 and the waste liquid outlet 6 is 0.45 mm; each single spiral microchannel has a cell inlet 1.
  • the fabrication of two kinds of spiral microchannel chips is mainly through the basic micro-machining technology.
  • the SU8 photoresist is used to make the mold on the silicon wafer by standard soft lithography technology. After the PDMS is demolded, the corresponding entrance and exit are punched. It was processed and bonded on the corresponding slide. In order to increase the firmness of the bond, it was placed in a 70 ° C oven and baked for 30 min.
  • the bonded spiral microchannel chip inlet and outlet are tightly connected with the inlet and outlet micro tubes by means of a magnetic clamp device and a precision guide pin connection, and the injection pump and the flow rate control system are used to achieve stable injection and real-time monitoring of flow rate changes. .
  • the sealing fluid was first tested under the high pressure of one atmosphere under the high pressure of the sheath fluid. The result showed no leakage, indicating that the chip bonding and the chip inlet and outlet connections were firm. The integrity of the chip and the elimination of the bubbles were observed under a microscope until the bubbles were completely removed.
  • the optimal parameters for the enrichment of circulating tumor cells by spiral microchannel sorting were optimized using standard polystyrene colored microbeads.
  • microspheres Three to 6 ⁇ m microspheres, 7 to 10 ⁇ m microspheres, and 15 to 18 ⁇ m microspheres were mixed to simulate red blood cells, white blood cells, and circulating tumor cells, respectively, and phosphate buffers were used for microspheres of 3 to 6 ⁇ m, 7 to 10 ⁇ m, and 15 to 18 ⁇ m. Dilute to 1.0*10 7 particles/ml, 1.0*10 6 particles/ml, 1.0*10 2 particles/ml, respectively, and mix according to an equal volume ratio.
  • the syringe pump loaded with the mixed microspheres and the syringe pump loaded with the phosphate buffer sheath fluid are respectively connected to the outer side of the spiral chip and the inlet of the inner side wall after the bubble elimination through the precision guide needle, and the flow rate is adjusted by the flow rate monitoring system and the pressure pump control.
  • the double helix microchannels (shown in Figure 1) and the conventional single spiral microchannels (shown in Figure 11) of the present invention were separately recorded and collected at different flow rates (i.e., 150 ⁇ l/min, 300 ⁇ l/ Min, 450 ⁇ l/min, 600 ⁇ l/min, 750 ⁇ l/min, 900 ⁇ l/min) microspheres were sorted and enriched at each outlet.
  • FIG. 8 An example of the result of microsphere separation at the cell exit is shown in Fig. 8. Since the ordinary camera cannot clearly capture the motion state of a single rare cell or particle, a smaller diameter circle is expressed for the clear and intuitive expression of the movement trajectory of rare cells or particles.
  • the dots represent rare cells or particles of 3 to 6 ⁇ m and 7 to 10 ⁇ m, and the dots of larger diameter represent the trajectories of rare cells or particles of 15 to 18 ⁇ m.
  • FIG. 9 An example of the microscopic count observation of the separation of the microspheres at the cell outlet 8 and the waste liquid outlet 7 is shown in Fig. 9.
  • Figure 9-a is a micrograph of the exit of a conventional single-spiral microchannel cell, with more microspheres, suggesting that the larger particles are poorly sorted and enriched, and the purity is low;
  • Figure 9-b shows The micrographs at the outlet of the double-spiral microchannel cells of the invention are almost all of the larger diameter microspheres, suggesting that the sorting and enrichment effect of the larger particles is very good and the purity is extremely high;
  • FIG. 9-c is the present invention
  • the microscopic picture of the double spiral microchannel waste liquid outlet 7 shows that almost all of the smaller diameter microspheres have almost no larger diameter microspheres, suggesting that larger particles are enriched to the cell outlet, Good recovery rate.
  • the conventional single spiral microchannel takes 40 minutes when processing 8 ml of blood sample, while the double helix microchannel of the present invention takes only 10 minutes.
  • the purity of the circulating tumor cells obtained by the double helix microchannel of the present invention and the repeatability of the experimental results are higher (similar to those described in Table 1), and the two have statistically significant differences.
  • the above research results suggest that the double helix microchannel of the present invention can meet and meet the clinical actual detection requirements of circulating tumor cells and the harsh conditions for subsequent detection and analysis (such as circulating tumor cell orders) compared with the conventional single spiral microchannels. Cell level gene detection).
  • HCC827, MCF-7, and SW480 tumor cell lines were diluted to 10 2 cells/ml, respectively, and mixed into normal blood cells treated with erythrocyte lysate according to a volume ratio of 1:5, using the double of the present invention.
  • Spiral microchannels were sorted and enriched.
  • the results of the staining smear using immunofluorescence technique are shown in Table 2. The results show that the double helix microchannels of the present invention have a recovery rate of at least 98% for a plurality of tumor cells, and the purity is similar to that of the above double helix microchannels, and both are about 99%.
  • the double helical microchannel detection time of the invention is shortened to 1/4 of the conventional single spiral microchannel, and the purity of the rare cells or particles enriched by sorting is shortened.
  • the methodological parameters such as the repeatability of the experimental results and the recovery rate are far superior to the conventional single spiral microchannels, indicating that the double helix microchannels of the present invention can better satisfy the conventional single spiral microchannels. The actual needs of clinical applications.
  • the pressure pump is disposed at the waste liquid outlet 6 of the first single spiral microchannel.
  • the entire spiral microchannel is composed of four single spiral microchannels.
  • the two-channel spiral flow channel of the present invention can be further evolved into a triple spiral microchannel, a quadrilateral microchannel, or a plurality of spiral microchannels.
  • the number of single spiral microchannels included in the multi-stage spiral microchannel is at least two, and the center of the plurality of single spiral microchannels is located at the same point, the (M-1)th single
  • the cell outlet of the spiral microchannel is the cell inlet of the Mth single spiral microchannel, and the waste liquid outlet of the (M-1) single spiral microchannel is directly connected to the pressure balance channel.
  • the pressure equalization channel functions to make the flow rate and direction of the cell inlet of the (M-1) single spiral microchannel or the cell inlet of the Mth single spiral microchannel and the (M-1) spiral
  • the flow rate and direction of the cell inlet of the microchannel and the cell outlet of the Mth single spiral microchannel remain the same or very close, and at the same time, the flow rate and direction of the waste liquid outlet of the Mth single spiral microchannel are compared with the first (M- 1)
  • the flow rate and direction of the waste liquid outlet of the spiral microchannel remain the same or very close, so that the hydrodynamic characteristics of each spiral microchannel in the multi-stage spiral microchannel to the target cell remain the same or similar .
  • a four-helical microchannel is taken as an example.
  • the cell outlet 4 of the first single spiral microchannel is the cell inlet of the second single spiral microchannel, and the second single spiral micro.
  • the cell outlet of the channel is the cell inlet of the third single-spiral microchannel
  • the cell outlet of the third single-spiral microchannel is the cell inlet of the fourth single-spiral microchannel
  • the first and second And the waste liquid outlet of the third single spiral microchannel has a corresponding pressure equalization channel 3, respectively.
  • the sheath fluid is from the sheath fluid inlet of the first single spiral microchannel, the sheath fluid inlet 5 of the second single spiral microchannel, the sheath fluid inlet 15 of the third single spiral microchannel, and the fourth
  • the sheath liquid inlet 17 of the single spiral microchannel is introduced; the waste liquid is discharged from the waste liquid outlet 6 of the first single spiral microchannel, the waste liquid outlet of the second single spiral microchannel, and the third single spiral
  • the waste liquid outlet 16 of the microchannel and the waste liquid outlet 19 of the fourth single spiral microchannel are derived; finally, the sorted and enriched rare cells or particles are collected at the cell outlet 18 of the fourth single spiral microchannel.
  • two spiral microchannels are connected in series, the cell outlet 8 of the second single spiral microchannel of the first spiral microchannel and the first single of the second spiral microchannel
  • the cell inlets 14 of the helical microchannels are joined to form a series arrangement of two helical microchannels, and a pressure equalization channel is provided at a location where each spiral microchannel is mounted in series.
  • the sheath fluid is from the sheath fluid inlet of the first single spiral microchannel of the first spiral microchannel, the sheath fluid inlet 5 of the second single spiral microchannel of the first spiral microchannel, The sheath liquid inlet 9 of the first single spiral microchannel of the two spiral microchannels and the sheath liquid inlet 11 of the second single spiral microchannel of the second spiral microchannel are introduced;
  • the waste liquid is from the first a waste liquid outlet 6 of the first single spiral microchannel of the spiral microchannel, a waste liquid outlet 7 of the second single spiral microchannel of the first spiral microchannel, and a second spiral microchannel
  • the waste liquid outlet 10 of the first single spiral microchannel and the waste liquid outlet 13 of the second single spiral microchannel of the second spiral microchannel are led out.
  • the sorted and enriched rare cells or particles are collected from the cell outlet 12 of the second single helical microchannel of the second helical microchannel.
  • the present invention can directly or cascadely use a plurality of N-stage spiral flow paths without using any external microtubes, which simplifies the manufacturing process and also makes the N-stage spiral flow path. Reduced complexity, improved work efficiency and accuracy of test results.
  • two double spiral microchannels are connected in parallel.
  • the sample solution enters from the cell inlet 1 of the first single helical microchannel of the two double helix microchannels, and the rare cell or particle exits from the cell outlet of the second single helical microchannel of the two double helix microchannels 12 Flow out.
  • the center projections of the double spiral microchannels participating in the parallel are coincident. Two, three or more samples to be detected can be simultaneously processed on the same microfluidic chip, thereby improving the detection efficiency while effectively reducing the detection cost and the processing time of the sample.
  • This embodiment differs from the first embodiment in that all the inlets and outlets (including the cell inlet and outlet, the sheath liquid inlet and the waste liquid outlet) in the present embodiment are disposed outside the geometry of the entire spiral microchannel, and the waste liquid outlet passes through.
  • the pressure-balanced microchannel flows to the outside of the geometry of the entire spiral microchannel along the extending direction of the other microchannels, and the flow rate and flow direction of the cell outlet contents are adjusted by adjusting the width, depth and length of the pressure-balanced microchannel. Adjustment.
  • a part of the inlet and outlet (such as the second-stage sheath liquid inlet 5 and the first-stage waste liquid waste liquid 6 shown in FIG. 1) is disposed inside the spiral microchannel, and the structure is designed as a chip. Manufacturing brings great inconvenience, and the operation of perforating and introducing or introducing fluids and/or cells in the center of the chip is also complicated, which makes the related chip fixture difficult to manufacture, and it is difficult to observe the intersection of the first-level exit and the second-level entrance. The flow state of fluids and/or cells. Therefore, in the present embodiment, all the inlets and outlets (including the cell inlet and outlet, the sheath liquid inlet, and the waste liquid outlet) are disposed outside the entire spiral microchannel.
  • COMSOL Multiphisics multi-physical simulation software was used to build a standardized single-helical chip, further improve the chip structure and successfully construct a standardized double-spiral micro-channel chip.
  • the key objective of the designed and constructed double helix chip is to focus the larger target cells (CTC) along the inner wall of the channel by inertial focusing, and the smaller blood cells avoid the sheath fluid separation from the outer side of the spiral channel.
  • the double-helical chip can capture the CTC in the blood sample more quickly and efficiently by cascading, meet the clinical requirements, and achieve the purpose of dynamic real-time monitoring of the tumor patient and selection of the optimal treatment plan.
  • further research on related mechanisms such as tumor metastasis can be conducted.
  • the position of the inertial focus of different cells is affected by the three-dimensional size of the spiral microchannel.
  • the three-dimensional size of the spiral micro-channel chip which can separate CTC ( ⁇ 15um) and blood cells (3 ⁇ 15um) is determined, which forces the sample to be from the first single spiral.
  • the cell inlet of the microchannel completely enters the spiral microchannel and begins its lateral displacement within the flow channel.
  • the difference in the lateral displacement focus position of different size cells CTC, RBC, WBC
  • the dual-channel spiral chip increases the in-chip flow path length based on the single-channel spiral chip, and avoids the problem of low cell purity caused by excessive lateral displacement of small particles due to short flow path at high flow rate and high flux.
  • simulation analysis of the first stage inlet and outlet of the double helix microchannel under conditions of pressure equalization channel 3 of different widths including: cell inlet 1, sheath inlet 2, waste outlet 6, cell
  • the volumetric flow characteristics and related ratios of the outlet 4) and the second-stage inlet and outlet including: the cell inlet 4, the sheath fluid inlet 5, the waste liquid outlet 7, and the cell outlet 8).
  • the simulation conditions were that the initial condition of the cell inlet was 150 ul/min and the initial condition of the sheath fluid inlet was 750 ul/min.
  • the simulation results show that the flow rate of the first-stage cell outlet 4 is equivalent to the flow rate of the first-stage cell inlet 1 only when the width of the pressure-balanced channel 3 is 350 ⁇ m. Under this condition, the flow rate is 144.18 ul/min (Table) three). This result indicates that the width of the pressure-equalizing channel 3 has an important regulating effect on the flow velocity of the first-stage cell outlet 4, and that the flow rate of the first-stage cell outlet 4 and the first-stage can be ensured only under the appropriate width conditions. The flow rate of the cell inlet 1 is comparable.
  • the spiral micro-flow channel chip is mainly made by the basic micro-machining technology, and the standard soft lithography technology is used to fabricate the mold on the silicon wafer by using the SU8 photoresist.
  • the PDMS is demolded, the corresponding entrance and exit are punched, and Bonded on the corresponding slide (45mm X45mm), in order to increase the firmness of the bond, place it in a 70 °C oven and bake for 30 minutes.
  • the bonded spiral microchannel chip inlet and outlet are tightly connected to the sample tube by a magnetic probe (Corsolution) device and a Fluidic Connect (Micronit) clamp connection, and the injection is achieved by a LongerPump syringe pump and a MCFS pressure flow rate control system. Stable and real-time monitoring of flow rate changes.
  • the sealing fluid was first tested under the high pressure of 1 bar. The result was no leakage, indicating that the chip bonding and the chip inlet and outlet were firmly connected. Observe the integrity of the chip and the bubble elimination, after the bubble is completely removed.
  • the optimal parameters for the enrichment of CTC by spiral microchannel sorting were optimized using standard polystyrene colored microbeads.
  • the cells were mixed with 3-6 ⁇ m microspheres, 7-10 ⁇ m microspheres and 15-18 ⁇ m microspheres to simulate red blood cells, white blood cells and CTC, respectively, and the microspheres of 3-6 ⁇ m, 7-10 ⁇ m and 15-18 ⁇ m were diluted with PBS buffer.
  • PBS buffer a buffer for PBS buffer.
  • the syringe pump loaded with the mixed microspheres and the syringe pump loaded with the PBS sheath fluid are respectively connected to the cell inlet 1 and the sheath liquid inlet 2 of the spiral chip after the bubble elimination by the chip holder, and the flow rate is adjusted by the flow rate monitoring system and the pressure pump control.
  • pressure, separately recorded and collected single and double spiral type chip (single spiral type chip in the first embodiment and the present embodiment) at different flow rates (200 ⁇ l / min, 350 ⁇ l / min, 500 ⁇ l / min, 650 ⁇ l / min, 800 ⁇ l /min, 950 ⁇ l/min)
  • the separation of colored microspheres at the exit of the chip is shown in Fig. 23, and the observation result by microscopic counting is shown in Fig. 24.
  • the flow rate is less than 650 ⁇ l/min.
  • the larger size of the particles cannot be focused near the cell outlet.
  • the purity of CTC sorting enrichment will be affected.
  • the number and size of the dots do not reflect the actual particle size. The number and size of the.
  • the two-channel spiral type chip was tested. The results are shown in Table 5. It reflects that the purity of sorting and enrichment of the two-channel spiral type chip is much higher than that of the single-channel spiral type chip. At the same time, due to the limitation of the flow length of the single spiral chip, when the flow rate is greater than 800 ⁇ l/min, the lateral displacement of the smaller size particles under the action of large inertial migration force tends to affect the purity of the sorted and enriched CTC cells. However, the double-helical chip increases the length of the flow path and performs the second-stage separation to greatly increase the detection flux.
  • the artificially cultured HCC827, MCF-7, and SW480 cell lines were diluted to 10 2 cells/ml, respectively, and the normal blood cells treated with the red blood cell lysate were mixed in a volume ratio of 1:5, and the chip in the present example was used. Detection, detection results using immunofluorescence technique staining smear counts are shown in Table 6. The results showed that the recovery rate of multiple tumor cells by the double helix microchannel chip was over 90%.
  • Figure 25 shows the SW480 immunofluorescence staining by sorting and enrichment. The results were observed by mixing the tumor cells with the captured cells. Very good linear correlation, while the double helix chip captures 90% of the capture efficiency of multiple tumor cells, see Figure 26.
  • the purity of sorting and enrichment CTC is also over 98%, but because The difference between tumor patients is large (such as WBC is significantly increased after chemotherapy), so its purity is often a variable.
  • the purity of CTC cells enriched by double helix chip is much higher than that of single helix.
  • the chip, its cell purity is critical for biological manipulation analysis such as CTC cell follow-up genetics.
  • This embodiment is similar in nature to the fifth embodiment, but all the inlets and outlets in the solution (including: cell inlet and outlet, sheath liquid inlet and waste liquid outlet) are disposed outside the geometry of the overall spiral microchannel, and easily observe and analyze results, and at the same time, it is easier to standardize fixtures, which facilitates batch processing and operation.
  • This embodiment is similar in nature to the third embodiment, but the design has the cell inlet of the first single spiral microchannel, the sheath fluid inlet of the first single spiral microchannel, and the second single spiral microchannel.
  • the sheath liquid inlet, the waste liquid outlet of the first single spiral microchannel are disposed at a relatively close position, and the two single spiral microchannels are arranged alternately in a concentric manner, and at the same time, all the inlets and exits (including: cell inlet) And the outlet, the sheath liquid inlet and the waste liquid outlet are all arranged outside the geometry of the overall spiral microchannel, and the overall structure is more compact and easy to use.
  • the double spiral microchannels in the two embodiments are connected in a parallel manner.
  • the parallel configuration can be used to simultaneously perform the same sample on two double spiral microchannels in a unit time. Processing, or speaking, can simultaneously process more fluids in one screening time, which can effectively increase the screening efficiency.
  • the detection time of the clinical sample can be shortened by at least one time.
  • only the parallel form of two double spiral microchannels is shown. In actual use, three, four or more double spiral microchannels can be connected in parallel according to requirements.
  • This embodiment is similar in nature to the seventh embodiment, but the design introduces the cell inlet of the first single spiral microchannel of the first double helix microchannel and the sheath fluid inlet of the first single spiral microchannel.
  • the sheath liquid inlet of the second single spiral microchannel, the waste liquid outlet of the first single spiral microchannel is disposed at a relatively close position; and the first single spiral of the second double spiral microchannel
  • the cell inlet of the microchannel, the sheath inlet of the first single spiral microchannel, the sheath inlet of the second single spiral microchannel, and the waste outlet of the first single spiral microchannel are disposed in close proximity
  • the two single spiral microchannels are arranged alternately in a concentric manner, and the overall structure is more compact and easy to use.
  • the spiral microchannels (ie, the series double helix microchannels) in the tenth embodiment are connected in parallel, and the parallel sample is used to make the same sample simultaneously in unit time.
  • the spiral microchannels shown in the two embodiments are processed, or more simultaneous treatment of more fluids can be realized in one screening time, which can effectively increase the screening efficiency.
  • the detection time of the clinical sample can be shortened by at least one time.
  • This embodiment not only has the advantages of ensuring multi-stage flow channel multiple screening through series connection, improving cell purity, but also has the advantage of improving screening efficiency by parallel form.
  • only the parallel forms of the spiral microchannels shown in the two embodiments are shown. In actual use, three, four, and more parallel spiral microchannels shown in Embodiment 10 can be performed as needed.
  • FIG. 11 Another embodiment of the basic design-single helical microchannel is shown in this embodiment, in which both the cell outlet and the waste outlet are disposed outside of the overall helical microchannel.
  • the structural design of the cell outlet and the waste liquid outlet disposed inside the spiral microchannel brings great inconvenience to the molding and manufacturing of the chip, and is perforated at the center of the chip.
  • the operation of exporting or introducing fluids and/or cells is also complicated, resulting in difficulty in making related chip holders, and it is difficult to observe the fluid and/or cell flow state at the junction of the sheath fluid and the cell inlet. Therefore, in the present embodiment, the cell outlet and the waste liquid outlet are both disposed outside the entire spiral microchannel, which is easier to observe and analyze, as well as standardized fixtures and simplified operations.
  • the schematic diagrams of the single spiral microchannels in the fourteenth embodiment are connected in a series manner, and the serial connection can realize multiple screening in one flow process, which can further improve the purity of the screening cells.
  • the serial connection can realize multiple screening in one flow process, which can further improve the purity of the screening cells.
  • only the series form of two single spiral microchannels is shown. In actual use, three, four, and more single spiral microchannels can be connected in series as needed.
  • the two single spiral microchannels are arranged in a concentric juxtaposed arrangement, and the spiral folding spiral is wound into a ring, which can be realized in a single flow process in a series connection manner. Screening can further improve the purity of the screened cells.
  • this embodiment only two series of single spiral microchannels are shown. In actual use, three, four, and more single spiral microchannels can be connected in series as needed.
  • the schematic diagrams of the single spiral microchannels in the fourteenth embodiment are connected in parallel, and the same sample can be used in the same time in the unit time in two embodiments.
  • the single spiral microchannels are shown to be processed, or more fluids can be processed simultaneously in one screening time, which can effectively increase the screening efficiency.
  • the detection time of clinical samples can be reduced by at least 1 time.
  • only two parallel forms of single spiral microchannels are shown. In actual use, three, four, and more single spiral microchannels can be connected in parallel according to requirements.
  • FIG. 17 Another embodiment of the embodiment 17 is shown in the embodiment.
  • the two single spiral microchannels are arranged in a concentric juxtaposition, and the spiral folding spiral is wound into a ring, and the parallel form can realize more in one screening time.
  • the simultaneous treatment of the fluid can effectively increase the screening efficiency.
  • only the parallel form of two single spiral microchannels is shown.
  • three, four or more single spiral microchannels can be connected in parallel according to requirements. .
  • the basic design of the present invention is a single spiral microchannel, the extension of which is designed as a series, parallel, hybrid, and multi-spiral microchannel of a single spiral microchannel (for example, a double spiral microchannel in the present specification)
  • the various implementation forms such as series, parallel, hybrid, etc., all of the above expressions are extended designs of the scheme, and the basic principles are all explained in the present specification, so the above various expressions should also fall under the protection of the present invention. In the range.

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Abstract

A spiral microchannel, a use method thereof, and a series/parallel-connected installation structure. The invention addresses the technical problem in the field of separating or enriching circulating tumor cells in peripheral blood of a malignant tumor patient by means of a spiral microchannel, and designs an inertial separator structure based on series-connected and parallel-connected spiral microchannels that applies, in a cascaded manner, multiple spiral microchannels. Compared to prior art techniques in the same field, the present invention has superior capturing efficiency and processing throughput for separation and enrichment of circulating tumor cells in peripheral blood, maximizes the purity of the circulating tumor cells, minimizes the damage thereto, and retains the original phenotype of the circulating tumor cells. A cell suspension containing high-purity circulating tumor cells obtained by using the invention is especially suitable for subsequent biomedical testing, genetic analysis, cell cultivation, xenograft tumor preparation and such, and has broad and far reaching practical application value in clinical medicine.

Description

一种螺旋形微通道及其使用方法与串、并联安装结构Spiral microchannel and its use method and series and parallel installation structure 技术领域Technical field
本发明属于稀有细胞或颗粒富集筛选领域，涉及一种螺旋形微通道及其使用方法与串、并联安装结构。The invention belongs to the field of rare cell or particle enrichment screening, and relates to a spiral microchannel and a method for using the same, and a series and parallel installation structure.
背景技术Background technique
侵袭和转移是肿瘤患者的主要临床致死原因，肿瘤转移疾病所引起的死亡占实体瘤所致死亡的90％左右，因此，肿瘤转移的早期精准检测显得优为重要。循环肿瘤细胞是指脱离原发和转移肿瘤病灶，侵袭并进入***、血液等循环***的肿瘤细胞。循环***中检测到循环肿瘤细胞提示可能有肿瘤微转移的存在，并有进一步发展成为远处转移的可能性。近年来，许多研究报告已经证实循环肿瘤细胞在肿瘤转移的早期诊断、个体化治疗、探索肿瘤转移机制等方面具有潜在的重要价值。相对于实体瘤的肿瘤组织细胞，外周血循环肿瘤细胞的检测具有可重复检测、取样简单、无创伤等优点，被称为液体活检，在近年来成为肿瘤精准诊断的热点。Invasion and metastasis are the main causes of death in cancer patients. The death caused by tumor metastasis accounts for about 90% of deaths caused by solid tumors. Therefore, accurate detection of early tumor metastasis is important. Circulating tumor cells refer to tumor cells that are detached from the primary and metastatic tumor lesions, invade and enter the circulatory system such as lymphatic vessels and blood. Circulating tumor cells detected in the circulatory system suggest the presence of tumor micrometastasis and the possibility of further development into distant metastases. In recent years, many research reports have confirmed that circulating tumor cells have potential value in early diagnosis, individualized treatment, and exploration of tumor metastasis mechanisms. Compared with tumor tissue cells of solid tumors, the detection of circulating blood cells of peripheral blood has the advantages of reproducible detection, simple sampling, and no trauma. It is called liquid biopsy and has become a hot spot for accurate diagnosis of tumors in recent years.
循环***中的循环肿瘤细胞不仅有形态学和遗传学异质性，而且十分稀少，即使是晚期肿瘤患者，每毫升全血中的循环肿瘤细胞可仅有1-10个，因此，要实现循环肿瘤细胞的检测，对其进行分选富集是一个必不可少的步骤，并且，循环肿瘤细胞分选富集的优劣将直接影响其后续的检测效果(如：计数、基因扩增、基因测序、细胞培养)。Circulating tumor cells in the circulatory system are not only morphologically and genetically heterogeneous, but also very rare. Even in patients with advanced tumors, there are only 1-10 circulating tumor cells per milliliter of whole blood. Therefore, it is necessary to achieve circulation. The detection of tumor cells is an indispensable step for sorting and enriching, and the advantages and disadvantages of circulating tumor cell sorting and enrichment will directly affect its subsequent detection effects (eg counting, gene amplification, genes). Sequencing, cell culture).
基于惯性微流原理的微流控芯片近年来成为无标记分选富集循环肿瘤细胞的常用方法之一，其主要特点是无需施加任何外力，如电力、磁力，就可以在微通道内实现包括细胞在内的一定尺寸粒子的聚焦流动。按芯片结构特征来划分，基于惯性微流原理的微流控芯片可主要分为直线形微通道、弯弧形微通道、螺旋形微通道等3种，其中，螺旋形微通道是弯弧通道的一种特殊形式。当流 体在直线形通道中呈层流状态时，流体的流速呈现一种抛物线状分布：通道中间的流体层速度最大，流体层越靠近通道壁，其流速越低，并且，随着流体层靠近通道壁的距离降低，其流速也按照一定比例降低，离通道壁最近的流体速度最小。伴随这种抛物线状的流速分布产生了一种剪切力梯度，这种剪切力梯度诱导产生的升力会将悬浮在流体中的粒子推向通道壁。当粒子迁移到距离通道壁足够近的位置时，通道壁诱导产生的升力又会将粒子推离通道壁。上述两种方向相反的升力的合力被称为惯性升力F L。惯性升力作用在粒子上，会使粒子在通道横截面中产生相对移动。当粒子移动到横截面的平衡位置，即粒子在横截面这一位置时，受到的惯性升力为零，粒子就会稳定在横截面这一位置，从而使粒子聚焦在横截面中稳定位置，形成聚焦流动，流向下游。粒子在直线形微通道横截面中受力平衡的位置与微通道横截面的形状有关：在圆形横截面的微通道中，粒子会聚焦形成一个环形；在正方形横截面的微通道中，粒子聚焦的位置会减少到四个，分别位于四个面的中间；在矩形横截面的微通道中，当微通道的纵横比较大时，即微通道特别宽或者特别高时，粒子聚焦的位置会减少到两个，分别位于两个较长面的中间。 Microfluidic chip based on inertial microfluidic principle has become one of the commonly used methods for label-free sorting and enrichment of circulating tumor cells in recent years. Its main feature is that it can be implemented in microchannels without applying any external force, such as electric power and magnetic force. The focused flow of certain size particles, including cells. According to the structure characteristics of the chip, the microfluidic chip based on the inertial microfluidic principle can be mainly divided into three types: a linear microchannel, a curved microchannel, and a spiral microchannel. Among them, the spiral microchannel is a curved channel. a special form. When the fluid is laminar in a linear channel, the flow velocity of the fluid exhibits a parabolic distribution: the velocity of the fluid layer in the middle of the channel is the largest, the closer the fluid layer is to the channel wall, the lower the flow rate, and as the fluid layer approaches The distance of the channel wall is reduced, the flow rate is also reduced by a certain ratio, and the fluid velocity closest to the channel wall is the smallest. This parabolic flow velocity distribution produces a shear force gradient that induces lift that pushes particles suspended in the fluid toward the channel walls. When the particles migrate to a position sufficiently close to the channel wall, the lift induced by the channel walls pushes the particles away from the channel walls. The resultant force of the above two opposite lift forces is referred to as the inertia lift F L . The inertial lift acts on the particles, causing the particles to move relative to each other in the cross section of the channel. When the particle moves to the equilibrium position of the cross-section, that is, when the particle is in the cross-section, the inertial lift is zero, and the particle is stabilized at the cross-section, so that the particle is focused in a stable position in the cross-section. Focus on the flow and flow downstream. The position at which the particles are balanced in the cross section of the linear microchannel is related to the shape of the cross section of the microchannel: in the microchannel of the circular cross section, the particles are focused to form a ring; in the microchannel of the square cross section, the particles The position of the focus is reduced to four, located in the middle of the four faces; in the microchannel of the rectangular cross section, when the vertical and horizontal of the microchannel is relatively large, that is, when the microchannel is particularly wide or particularly high, the position of the particle focus will be Reduce to two, located in the middle of the two longer faces.
当流体在弯弧形微通道中流动时，情况比直线形微通道中的更复杂。呈抛物线流动的流体在通道中间速度最大。在经过微通道转弯处时，微通道中间的流体因其流速最大而受到的离心力最大，从而流向弯弧形微通道的外侧壁。靠近微通道壁的流体流速最小，所受离心力也最小，从而受到中间高流速流体的挤压。为了保持流体中各处质量守恒，在垂直于流体流动的方向上，形成一对反向旋转且对称的涡流，分别位于微通道横截面的上部和下部，由此产生一种被称作迪恩涡流的二次流。迪恩涡流会对流体中的颗粒产生曳力作用，被称为迪恩曳力F D。因此在弯弧形微通道中，流动的粒子会同时受到惯性升力和迪恩曳力的作用，这两种力的相对大小决定粒子在弯弧形通道中流动的聚焦流动情况。 When the fluid flows in a curved microchannel, the situation is more complicated than in a linear microchannel. The fluid flowing in a parabola has the highest velocity in the middle of the channel. When passing through the microchannel turn, the fluid in the middle of the microchannel is subjected to the largest centrifugal force due to its maximum flow rate, thereby flowing to the outer side wall of the curved microchannel. The fluid flow near the walls of the microchannel is minimal and the centrifugal force is minimal, which is squeezed by the intermediate high flow rate fluid. In order to maintain mass conservation throughout the fluid, a pair of counter-rotating and symmetrical vortices are formed in a direction perpendicular to the flow of the fluid, respectively located at the upper and lower portions of the cross-section of the microchannel, thereby creating a type of Dean The secondary flow of the eddy current. The Dean vortex acts as a drag force on particles in the fluid, known as Dean drag F D . Therefore, in a curved arc-shaped microchannel, the flowing particles are simultaneously subjected to inertial lift and Dean drag, and the relative magnitudes of the two forces determine the focused flow of the particles in the curved passage.
微通道螺旋形微通道是一种特殊的弯弧形微通道，它既具有弯弧形微通道 的上述聚焦流动的特性，也具有自身的独有特征，因此，相对于上述的直线形和弯弧形微通道，螺旋形微通道在惯性微流芯片中具有独特的应用优势。在螺旋形微通道中层流流体的惯性迁移力和迪恩曳力共同作用下，粒子在管道内侧壁和外侧壁之间来回移动并形成迪恩涡流。上述横向迁移被称之为迪恩周期：当一个最初在外侧壁的粒子经过一定长度的微通道横向移动至内侧壁时，被称之为完成了0.5个迪恩周期；当此相同粒子再经过一定长度的下游微通道返回至初始的外侧壁时，被称之为完成了一个完整的迪恩周期，或者说，完成了1个迪恩周期。如上所述，除了迪恩曳力外，较大的粒子(特别是其直径达到a/h≥0.07)还会同时受到惯性升力的作用，从而使粒子的聚焦位置极度依赖于惯性升力与迪恩曳力的比值(F L/F D)，上述比值与粒子的尺寸呈指数关系，粒子的聚焦主要依赖于最小的微通道尺度参数(如螺旋形微通道的高度h)。较大的粒子(即：a/h≥0.07)，迪恩曳力和惯性升力处于一个平衡状态，会经过一定长度的微通道后聚焦于螺旋形通道的内侧壁，并在下游的微通道中始终保持在微通道的内侧壁；而较小粒子(即：a/h<0.07)，其迪恩曳力占优势，粒子以迪恩涡流形式不断地在微通道内的外侧壁和内侧壁之间来回横移。 The microchannel spiral microchannel is a special curved microchannel which has the above-mentioned characteristic characteristics of the curved flow of the curved microchannel and also has its own unique characteristics, and therefore, is relatively straight and curved with respect to the above. Curved microchannels, spiral microchannels have unique application advantages in inertial microfluidic chips. In combination with the inertial migration force of the laminar fluid in the spiral microchannel and the Dean drag, the particles move back and forth between the inner and outer sidewalls of the pipe and form a Dean vortex. The above lateral migration is called the Dean cycle: when a particle initially on the outer sidewall moves laterally to the inner sidewall through a length of microchannel, it is said to complete 0.5 Dean cycles; when the same particle passes through When a certain length of downstream microchannel returns to the original outer sidewall, it is said to complete a complete Dean cycle, or complete one Dean cycle. As mentioned above, in addition to Dean's drag, larger particles (especially a diameter of a/h ≥ 0.07) are also subjected to inertial lift, so that the focus position of the particles is extremely dependent on inertial lift and Dean. The ratio of the drag force (F L /F D ), the ratio is exponentially related to the size of the particle, and the focus of the particle is mainly dependent on the smallest microchannel scale parameter (such as the height h of the spiral microchannel). Larger particles (ie: a/h ≥ 0.07), Dean drag and inertial lift are in an equilibrium state, after a certain length of microchannels, they are focused on the inner side wall of the spiral channel and in the downstream microchannel It is always kept on the inner side wall of the microchannel; while the smaller particles (ie: a/h<0.07), the Dean drag force predominates, and the particles are continuously in the form of Dean vortex in the outer and inner sidewalls of the microchannel. Move back and forth.
在螺旋形微通道中，可通过在微通道的外侧壁部分设计粒子入口，在微通道的内侧壁部分设计鞘液入口。在此条件下，具有较高速度的鞘液流体会将粒子流挤压至微通道的外侧壁；当粒子在微通道中流动时，所有初始状态(即入口处)的粒子在迪恩曳力的作用下，由外侧壁经0.5个迪恩周期的微通道长度移动至内侧壁；当到达近内侧壁后，较大的粒子(即：a/h≥0.07)因受到强烈的惯性升力而强力聚焦于内侧壁，并进一步在迪恩曳力的作用下阻止其进一步向微通道的外侧壁移动，因此，在上述惯性升力和迪恩曳力达到某个平衡点的时候，较大的粒子始终聚焦在微通道的内侧壁，并在后续的下游微通道中始终保持在微通道内侧壁的聚焦流；但是，经0.5个迪恩周期移动到内侧壁的较小粒子(即：a/h<0.07)受到的惯性升力的小，主要承受的是迪恩曳力，因此，会在迪恩涡流的作用下在下游微通道中经1个迪恩周期长度的微通道横向迁移动至外侧壁， 若下游的微通道足够长，则较小粒子以迪恩涡流形式在微通道壁的外侧壁和内侧壁来回横向迁移。可见，较大粒子始终在螺旋形微通道的内侧壁呈惯性聚焦流束，而较小粒子则以迪恩涡流形式在微通道的外侧壁和内侧壁来回横向迁移。当设计特定长度的螺旋形微通道，其出口处正好使较小的粒子位于或接近于外侧壁，就可通过适当的出口将较大粒子与较小粒子分离，实现特定尺寸粒子的富集。In the spiral microchannel, a sheath inlet can be designed in the inner sidewall portion of the microchannel by designing a particle inlet in the outer sidewall portion of the microchannel. Under this condition, the sheath fluid with higher velocity will squeeze the particle flow to the outer sidewall of the microchannel; when the particles flow in the microchannel, all the initial state (ie, the entrance) of the particles at Dean drag By the action of the microchannel length of 0.5 Dein cycle from the outer sidewall to the inner sidewall; when reaching the inner sidewall, the larger particles (ie: a/h ≥ 0.07) are strongly resisted by strong inertial lift. Focusing on the inner side wall and further preventing it from moving further to the outer side wall of the microchannel under the action of Dean's drag force, therefore, when the inertial lift and the Dean drag force reach a certain equilibrium point, the larger particles are always Focusing on the inner sidewall of the microchannel and maintaining the focused flow in the inner sidewall of the microchannel in subsequent downstream microchannels; however, smaller particles moving to the inner sidewall over 0.5 Dein cycles (ie: a/h< 0.07) The inertial lift received is mainly due to the Dean drag force. Therefore, it will move laterally to the outer side wall through the microchannel of the Dean period length in the downstream microchannel under the action of Dean vortex. If the downstream microchannel is sufficient , Dean vortices in the form of smaller particles migrate back and forth laterally outer and inner sidewalls of the microchannel wall. It can be seen that the larger particles always have an inertial focusing stream on the inner side wall of the spiral microchannel, while the smaller particles migrate laterally back and forth in the outer and inner sidewalls of the microchannel in the form of a Dean vortex. When a spiral microchannel of a particular length is designed with its exit just at or near the outer sidewall, larger particles can be separated from the smaller particles by appropriate outlets to achieve enrichment of particles of a particular size.
螺旋形微通道的上述特性被广泛应用于恶性肿瘤患者外周血循环肿瘤细胞的分选富集。其主要原理在于：相对于外周血中的红细胞(直径约为6-8μm)和白细胞(直径约为8-12μm)，循环肿瘤细胞(直径约为20-30μm)的尺寸更大，通过限定纵横比的矩形横截面，在特定流体流速条件下，外周血循环肿瘤细胞将沿着螺旋形微通道的内侧壁聚焦层流至细胞出口，样本中的其余细胞则沿着所述通道的外侧壁层流至废液出口，从而实现循环肿瘤细胞的分离。同时，还可通过进一步设计多个出口，使具有不同尺寸的细胞自各个特定出口分流富集。The above characteristics of the spiral microchannel are widely used for the sorting and enrichment of peripheral blood circulating tumor cells in patients with malignant tumors. The main principle is that the size of circulating tumor cells (about 20-30 μm in diameter) is larger than that of red blood cells (about 6-8 μm in diameter) and white blood cells (about 8-12 μm in diameter) in peripheral blood. Ratio of rectangular cross-section, at a specific fluid flow rate, peripheral blood circulating tumor cells will flow along the inner sidewall of the spiral microchannel to the cell outlet, and the remaining cells in the sample will flow along the outer wall of the channel. To the waste liquid outlet, thereby achieving the separation of circulating tumor cells. At the same time, cells with different sizes can be shunted from each particular outlet by further designing multiple outlets.
相对于直线型和弯弧形惯性微流芯片，螺旋形惯性微流芯片在分选富集循环肿瘤细胞领域具有独特的优势，并主要表现在能够快速地处理全血样本，达到3mL/min的速度。但是， 现有的螺旋形惯性微流芯片主要是单通道形式的单螺旋形微通道惯性微流芯片其主要弊端是所获得的循环肿瘤细胞纯度低，从而导致循环肿瘤细胞的后续鉴别和临床实际应用存在较大的困难，其实际临床应用价值较低。尽管现有的螺旋形微通道在分选富集循环肿瘤细胞方面仍然存在较大的不足和相关的技术瓶颈，但是，已有的研究结果提示，如果能够巧妙地“级联”应用多个单个螺旋形微通道，就能在确保良好捕获效率和处理通量的前提条件下，极大地提高的分选富集到的细胞混合液中循环肿瘤细胞纯度，使其满足临床的实际需求。 Compared with linear and curved inertial microfluidic chips, spiral inertial microfluidic chips have unique advantages in the field of sorting and enriching circulating tumor cells, and are mainly manifested in the ability to rapidly process whole blood samples to 3 mL/min. speed. However, the existing spiral inertial microfluidic chip is mainly a single-channel single-spiral microchannel inertial microfluidic chip. The main drawback is that the obtained circulating tumor cells have low purity, which leads to subsequent identification and clinical practice of circulating tumor cells. There are major difficulties in application, and its actual clinical application value is low. Although the existing spiral microchannels still have large deficiencies and related technical bottlenecks in sorting and enriching circulating tumor cells, the existing research results suggest that if you can subtly "cascade" multiple individual applications The spiral microchannel can greatly improve the purity of circulating tumor cells in the sorted and enriched cell mixture under the premise of ensuring good capture efficiency and processing flux, so as to meet the actual clinical needs.
发明内容Summary of the invention
有鉴于此，本发明的目的在于提供一种螺旋形微通道及其使用方法与串、 并联安装结构。In view of the above, an object of the present invention is to provide a spiral microchannel, a method of using the same, and a series and parallel mounting structure.
为达到上述目的，本发明提供如下技术方案：In order to achieve the above object, the present invention provides the following technical solutions:
一种螺旋形微通道，用于分选富集稀有细胞或颗粒，包括N个单螺旋形微通道，单个的单螺旋形微通道包括入口、与入口密封连接的微通道以及与通道远离入口的一端密封连接的出口，所述的入口由细胞入口及鞘液入口汇聚而成，所述的出口由废液出口及细胞出口汇聚而成；第(M-1)个单螺旋形微通道的细胞出口与第M个单螺旋形微通道的细胞入口密封连接成第(M-1)个分级循环点，由第(M-1)个单螺旋形微通道的废液出口、第M个单螺旋形微通道的鞘液入口以及第(M-1)个分级循环点构成第(M-1)个分级循环处；A spiral microchannel for sorting and enriching rare cells or particles, comprising N single spiral microchannels, the single single spiral microchannel including an inlet, a microchannel sealingly connected to the inlet, and a channel away from the inlet An outlet that is sealed at one end, the inlet is formed by a cell inlet and a sheath inlet, and the outlet is formed by a waste liquid outlet and a cell outlet; the (M-1) single spiral microchannel cells The outlet is sealingly connected to the cell inlet of the Mth single spiral microchannel into the (M-1)th graded circulation point, the waste liquid outlet of the (M-1) single spiral microchannel, the Mth single helix The sheath liquid inlet of the microchannel and the (M-1)th graded circulation point constitute the (M-1)th classification cycle;
在第(M-1)个分级循环处内的第(M-1)个单螺旋形微通道的废液出口设有压力平衡模块，第M个单螺旋形微通道的细胞入口的液体流速为v1，第(M-1)个单螺旋形微通道的细胞入口的液体流速为v2；第M个单螺旋形微通道的细胞出口的液体流速为v3，第(M-1)个单螺旋形微通道的细胞出口的液体流速为v4；第M个单螺旋形微通道的废液出口的液体流速为v5，第(M-1)个单螺旋形微通道的废液出口的液体流速为v6；A liquid pressure flow rate is provided at a waste liquid outlet of the (M-1) single spiral microchannel in the (M-1)th classification cycle, and a liquid flow rate of the cell inlet of the Mth single spiral microchannel is V1, the liquid flow rate of the cell inlet of the (M-1) single spiral microchannel is v2; the liquid flow rate of the cell outlet of the Mth single spiral microchannel is v3, the (M-1) single spiral The liquid flow rate of the cell outlet of the microchannel is v4; the liquid flow rate of the waste liquid outlet of the Mth single spiral microchannel is v5, and the liquid flow rate of the waste liquid outlet of the (M-1) single spiral microchannel is v6 ;
该压力平衡模块用于使第M个单螺旋形微通道的细胞入口的液体流动方向和第(M-1)个单螺旋形微通道的细胞入口的液体流动方向一致，且0.7*v2≤v1≤1.3*v2；使第M个单螺旋形微通道的细胞出口的液体流动方向和第(M-1)个单螺旋形微通道的细胞出口的液体流动方向一致，且0.7*v4≤v3≤1.3*v4，且v1＝v4；使第M个单螺旋形微通道的废液出口的液体流动方向和第(M-1)个单螺旋形微通道的废液出口的液体流动方向一致，且0.7*v6≤v5≤1.3*v6；The pressure balance module is configured to make the liquid flow direction of the cell inlet of the Mth single spiral microchannel coincide with the liquid flow direction of the cell inlet of the (M-1) single spiral microchannel, and 0.7*v2≤v1 ≤1.3*v2; the liquid flow direction of the cell outlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the cell outlet of the (M-1) single spiral microchannel, and 0.7*v4≤v3≤ 1.3*v4, and v1=v4; the liquid flow direction of the waste liquid outlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the waste liquid outlet of the (M-1) single spiral microchannel, and 0.7*v6≤v5≤1.3*v6;
所述的N个单螺旋形微通道形成互不交叉的螺旋形微通道；The N single spiral microchannels form spiral microchannels that do not intersect each other;
其中，第一个单螺旋形微通道的细胞入口用于进入含稀有细胞或颗粒的样本液体；第N个单螺旋形微通道的细胞出口用于收集分选富集后的稀有细胞或颗粒；所述鞘液入口用于添加带有渗透压，保证稀有细胞或颗粒完整的稀释液；1＜M≤N，M、N为正整数。Wherein the cell inlet of the first single spiral microchannel is used to enter a sample liquid containing rare cells or particles; the cell outlet of the Nth single spiral microchannel is used to collect rare cells or particles after sorting and enrichment; The sheath fluid inlet is used to add a diluent with osmotic pressure to ensure the integrity of rare cells or particles; 1 < M ≤ N, M, N are positive integers.
可选地，所述压力平衡模块为设置在第(M-1)个分级循环处内的第(M-1)个单螺旋形微通道的废液出口处且与该废液出口密封连接的压力平衡微通道；所述压力平衡微通道设有(N-1)个。Optionally, the pressure balance module is disposed at a waste liquid outlet of the (M-1) single spiral microchannel disposed in the (M-1)th classification cycle and is sealingly connected to the waste liquid outlet. Pressure-balanced microchannels; (N-1) of the pressure-balanced microchannels.
可选地，所述压力平衡微通道与N个单螺旋形微通道设置在同一平面内。Optionally, the pressure equalization microchannel is disposed in the same plane as the N single spiral microchannels.
可选地，所述压力平衡模块为设置在第(M-1)个分级循环处内的第(M-1)个单螺旋形微通道的废液出口处的压力泵。Optionally, the pressure balance module is a pressure pump disposed at a waste liquid outlet of the (M-1) single spiral microchannel in the (M-1)th classification cycle.
可选地，第一个单螺旋形微通道的入口及第N个单螺旋形微通道的出口位于螺旋形微通道几何形状的外侧。Optionally, the inlet of the first single helical microchannel and the outlet of the Nth single helical microchannel are located outside of the helical microchannel geometry.
可选地，所述的螺旋形微通道由N个单螺旋形微通道形成相互平行的环状。Optionally, the spiral microchannels are formed by N single spiral microchannels forming mutually parallel loops.
可选地，所述的螺旋形微通道之间的单螺旋形微通道和与之相邻且平行的单螺旋形微通道之间的水平距离为0.1～1000μm。Optionally, the horizontal distance between the single spiral microchannel between the spiral microchannels and the single spiral microchannel adjacent thereto and parallel is 0.1-1000 μm.
可选地，单个的单螺旋形微通道的截面为矩形或梯形。Optionally, the single single spiral microchannel has a rectangular or trapezoidal cross section.
可选地，所述的单螺旋形微通道的截面为矩形，该矩形的宽度w与高度h的比值w/h＝1.2～10。Optionally, the single spiral microchannel has a rectangular cross section, and the ratio of the width w to the height h of the rectangle is w/h=1.2-10.
可选地，所述的单螺旋形微通道的截面为梯形，所述该梯形靠近螺旋形微通道的几何中心的一侧高度低于该梯形远离螺旋形微通道的几何中心的一侧高度。Optionally, the single spiral microchannel has a trapezoidal cross section, and the height of one side of the trapezoid near the geometric center of the spiral microchannel is lower than the height of the trapezoid away from the geometric center of the spiral microchannel.
可选地，微通道的宽度w为0.1-1000μm，高度为0.1-800μm。Optionally, the microchannel has a width w of 0.1-1000 μm and a height of 0.1-800 μm.
可选地，N＝2，所述的螺旋形微通道由第一个单螺旋形微通道及第二个单螺旋形微通道组成。Optionally, N=2, the spiral microchannel consists of a first single spiral microchannel and a second single spiral microchannel.
可选地，单个的单螺旋形微通道的入口及该单螺旋形微通道的出口之间的距离为非靶细胞或非靶颗粒流体力学特征的1.7-2.0个迪恩周期，或者，2.7-3.0个迪恩周期，或者，3.7-4.0个迪恩周期，或者，4.7-5.0个迪恩周期，或者，5.7-6.0个迪恩周期，或者，6.7-7.0个迪恩周期，或者，7.7-8.0个迪恩周期，或者，8.7-9.0个迪恩周期，或者，9.7-10.0个迪恩周期。Optionally, the distance between the entrance of the single single helical microchannel and the exit of the single helical microchannel is 1.7-2.0 Dean cycles of non-target or non-target particle hydrodynamic characteristics, or, 2.7- 3.0 Dean cycles, or 3.7-4.0 Dean cycles, or 4.7-5.0 Dean cycles, or 5.7-6.0 Dean cycles, or 6.7-7.0 Dean cycles, or 7.7- 8.0 Dean cycles, or 8.7-9.0 Dean cycles, or 9.7-10.0 Dean cycles.
可选地，所述单螺旋形微通道的通道截面宽度最大处宽度为l1，该单螺旋 形微通道的细胞入口和/或细胞出口的宽度为l2，0.1*l1≤l2≤0.4*l1；该单螺旋形微通道的鞘液入口和/或废液出口的宽度为l3，0.1*l1≤l3≤0.9*l1。Optionally, the single-spiral microchannel has a channel cross-sectional width at a maximum width of l1, and the single-spiral microchannel has a cell inlet and/or a cell outlet having a width of l2, 0.1*l1≤l2≤0.4*l1; The width of the sheath liquid inlet and/or the waste liquid outlet of the single spiral microchannel is l3, 0.1*l1≤l3≤0.9*l1.
可选地，N＝4，所述的螺旋形微通道由第一个单螺旋形微通道、第二个单螺旋形微通道、第三个单螺旋形微通道、以及第四个单螺旋形微通道组成。Optionally, N=4, the spiral microchannel comprises a first single spiral microchannel, a second single spiral microchannel, a third single spiral microchannel, and a fourth single spiral Microchannel composition.
可选地，所述稀有细胞或颗粒的直径为a，所述微通道的高度为h，0.07≤a/h≤0.5。Optionally, the rare cells or particles have a diameter of a, and the height of the microchannels is h, 0.07≤a/h≤0.5.
可选地，所述稀有细胞或颗粒为循环肿瘤细胞、循环祖细胞、循环胎儿细胞、细胞囊泡、外泌体或病原体。Optionally, the rare cells or particles are circulating tumor cells, circulating progenitor cells, circulating fetal cells, cell vesicles, exosomes or pathogens.
可选地，还包括用于对该螺旋形通道所分选富集到的稀有细胞或颗粒进行鉴定和/或计数的检测单元；所述检测单元设置在第N个单螺旋形微通道的细胞出口处。Optionally, further comprising a detection unit for identifying and/or counting rare cells or particles sorted and enriched for the spiral channel; the detection unit being disposed in the cell of the Nth single helical microchannel exit.
可选地，所述检测单元通过特定技术手段进行稀有细胞或颗粒的性状鉴定；所述的特定技术手段选自由生物光学、生物电学、生物声学、生物磁学及其任意组合组成的组。Optionally, the detecting unit performs the identification of the characteristics of the rare cells or particles by a specific technical means; the specific technical means is selected from the group consisting of bio-optics, bioelectrics, bioacoustics, biomagnetism and any combination thereof.
可选地，所述检测单元通过对特定特征的检测进行稀有细胞或颗粒性状的识别，所述的特定特征选自由稀有细胞或颗粒的太赫兹波谱、拉曼光谱、阻抗、差分阻抗及其任意组合组成的组。Optionally, the detecting unit performs identification of a rare cell or particle trait by detecting a specific feature selected from a terahertz spectrum, a Raman spectrum, an impedance, a differential impedance, and any of the rare cells or particles. A group consisting of combinations.
可选地，(N-1)个所述的压力平衡微通道与N个单螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整第(M-1)个压力平衡微通道的长度与内径来调整第(M-1)个细胞出口内通过内容物的流速。Optionally, (N-1) the pressure-balanced microchannels and the N single-spiral microchannels form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed in a spiral micro-channel Outside the geometry of the channel, the flow rate through the contents of the (M-1) cell outlet is adjusted by adjusting the length and inner diameter of the (M-1)th pressure-balanced microchannel.
可选地，(N-1)个所述的压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。Optionally, the outlets of the (N-1) pressure equalization microchannels are each disposed outside of the geometry of the spiral microchannel.
可选地，N个所述细胞出口中的一个或多个设置在螺旋形微通道的几何图形外部。Optionally, one or more of the N of said cell outlets are disposed outside of the geometry of the helical microchannel.
可选地，所有细胞出口均设置在螺旋形微通道的几何图形外部。Optionally, all cell outlets are disposed outside of the geometry of the helical microchannel.
可选地，N个入口中的一个或多个设置在螺旋形微通道的几何图形外部。Optionally, one or more of the N inlets are disposed outside of the geometry of the spiral microchannel.
可选地，所有细胞入口均设置在螺旋形微通道的几何图形外部。Optionally, all of the cell inlets are disposed outside of the geometry of the spiral microchannel.
可选地，所有鞘液入口均设置在螺旋形微通道的几何图形外部。Optionally, all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
一种螺旋形微通道的使用方法，包括以下步骤：A method of using a spiral microchannel, comprising the steps of:
S1：将样本液体从第一个单螺旋形微通道的细胞入口导入，将稀释液从第一个单螺旋形微通道的鞘液入口至第N个单螺旋形微通道的鞘液入口导入；S1: introducing the sample liquid from the cell inlet of the first single spiral microchannel, and introducing the diluent from the sheath liquid inlet of the first single spiral microchannel to the sheath liquid inlet of the Nth single spiral microchannel;
S2：在第N个单螺旋形微通道的细胞出口收集富含稀有细胞或颗粒的流体；在第一个单螺旋形微通道的废液出口至第N个单螺旋形微通道的废液出口排出废液。S2: collecting a fluid rich in rare cells or particles at the cell outlet of the Nth single spiral microchannel; discharging the waste liquid in the first single spiral microchannel to the waste liquid outlet of the Nth single spiral microchannel Drain the waste liquid.
可选地，步骤S1中采用的样本液体为具有一定红细胞比积的的血液样本，或通过溶血方式去除大量红细胞的血液样本；所述血液样本中的红细胞比积为0％～70％。Optionally, the sample liquid used in step S1 is a blood sample having a certain ratio of red blood cells, or a blood sample in which a large amount of red blood cells is removed by hemolysis; the ratio of red blood cells in the blood sample is 0% to 70%.
可选地，步骤S1中采用的稀释液具有一定渗透压，用于保护血细胞完整性，为磷酸盐缓冲液、培养血清或生理盐水。Optionally, the diluent used in step S1 has a certain osmotic pressure for protecting blood cell integrity, and is phosphate buffer, culture serum or physiological saline.
可选地，还包括步骤S1.1；Optionally, further comprising step S1.1;
S1.1：调整压力平衡模块，使所述第M个单螺旋形微通道的细胞入口的液体流动方向和第(M-1)个单螺旋形微通道的细胞入口的液体流动方向一致，且0.7*v2≤v1≤1.3*v2；使第M个单螺旋形微通道的细胞出口的液体流动方向和第(M-1)个单螺旋形微通道的细胞出口的液体流动方向一致，且0.7*v4≤v3≤1.3*v4，且v1＝v4；使第M个单螺旋形微通道的废液出口的液体流动方向和第(M-1)个单螺旋形微通道的废液出口的液体流动方向一致，且0.7*v6≤v5≤1.3*v6。S1.1: adjusting the pressure balance module such that the liquid flow direction of the cell inlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the cell inlet of the (M-1) single spiral microchannel, and 0.7*v2≤v1≤1.3*v2; the liquid flow direction of the cell outlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the cell outlet of the (M-1) single spiral microchannel, and 0.7 *v4 ≤ v3 ≤ 1.3 * v4, and v1 = v4; liquid flow direction of the waste liquid outlet of the Mth single spiral microchannel and liquid of the waste liquid outlet of the (M-1) single spiral microchannel The flow direction is consistent, and 0.7*v6≤v5≤1.3*v6.
一种螺旋形微通道的串联安装结构，其特征在于，采用P个螺旋形微通道串联设置的方式，第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的细胞出口与第Q个螺旋形微通道的第一个单螺旋形微通道入口相连接，形成螺旋形微通道串联安装处，1＜Q≤P。A series installation structure of spiral microchannels, characterized in that, by means of P spiral microchannels arranged in series, the cell outlet of the Nth single spiral microchannel of the (Q-1) spiral microchannel Connected to the first single spiral microchannel inlet of the Qth spiral microchannel to form a spiral microchannel series installation, 1 < Q ≤ P.
可选地，在螺旋形微通道串联安装处的第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的废液出口处也设有压力平衡模块。Optionally, a pressure equalization module is also provided at the waste liquid outlet of the Nth single spiral microchannel of the (Q-1)th spiral microchannel at the spiral microchannel series installation.
可选地，所述压力平衡模块为与螺旋形微通道串联安装处的第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的废液出口密封连接的压力平衡微通道。Optionally, the pressure balance module is a pressure balanced microchannel sealingly connected to a waste liquid outlet of the Nth single spiral microchannel of the (Q-1)th spiral microchannel in a series installation of the spiral microchannel. .
可选地，所述的压力平衡微通道和与其相匹配的螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。Optionally, the pressure-balanced microchannel and the matching helical microchannel form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed outside the geometry of the spiral microchannel By adjusting the length and inner diameter of the pressure-balanced microchannel to adjust the flow rate through the contents in the cell outlet that matches it.
可选地，所有压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。Optionally, the outlets of all of the pressure balanced microchannels are disposed outside of the geometry of the helical microchannels.
可选地，所述细胞出口中的一个或多个设置在螺旋形微通道的几何图形外部。Optionally, one or more of the cell outlets are disposed outside of the geometry of the helical microchannel.
可选地，所有细胞出口均设置在螺旋形微通道的几何图形外部。Optionally, all cell outlets are disposed outside of the geometry of the helical microchannel.
可选地，所述入口中的一个或多个设置在螺旋形微通道的几何图形外部。Optionally, one or more of the inlets are disposed outside of the geometry of the spiral microchannel.
可选地，所有细胞入口均设置在螺旋形微通道的几何图形外部。Optionally, all of the cell inlets are disposed outside of the geometry of the spiral microchannel.
可选地，所有鞘液入口均设置在螺旋形微通道的几何图形外部。Optionally, all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
可选地，所述压力平衡模块为设置在螺旋形微通道串联安装处的第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的废液出口处的压力泵。Optionally, the pressure equalization module is a pressure pump disposed at the waste liquid outlet of the Nth single spiral microchannel of the (Q-1)th spiral microchannel at the spiral microchannel series installation.
可选地，N＝2，所述螺旋形微通道为双螺旋形微通道。Optionally, N=2, the spiral microchannel is a double helix microchannel.
可选地，P＝2，采用2个螺旋形微通道串联设置的方式，第一个螺旋形微通道的第N个单螺旋形微通道的细胞出口与第二个螺旋形微通道的第一个单螺旋形微通道入口相连接。Optionally, P=2, using two spiral microchannels arranged in series, the cell outlet of the Nth single spiral microchannel of the first spiral microchannel and the first of the second spiral microchannel The single spiral microchannel inlets are connected.
一种螺旋形微通道的并联安装结构，采用X个螺旋形微通道并联设置的方式，样本溶液从X个螺旋形微通道的第一个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从X个螺旋形微通道的第N个单螺旋形微通道的细胞出口流出；X＞1。A parallel installation structure of spiral microchannels adopts a method in which X spiral microchannels are arranged in parallel, and sample solution enters from the cell inlet of the first single spiral microchannel of the X spiral microchannels, and rare cells or particles Flowing out of the cell outlet of the Nth single helical microchannel of the X helical microchannels; X>1.
可选地，将X个螺旋形微通道的第一个单螺旋形微通道的细胞入口相互连通。Optionally, the cell inlets of the first single helical microchannel of the X helical microchannels are interconnected.
可选地，将X个螺旋形微通道的第N个单螺旋形微通道的细胞出口相互连通。Alternatively, the cell outlets of the Nth single helical microchannels of the X helical microchannels are interconnected.
可选地，所述的X个螺旋形微通道设置在同一平面内。Optionally, the X spiral microchannels are disposed in the same plane.
可选地，所述的X个螺旋形微通道同心设置在同一平面内。Optionally, the X spiral microchannels are concentrically disposed in the same plane.
可选地，所述的X个螺旋形微通道同心且沿垂直于单个螺旋形微通道所在平面的方向设置。Optionally, the X spiral microchannels are concentric and arranged in a direction perpendicular to the plane of the single spiral microchannel.
可选地，X＝2，采用两个螺旋形微通道并联设置的方式，样本溶液从两个螺旋形微通道的第一个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从两个螺旋形微通道的第N个单螺旋形微通道的细胞出口流出。Optionally, X=2, using two spiral microchannels arranged in parallel, the sample solution enters from the cell inlet of the first single spiral microchannel of the two helical microchannels, and the rare cells or particles are from two The cell outlet of the Nth single helical microchannel of the helical microchannel flows out.
可选地，N＝2，采用X个双螺旋形微通道并联设置的方式，样本溶液从X个双螺旋形微通道的第一个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从X个双螺旋形微通道的第二个单螺旋形微通道的细胞出口流出。Optionally, N=2, using X double helix microchannels in parallel, the sample solution enters from the cell inlet of the first single spiral microchannel of the X double helix microchannels, and the rare cells or particles are The cell outlets of the second single helical microchannel of the X double helix microchannels flow out.
可选地，在螺旋形微通道中的第X个螺旋形微通道的第N个单螺旋形微通道的废液出口处设有压力平衡模块。Optionally, a pressure equalization module is provided at the waste liquid outlet of the Nth single spiral microchannel of the Xth spiral microchannel in the spiral microchannel.
可选地，所述压力平衡模块为压力泵。Optionally, the pressure balance module is a pressure pump.
可选地，所述压力平衡模块为压力平衡微通道。Optionally, the pressure balance module is a pressure balanced microchannel.
可选地，所述的压力平衡微通道和与其相匹配的螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。Optionally, the pressure-balanced microchannel and the matching helical microchannel form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed outside the geometry of the spiral microchannel By adjusting the length and inner diameter of the pressure-balanced microchannel to adjust the flow rate through the contents in the cell outlet that matches it.
可选地，所有压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。Optionally, the outlets of all of the pressure balanced microchannels are disposed outside of the geometry of the helical microchannels.
可选地，所述细胞出口中的一个或多个设置在螺旋形微通道的几何图形外部。Optionally, one or more of the cell outlets are disposed outside of the geometry of the helical microchannel.
可选地，所有细胞出口均设置在螺旋形微通道的几何图形外部。Optionally, all cell outlets are disposed outside of the geometry of the helical microchannel.
可选地，所述入口中的一个或多个设置在螺旋形微通道的几何图形外部。Optionally, one or more of the inlets are disposed outside of the geometry of the spiral microchannel.
可选地，所有细胞入口均设置在螺旋形微通道的几何图形外部。Optionally, all of the cell inlets are disposed outside of the geometry of the spiral microchannel.
可选地，所有鞘液入口均设置在螺旋形微通道的几何图形外部。Optionally, all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
一种螺旋形微通道道的组合连接安装结构，包括K个微通道单体，所述微通道单体为权利要求1～27任一项中所述的螺旋形微通道；所述微通道单体采用串联、并联、多次串联、多次并联、或串并联组合的连接形式相互组合，其中K为正整数且K≥2；串联的连接形式为将第(K-1)个微通道单体的第N个细胞出口与第K个微通道单体的第一个细胞入口密封连接，组成串联式螺旋形微通道；并联的连接形式为将第(K-1)个微通道单体的第一个细胞入口与第K个微通道单体的第一个细胞入口密封连接汇聚组合成统一的细胞入口；将第(K-1)个微通道单体的第一个鞘液入口与第K个微通道单体的第一个鞘液入口密封连接汇聚组合成统一的鞘液入口，组成并联式螺旋形微通道。A combined connection mounting structure of a spiral microchannel, comprising K microchannel monomers, the microchannel monomer being the spiral microchannel according to any one of claims 1 to 27; The body is combined with each other in series, parallel, multiple series, multiple parallel, or series-parallel combination, wherein K is a positive integer and K≥2; the connection form of the series is the (K-1) microchannel single The Nth cell outlet of the body is sealingly connected with the first cell inlet of the Kth microchannel monomer to form a series spiral microchannel; the parallel connection form is the (K-1) microchannel monomer The first cell inlet is combined with the first cell inlet of the Kth microchannel monomer to form a uniform cell inlet; the first sheath inlet of the (K-1) microchannel monomer is The first sheath fluid inlet sealing junction of the K microchannel monomers is combined into a uniform sheath fluid inlet to form a parallel spiral microchannel.
可选地，多次串联的连接形式为将多个串联式螺旋形微通道以串联的连接形式相互组合；或将串联式螺旋形微通道与微通道单体以串联的连接形式相互组合。Alternatively, the plurality of series connection forms are a combination of a plurality of series spiral microchannels connected in series in a series connection; or a series spiral microchannel and a microchannel monomer are combined with each other in a series connection.
可选地，并联的连接形式还包括将第(K-1)个微通道单体的第N个细胞出口与第K个微通道单体的第N个细胞出口密封连接汇聚组合成统一的细胞出口；将第(K-1)个微通道单体的第N个废液出口与第K个微通道单体的第N个废液出口密封连接汇聚组合成统一的废液出口。Optionally, the parallel connection form further comprises sealingly combining the Nth cell outlet of the (K-1) microchannel monomer with the Nth cell outlet of the Kth microchannel monomer to form a unified cell. The outlet; the Nth waste liquid outlet of the (K-1) microchannel monomer and the Nth waste liquid outlet of the Kth microchannel monomer are sealed and combined to form a unified waste liquid outlet.
可选地，多次并联的连接形式为将多个并联式螺旋形微通道以并联的连接形式相互组合；或将并联式螺旋形微通道与微通道单体以并联的形式相互组合。Alternatively, the multiple parallel connection forms are a combination of a plurality of parallel spiral microchannels in parallel connection; or a parallel spiral microchannel and a microchannel unit are combined in parallel.
可选地，串并联组合的连接形式为将一个或多个并联式螺旋形微通道和/或一个或多个串联式螺旋形微通道和/或一个或多个微通道单体以串联的连接形式或并联的连接形式相互组合，形成混联式螺旋形流道。Optionally, the series-parallel combination is in the form of a series connection of one or more parallel spiral microchannels and/or one or more series spiral microchannels and/or one or more microchannel monomers. Formal or parallel connection forms are combined with each other to form a hybrid spiral flow path.
一种单螺旋形微通道，包括入口、与入口密封连接的微通道以及与通道远 离入口的一端密封连接的出口，所述的入口由细胞入口及鞘液入口汇聚而成，所述的出口由废液出口及细胞出口汇聚而成，其中，细胞入口用于进入含稀有细胞或颗粒的样本液体；细胞出口用于收集分选富集后的稀有细胞或颗粒；鞘液入口用于添加带有渗透压，保证稀有细胞或颗粒完整的稀释液。A single spiral microchannel comprising an inlet, a microchannel sealingly connected to the inlet, and an outlet sealingly connected to an end of the channel remote from the inlet, the inlet being formed by a cell inlet and a sheath fluid inlet, the outlet being The waste liquid outlet and the cell outlet are aggregated, wherein the cell inlet is used to enter a sample liquid containing rare cells or particles; the cell outlet is used to collect the rare cells or particles after sorting and enrichment; the sheath liquid inlet is used for adding Osmotic pressure ensures a complete dilution of rare cells or particles.
可选地，所述细胞入口和/或鞘液入口和/或废液出口和/或细胞出口设置在微通道的几何外形外侧。Optionally, the cell inlet and/or sheath fluid inlet and/or waste outlet and/or cell outlet are disposed outside of the geometry of the microchannel.
一种单螺旋形微通道的串联安装结构，采用P个单螺旋形微通道串联设置的方式，第(Q-1)个单螺旋形微通道的细胞出口与第Q个单螺旋形微通道的细胞入口相连接，1＜Q≤P。A series installation structure of single spiral microchannels adopting P single spiral microchannels in series, the cell outlet of the (Q-1) single spiral microchannel and the Qth single spiral microchannel The cell inlets are connected, 1 < Q ≤ P.
可选地，第(Q-1)个单螺旋形微通道的废液出口处设有压力平衡模块。Optionally, a pressure equalization module is disposed at the waste liquid outlet of the (Q-1) single spiral microchannel.
可选地，所述压力平衡模块为压力泵或压力平衡微通道。Optionally, the pressure balance module is a pressure pump or a pressure balanced microchannel.
可选地，所述压力平衡模块为与第(Q-1)单个螺旋形微通道的废液出口密封连接的压力平衡微通道。Optionally, the pressure equalization module is a pressure balanced microchannel that is sealingly coupled to the waste liquid outlet of the (Q-1) single spiral microchannel.
可选地，所述的压力平衡微通道和与其相匹配的所有单螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。Optionally, the pressure-balanced microchannel and all of the single-helical microchannels matched thereto form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed in the geometry of the spiral microchannel Outside the graph, the flow rate through the contents of the cell outlet is matched by adjusting the length and inner diameter of the pressure-balanced microchannel.
可选地，所有压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。Optionally, the outlets of all of the pressure balanced microchannels are disposed outside of the geometry of the helical microchannels.
可选地，所述细胞入口和/或鞘液入口和/或废液出口和/或细胞出口设置在螺旋形微通道的几何外形外侧。Optionally, the cell inlet and/or sheath fluid inlet and/or waste outlet and/or cell outlet are disposed outside of the geometry of the helical microchannel.
一种单螺旋形微通道的并联安装结构，采用X个单螺旋形微通道并联设置的方式，样本溶液从X个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从X个单螺旋形微通道的细胞出口流出；X＞1。A parallel installation structure of single spiral microchannels adopts X single spiral microchannels in parallel, the sample solution enters from the cell inlet of X single spiral microchannels, and rare cells or particles from X single spirals The cell outlet of the microchannel flows out; X>1.
可选地，将X个单螺旋形微通道的细胞入口和/或细胞出口相互连通。Alternatively, the cell inlets and/or cell outlets of the X single helical microchannels are interconnected.
可选地，在第X个单螺旋形微通道的废液出口处设有压力平衡模块；所述 压力平衡模块为压力泵或压力平衡微通道。Optionally, a pressure equalization module is disposed at the waste liquid outlet of the Xth single spiral microchannel; the pressure balance module is a pressure pump or a pressure balanced microchannel.
可选地，所述的压力平衡微通道和与其相匹配的单螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。Optionally, the pressure-balanced microchannel and the matched single-helical microchannel form a spiral microchannel that does not intersect each other, and the outlet of the one or more pressure-balanced microchannels is disposed on the geometry of the spiral microchannel Externally, the flow rate through the contents of the cell outlet that matches it is adjusted by adjusting the length and inner diameter of the pressure-balanced microchannel.
可选地，所有细胞入口和/或鞘液入口和/或废液出口和/或细胞出口设置在微通道的几何外形外侧。Optionally, all cell inlets and/or sheath fluid inlets and/or waste outlets and/or cell outlets are disposed outside of the geometry of the microchannels.
可选地，所有细胞入口和/或鞘液入口均设置在螺旋形微通道的几何图形外部。Optionally, all cell inlets and/or sheath fluid inlets are disposed outside of the geometry of the helical microchannels.
本发明的有益效果在于：The beneficial effects of the invention are:
本发明针对螺旋形微通道在恶性肿瘤患者外周血循环肿瘤细胞的分选富集领域的技术难题，开发更简单有效的技术，巧妙地设计出了合理级联应用多个螺旋形微通道的串联及并联式螺旋形微通道惯性分离结构，实现了在单张螺旋形惯性微流芯片上的级联叠加式分级富集和/或纯化循环肿瘤细胞。相对于现有的同类技术，本发明对分选富集和/或外周血循环肿瘤细胞，不仅具有良好的循环肿瘤细胞的捕获效率和处理通量，同时，也可以最大化循环肿瘤细胞的纯度，最小化循环肿瘤细胞的损失，并维持循环肿瘤细胞的原始表型，极大地降低检测成本，缩短了处理时间和流程，所获得的含有高纯度循环肿瘤细胞的细胞悬液特别适宜于后续的生物医学检测、遗传学分析、细胞培养、异种移植瘤制备等，具有广泛而深远的临床实际应用价值。The invention aims at the technical problem of the spiral microchannel in the field of sorting and enriching the peripheral blood circulation tumor cells of malignant tumor patients, develops a simpler and more effective technology, and skillfully designs a series connection of a plurality of spiral microchannels by rational cascade. The parallel spiral microchannel inertial separation structure realizes cascading superposition hierarchical enrichment and/or purification of circulating tumor cells on a single spiral inertial microfluidic chip. Compared with the existing similar technology, the present invention not only has a good collection efficiency and processing flux of circulating tumor cells, but also maximizes the purity of circulating tumor cells, for sorting enrichment and/or peripheral blood circulating tumor cells. Minimize the loss of circulating tumor cells and maintain the original phenotype of circulating tumor cells, greatly reducing the cost of detection, shortening the processing time and flow, and obtaining cell suspensions containing high-purity circulating tumor cells are particularly suitable for subsequent organisms. Medical testing, genetic analysis, cell culture, xenograft tumor preparation, etc., have extensive and far-reaching clinical practical application value.
本发明的其他优点、目标和特征在某种程度上将在随后的说明书中进行阐述，并且在某种程度上，基于对下文的考察研究对本领域技术人员而言将是显而易见的，或者可以从本发明的实践中得到教导。本发明的目标和其他优点可以通过下面的说明书来实现和获得。Other advantages, objects, and features of the invention will be set forth in part in the description which follows, and in the <RTIgt; The teachings of the present invention are taught. The objectives and other advantages of the invention may be realized and obtained by the following description.
附图说明DRAWINGS
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作优选的详细描述，其中：In order to make the objects, technical solutions, and advantages of the present invention more comprehensible, the present invention
图1为本发明中实施例一的结构示意图；1 is a schematic structural view of Embodiment 1 of the present invention;
图2为本发明中实施例二的结构示意图；2 is a schematic structural view of Embodiment 2 of the present invention;
图3为本发明中实施例三的结构示意图；3 is a schematic structural view of Embodiment 3 of the present invention;
图4为本发明中实施例四的结构示意图；Figure 4 is a schematic structural view of Embodiment 4 of the present invention;
图5为本发明中实施例五的结构示意图；Figure 5 is a schematic structural view of Embodiment 5 of the present invention;
图6为本发明中实施例六的结构示意图；Figure 6 is a schematic structural view of Embodiment 6 of the present invention;
图7为本发明中实施例七的结构示意图；Figure 7 is a schematic structural view of Embodiment 7 of the present invention;
图8为本发明的实施例一中不同尺寸的颗粒在不同流速下的运动轨迹图；Figure 8 is a motion trajectory diagram of particles of different sizes at different flow rates in the first embodiment of the present invention;
图8a为流速为150μl/min的不同尺寸的颗粒在实施例一中的运动轨迹；Figure 8a is a motion trajectory of the particles of different sizes at a flow rate of 150 μl/min in the first embodiment;
图8b为流速为300μl/min的不同尺寸的颗粒在实施例一中的运动轨迹；Figure 8b is a motion trajectory of the particles of different sizes at a flow rate of 300 μl/min in the first embodiment;
图8c为流速为450μl/min的不同尺寸的颗粒在实施例一中的运动轨迹；Figure 8c is a motion trajectory of the particles of different sizes at a flow rate of 450 μl/min in the first embodiment;
图8d为流速为600μl/min的不同尺寸的颗粒在实施例一中的运动轨迹；Figure 8d is a motion trajectory of the particles of different sizes at a flow rate of 600 μl/min in the first embodiment;
图8e为流速为750μl/min的不同尺寸的颗粒在实施例一中的运动轨迹；Figure 8e is a motion trajectory of the particles of different sizes at a flow rate of 750 μl/min in the first embodiment;
图8f为流速为900μl/min的不同尺寸的颗粒在实施例一中的运动轨迹；Figure 8f is a motion trajectory of the particles of different sizes at a flow rate of 900 μl/min in the first embodiment;
图9为本发明的实施例一中不同螺旋形微通道出口处的显微镜图片；Figure 9 is a micrograph of the exit of different spiral microchannels in the first embodiment of the present invention;
图9a为传统的单螺旋形微通道细胞出口处的显微图片；Figure 9a is a photomicrograph at the exit of a conventional single-spiral microchannel cell;
图9b为本发明所述双螺旋形微通道细胞出口处的显微图片；Figure 9b is a micrograph of the outlet of the double helix microchannel cell of the present invention;
图9c为本发明所述双螺旋形微通道废液出口7处的显微图片；Figure 9c is a photomicrograph of the double spiral microchannel waste liquid outlet 7 of the present invention;
图10为本发明的实施例一中螺旋形微通道中流体流动速度标注；Figure 10 is a diagram showing the fluid flow velocity in the spiral microchannel in the first embodiment of the present invention;
图11为传统的单螺旋形微通道；Figure 11 is a conventional single spiral microchannel;
图12为本发明中实施例八的结构示意图；Figure 12 is a schematic structural view of Embodiment 8 of the present invention;
图13为本发明中实施例九的结构示意图；Figure 13 is a schematic structural view of Embodiment 9 of the present invention;
图14为本发明中实施例十的结构示意图；Figure 14 is a schematic structural view of Embodiment 10 of the present invention;
图15为本发明中实施例十一的结构示意图；Figure 15 is a schematic structural view of Embodiment 11 of the present invention;
图16为本发明中实施例十二的结构示意图；Figure 16 is a schematic structural view of Embodiment 12 of the present invention;
图17为本发明中实施例十三的结构示意图；Figure 17 is a schematic structural view of Embodiment 13 of the present invention;
图18为本发明中实施例十四的结构示意图；Figure 18 is a schematic structural view of Embodiment 14 of the present invention;
图19为本发明中实施例十五的结构示意图；Figure 19 is a schematic structural view of Embodiment 15 of the present invention;
图20为本发明中实施例十六的结构示意图；Figure 20 is a schematic structural view of Embodiment 16 of the present invention;
图21为本发明中实施例十七的结构示意图；Figure 21 is a schematic structural view of Embodiment 17 of the present invention;
图22为本发明中实施例十八的结构示意图；Figure 22 is a schematic structural view of Embodiment 18 of the present invention;
图23为本发明中实施例八的仿真结果，表示不同流速下聚苯乙烯微球的运动轨迹；Figure 23 is a simulation result of the eighth embodiment of the present invention, showing the trajectories of the polystyrene microspheres at different flow rates;
图23a中流速为200ul/min；The flow rate in Figure 23a is 200 ul / min;
图23b中流速为350ul/min；The flow rate in Figure 23b is 350 ul / min;
图23c中流速为500ul/min；The flow rate in Figure 23c is 500 ul / min;
图23d中流速为650ul/min；The flow rate in Figure 23d is 650 ul / min;
图23e中流速为800ul/min；The flow rate in Figure 23e is 800 ul / min;
图23f中流速为950ul/min；The flow rate in Figure 23f is 950 ul / min;
图24为本发明中实施例八与实施例十四的仿真结果对比图；Figure 24 is a comparison diagram of simulation results of Embodiment 8 and Embodiment 14 of the present invention;
图24a表示实施例八中第二个单螺旋形微通道的细胞出口处的富集情况；Figure 24a shows the enrichment at the cell exit of the second single helical microchannel in Example VIII;
图24b表示图11所示传统的单螺旋形微通道的细胞出口处的富集情况；Figure 24b shows the enrichment at the cell exit of the conventional single-spiral microchannel of Figure 11;
图24c表示实施例八中第一个单螺旋形微通道的细胞出口处的富集情况；Figure 24c shows the enrichment at the cell outlet of the first single helical microchannel in Example VIII;
图24d表示图11所示传统的单螺旋形微通道的废液出口处的富集情况；Figure 24d shows the enrichment of the waste liquid outlet at the conventional single spiral microchannel shown in Figure 11;
图24e表示实施例八中第二个单螺旋形微通道的废液出口处的富集情况；Figure 24e shows the enrichment at the waste liquid outlet of the second single spiral microchannel in the eighth embodiment;
图25表示实施例八中第二个单螺旋形微通道的废液出口及细胞出口处收集到的细胞的免疫荧光染色结果；Figure 25 is a view showing the results of immunofluorescence staining of the waste liquid outlet of the second single-spiral microchannel in the eighth embodiment and the cells collected at the cell outlet;
图25a为第二个单螺旋形微通道的废液出口处收集到的细胞在光镜40倍下的细胞形态；Figure 25a shows the cell morphology of the cells collected at the waste liquid outlet of the second single-spiral microchannel at 40 times the light microscope;
图25b为双螺旋芯片分选富集到的白细胞DAPI荧光染色结果；Figure 25b shows the results of DAPI fluorescence staining of leukocytes enriched by double-helical chip sorting;
图25c为双螺旋芯片分选富集到的白细胞CD45抗体荧光染色结果；Figure 25c is a result of fluorescent staining of leukocyte CD45 antibody enriched by double-helical chip sorting;
图25d为双螺旋芯片分选富集到的白细胞荧光染色Merge结果；Figure 25d shows the result of fluorescent staining of leukocytes by double-stranded chip sorting;
图25e为第二个单螺旋形微通道的细胞出口处收集到的细胞在光镜40倍下的细胞形态；Figure 25e is the cell morphology of the cells collected at the cell exit of the second single-spiral microchannel at 40 times the light microscope;
图25f为双螺旋芯片分选富集到的SW480DAPI荧光染色结果Figure 25f shows the SW480DAPI fluorescence staining results of the double-helical chip sorting and enrichment
图25g为双螺旋芯片分选富集到的SW480EPCAM荧光染色结果Figure 25g shows the results of fluorescent staining of SW480EPCAM enriched by double-helical chip sorting.
图25h为双螺旋芯片分选富集到的SW480荧光染色Merge结果；Figure 25h is a result of SW480 fluorescence staining Merge obtained by double-helical chip sorting and enrichment;
图26为双螺旋芯片对肿瘤细胞的捕获效率的分析结果；Figure 26 is a result of analysis of the capture efficiency of tumor cells by a double helix chip;
图26a为双螺旋芯片对全血样本中不同浓度肿瘤细胞回收情况；Figure 26a shows the recovery of different concentrations of tumor cells in a whole blood sample by a double helix chip;
图26b为双螺旋芯片对不同肿瘤细胞捕获效率直方图。Figure 26b is a histogram of the capture efficiency of different tumor cells by a double helix chip.
具体实施方式Detailed ways
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。需要说明的是，以下实施例中所提供的图示仅以示意方式说明本发明的基本构想，在不冲突的情况下，以下实施例及实施例中的特征可以相互组合。The embodiments of the present invention are described below by way of specific examples, and those skilled in the art can readily understand other advantages and effects of the present invention from the disclosure of the present disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes can be made without departing from the spirit and scope of the invention. It should be noted that the illustrations provided in the following embodiments merely illustrate the basic concept of the present invention in a schematic manner, and the features in the following embodiments and embodiments may be combined with each other without conflict.
请参阅图1-图26附图中的元件标号分别表示：第一个单螺旋形微通道的细胞入口1、第一个单螺旋形微通道的鞘液入口2、第一个单螺旋形微通道的入口A、压力平衡微通道3、第一个单螺旋形微通道的细胞出口4、第二个单螺旋形微通道的鞘液入口5、第一个单螺旋形微通道的废液出口6、第一个单螺旋形微通道的出口B、第二个单螺旋形微通道的废液出口7、第二个单螺旋形微通道的细胞出口8、第二个单螺旋形微通道的出口C、第二个螺旋形微通道中的第一个单螺旋形微通道的鞘液入口9、第二个螺旋形微通道的第一个单螺旋形微通道的 废液出口10、第二个螺旋形微通道的第二个单螺旋形微通道的鞘液入口11、第二个螺旋形微通道的第二个单螺旋形微通道的细胞出口12、第二个螺旋形微通道的第二个单螺旋形微通道的废液出口13、第二个螺旋形微通道的第一个单螺旋形微通道的细胞入口14、第三个单螺旋形微通道的鞘液入口15、第三个单螺旋形微通道的废液出口16、第四个单螺旋形微通道的鞘液入口17、第四个单螺旋形微通道的细胞出口18、第四个单螺旋形微通道的废液出口19。Referring to the reference numerals in the drawings of Figs. 1 to 26, respectively, the cell inlet 1 of the first single spiral microchannel, the sheath fluid inlet 2 of the first single spiral microchannel, and the first single spiral micro The inlet A of the channel, the pressure-balanced microchannel 3, the cell outlet 4 of the first single-spiral microchannel, the sheath inlet 5 of the second single-spiral microchannel, and the waste outlet of the first single-spiral microchannel 6. The outlet B of the first single spiral microchannel, the waste outlet 7 of the second single spiral microchannel, the cell outlet 8 of the second single spiral microchannel, and the second single spiral microchannel The outlet liquid C, the sheath liquid inlet 9 of the first single spiral microchannel in the second spiral microchannel, the waste liquid outlet 10 of the first single spiral microchannel of the second spiral microchannel, and the second The sheath flow inlet 11 of the second single spiral microchannel of the spiral microchannel, the cell outlet 12 of the second single spiral microchannel of the second spiral microchannel, and the second spiral microchannel The waste liquid outlet 13 of the two single spiral microchannels, the first single of the second spiral microchannel The cell inlet 14 of the spiral microchannel, the sheath inlet 15 of the third single helical microchannel, the waste outlet 16 of the third single helical microchannel, and the sheath inlet 17 of the fourth single helical microchannel The cell outlet 18 of the fourth single-spiral microchannel, and the waste outlet 19 of the fourth single-spiral microchannel.
实施例一 Embodiment 1
本发明涉及一种螺旋形微通道，用于分选富集稀有细胞或颗粒，在本实施例中包括两个单螺旋形微通道，该两个单螺旋形微通道设置在同一平面内且互不交叉地缠绕成环状，如图1所示。The present invention relates to a spiral microchannel for sorting and enriching rare cells or particles, in this embodiment comprising two single spiral microchannels, the two single spiral microchannels being disposed in the same plane and mutually It is wound into a ring without crossing, as shown in Figure 1.
单个的单螺旋形微通道包括入口A、与入口密封连接的微通道以及与通道远离入口的一端密封连接的出口B，所述的入口由细胞入口1及鞘液入口2汇聚而成，所述的出口B由废液出口6及细胞出口4汇聚而成；The single single spiral microchannel includes an inlet A, a microchannel sealingly connected to the inlet, and an outlet B sealingly connected to an end of the channel remote from the inlet, the inlet being aggregated by the cell inlet 1 and the sheath inlet 2, The outlet B is formed by the waste liquid outlet 6 and the cell outlet 4;
第一个单螺旋形微通道的细胞出口4与第二个单螺旋形微通道的细胞入口密封连接成第一个分级循环点，由第一个单螺旋形微通道的废液出口6、第二个单螺旋形微通道的鞘液入口5以及第一个分级循环点构成第一个分级循环处；The cell outlet 4 of the first single-spiral microchannel is sealingly connected to the cell inlet of the second single-spiral microchannel to form a first staged circulation point, and the waste liquid outlet of the first single-spiral microchannel is 6, The sheath liquid inlet 5 of the two single spiral microchannels and the first staged circulation point constitute the first classification cycle;
在第一个分级循环处内的第一个单螺旋形微通道的废液出口6设有压力平衡微通道3，第二个单螺旋形微通道的细胞入口的液体流速为v1，第一个单螺旋形微通道的细胞入口1的液体流速为v2；第二个单螺旋形微通道的细胞出口8的液体流速为v3，第一个单螺旋形微通道的细胞出口4的液体流速为v4；第二个单螺旋形微通道的废液出口7的液体流速为v5，第一个单螺旋形微通道的废液出口6的液体流速为v6；The waste liquid outlet 6 of the first single spiral microchannel in the first classification cycle is provided with a pressure equalization microchannel 3, and the liquid flow rate of the cell inlet of the second single spiral microchannel is v1, the first The liquid flow rate of the cell inlet 1 of the single spiral microchannel is v2; the liquid flow rate of the cell outlet 8 of the second single spiral microchannel is v3, and the liquid flow rate of the cell outlet 4 of the first single spiral microchannel is v4 The liquid flow rate of the waste liquid outlet 7 of the second single spiral microchannel is v5, and the liquid flow rate of the waste liquid outlet 6 of the first single spiral microchannel is v6;
该压力平衡微通道用于使第二个单螺旋形微通道的细胞入口的液体流动方向和第一个单螺旋形微通道的细胞入口1的液体流动方向一致，且0.7*v2≤v1≤1.3*v2；使第二个单螺旋形微通道的细胞出口8的液体流动方向和第一个单螺旋形微通道的细胞出口4的液体流动方向一致，且0.7*v4≤v3≤1.3*v4，且 v1＝v4；使第二个单螺旋形微通道的废液出口7的液体流动方向和第一个单螺旋形微通道的废液出口6的液体流动方向一致，且0.7*v6≤v5≤1.3*v6；The pressure-balanced microchannel is used to make the liquid flow direction of the cell inlet of the second single-spiral microchannel coincide with the liquid flow direction of the cell inlet 1 of the first single-spiral microchannel, and 0.7*v2≤v1≤1.3 *v2; making the liquid flow direction of the cell outlet 8 of the second single-spiral microchannel coincide with the liquid flow direction of the cell outlet 4 of the first single-spiral microchannel, and 0.7*v4≤v3≤1.3*v4, And v1=v4; the liquid flow direction of the waste liquid outlet 7 of the second single spiral microchannel is consistent with the liquid flow direction of the waste liquid outlet 6 of the first single spiral microchannel, and 0.7*v6≤v5≤ 1.3*v6;
其中，第一个单螺旋形微通道的细胞入口1用于进入含稀有细胞或颗粒的样本液体；第二个单螺旋形微通道的细胞出口8用于收集分选富集后的稀有细胞或颗粒；所述第一个单螺旋形微通道的鞘液入口2以及第二个单螺旋形微通道的鞘液入口5用于添加带有渗透压，保证稀有细胞或颗粒完整性和生物学活性的稀释液。Wherein the cell inlet 1 of the first single-spiral microchannel is used to enter a sample liquid containing rare cells or particles; the cell outlet 8 of the second single-spiral microchannel is used to collect rare cells after sorting and enrichment or Particles; the sheath fluid inlet 2 of the first single spiral microchannel and the sheath fluid inlet 5 of the second single spiral microchannel are used to add osmotic pressure to ensure rare cell or particle integrity and biological activity Diluent.
采用两个单螺旋形微通道的形式，第二个单螺旋形微通道能够直接对第一个单螺旋形微通道分选富集到的稀有细胞或颗粒进行进一步的分选富集，从而比单个的单螺旋形微通道捕获稀有细胞或颗粒的效率更高，相当于对稀有细胞或颗粒进行了两次分选富集和纯化，其对靶细胞的捕获效率、吞吐量、细胞纯度、细胞活性等四项参数均优于单螺旋形微通道。In the form of two single-helical microchannels, the second single-helical microchannel can directly sort and enrich the rare cells or particles enriched by the first single-spiral microchannel, thereby A single single-helical microchannel captures rare cells or particles more efficiently, which is equivalent to two sorts of enrichment and purification of rare cells or particles, which capture the target cells, throughput, cell purity, and cells. The four parameters of activity are superior to single spiral microchannels.
本发明所述双螺旋形微通道由第一个单螺旋形微通道和第二个单螺旋形微通道经首尾直接连接方式构成一个外观为平行且具有相同圆心点的双螺旋形微通道的整体几何结构，其特征是双螺旋形微通道的第一个单螺旋形微通道的入口A与第二个单螺旋形微通道的出口均位于微通道主体几何结构的外侧。The double helix microchannel of the present invention comprises a first single spiral microchannel and a second single spiral microchannel formed by a direct connection between the first spiral and the microchannel, and a double spiral microchannel having a parallel appearance and the same center point. The geometric structure is characterized in that the inlet A of the first single spiral microchannel of the double helix microchannel and the outlet of the second single spiral microchannel are located outside the geometry of the microchannel body.
本发明所述双螺旋形微通道的每个单螺旋形微通道均有细胞入口1、鞘液入口2、细胞出口4和废液出口6，其中，细胞入口4和鞘液入口2汇聚成每个单螺旋形微通道的入口A，细胞出口4和废液出口6汇聚成每个单螺旋形微通道的出口B。细胞入口1通常位于既定单螺旋形微通道入口的外侧壁部分，鞘液入口2通常位于既定单螺旋形微通道入口的内侧壁部分；细胞出口4通常位于既定单螺旋形微通道出口的内侧壁部分，废液出口6通常位于既定单螺旋形微通道出口的外侧壁部分。Each of the single spiral microchannels of the double helix microchannel of the present invention has a cell inlet 1, a sheath inlet 2, a cell outlet 4, and a waste liquid outlet 6, wherein the cell inlet 4 and the sheath fluid inlet 2 converge into each The inlet A of the single spiral microchannel, the cell outlet 4 and the waste outlet 6 converge into the outlet B of each single spiral microchannel. The cell inlet 1 is typically located at the outer sidewall portion of the inlet of the intended single helical microchannel, the sheath fluid inlet 2 is typically located at the inner sidewall portion of the inlet of the intended single helical microchannel; the cell outlet 4 is typically located at the inner sidewall of the exit of the intended single helical microchannel. In part, the waste liquid outlet 6 is typically located at the outer sidewall portion of the outlet of the intended single spiral microchannel.
本发明所述双螺旋形微通道的两个单螺旋形微通道具有相同或相近的限定纵横比的矩形横载面，也可为内侧壁高度低于外侧壁高度的梯形截面，用于因靶细胞与非靶细胞存在的尺寸差异而仅使靶细胞在微通道内侧壁形成聚焦流， 非靶细胞则以迪恩涡流方式在微通道内侧壁和外侧壁之间来回横向迁移。更进一步，微通道的宽度是0.1-1000μm，深度是0.1-800μm，或更进一步，微通道的宽度是100-800μm，深度是50-500μm，或更进一步，微通道的宽度是600μm，深度是140-180μm。The two single spiral microchannels of the double spiral microchannel of the present invention have the same or similar rectangular cross-sectional planes defining the aspect ratio, and may also be a trapezoidal cross-section with an inner sidewall height lower than the outer sidewall height for the target The difference in size between the cells and the non-target cells allows only the target cells to form a focused flow in the inner walls of the microchannels, while the non-target cells migrate laterally back and forth between the inner and outer sidewalls of the microchannels in a Dean vortex. Further, the width of the microchannel is 0.1-1000 μm, the depth is 0.1-800 μm, or further, the width of the microchannel is 100-800 μm, the depth is 50-500 μm, or further, the width of the microchannel is 600 μm, and the depth is 140-180 μm.
本发明所述双螺旋形微通道的两个单螺旋形微通道具有相同或相近的长度，其特征是第1个和第2个单螺旋形微通道的螺旋形微通道起始点至终点的长度是非靶细胞流体力学特征的1.7-2.0个迪恩周期，或者，2.7-3.0个迪恩周期，或者，3.7-4.0个迪恩周期，或者，4.7-5.0个迪恩周期，或者，5.7-6.0个迪恩周期，或者，6.7-7.0个迪恩周期，或者，7.7-8.0个迪恩周期，或者，8.7-9.0个迪恩周期，或者，9.7-10.0个迪恩周期。在此条件下，在第一个和第二个单螺旋形微通道的终点处，非靶细胞沿迪恩涡流迁移至螺旋形微通道的外侧壁部分。The two single helical microchannels of the double helix microchannel of the present invention have the same or similar length and are characterized by the length from the start point to the end point of the spiral microchannel of the first and second single spiral microchannels 1.7-2.0 Dean cycles for non-target cell hydrodynamics, or 2.7-3.0 Dean cycles, or 3.7-4.0 Dean cycles, or 4.7-5.0 Dean cycles, or 5.7-6.0 A Dean cycle, or 6.7-7.0 Dean cycles, or 7.7-8.0 Dean cycles, or 8.7-9.0 Dean cycles, or 9.7-10.0 Dean cycles. Under this condition, at the end of the first and second single-spiral microchannels, the non-target cells migrate along the Dean vortex to the outer sidewall portion of the helical microchannel.
本发明所述双螺旋形微通道的第一个单螺旋形微通道的废液出口有压力平衡微通道3，本发明所上述的压力平衡微通道3的尺寸与外形无特殊要求，对其纵横比、曲率半径、宽度、高度和长度均无特殊要求，其作用是平衡第一个单螺旋形微通道的细胞出口1的压力，使第二个单螺旋形微通道的细胞入口或者第一个单螺旋形微通道的细胞出口4的流速与方向与第一个单螺旋形微通道的细胞入口1和第二个单螺旋形微通道的细胞出口4的流速与方向保持相同或极相近，同时，使第一个单螺旋形微通道的废液出口6的流速与方向与第二个单螺旋形微通道的废液出口7的流速与方向保持相同或极相近。The waste liquid outlet of the first single spiral microchannel of the double spiral microchannel of the present invention has a pressure balance microchannel 3. The size and shape of the pressure balance microchannel 3 of the present invention have no special requirements, and There is no special requirement for the ratio, radius of curvature, width, height and length. The function is to balance the pressure of the cell outlet 1 of the first single-spiral microchannel, so that the cell inlet of the second single-spiral microchannel or the first one The flow rate and direction of the cell outlet 4 of the single-spiral microchannel are the same or very similar to the flow rate and direction of the cell inlet 1 of the first single-spiral microchannel and the cell outlet 4 of the second single-spiral microchannel, while The flow rate and direction of the waste liquid outlet 6 of the first single spiral microchannel are kept the same or very close to the flow rate and direction of the waste liquid outlet 7 of the second single spiral microchannel.
本发明所述双螺旋形微通道的第一个和第二个螺旋形微通道的水平平行间距是0.1-1000μm。The horizontal parallel spacing of the first and second helical microchannels of the double helix microchannel of the present invention is from 0.1 to 1000 μm.
本发明所述双螺旋形微通道的第一个和/或第二个螺旋形微通道的细胞入口1微通道的宽度是微通道整体宽度的10-40％，第一个和/或第二个螺旋形微通道的废液出口6的微通道的宽度是微通道整体宽度的40-90％。The width of the cell inlet 1 microchannel of the first and/or second helical microchannel of the double helix microchannel of the present invention is 10-40% of the overall width of the microchannel, the first and/or the second The width of the microchannel of the waste liquid outlet 6 of the spiral microchannel is 40-90% of the overall width of the microchannel.
本发明所述的血液样本是指具有一定红细胞比积的血液样本，或者，通过溶血方式去除大量红细胞的血液样本，或者，通过其他本领域公知方法或装置 获取的无红细胞或仅有少量或极少量红细胞的血液样本。The blood sample according to the present invention refers to a blood sample having a certain ratio of red blood cells, or a blood sample for removing a large amount of red blood cells by hemolysis, or a red blood cell or only a small amount or a pole obtained by other methods or devices known in the art. A small blood sample of red blood cells.
本发明所述鞘液是指具有一定渗透压，且不破坏血细胞完整性的任何一种本领域公知的各种溶液，如磷酸盐缓冲液、培养血清、生理盐水等。The sheath fluid of the present invention refers to any of various solutions known in the art having a certain osmotic pressure and not destroying the integrity of blood cells, such as phosphate buffer, culture serum, physiological saline and the like.
粒子在弯弧形微通道中能否形成惯性聚焦流动与微通道的尺寸和曲率半径、粒子的尺寸和流速等因素都有关系。已有的研究表明，在通道的宽度大于高度的矩形截面通道中，一定尺寸的粒子形成惯性聚焦流动通常需要满足条件a/h≥0.07，a是粒子的直径，h是微通道的最小尺寸，惯性微流芯片中微通道的高度通常是最小尺寸。如果粒子尺寸过小，可能导致粒子无法在有限长度的微通道内惯性聚焦。也有研究表明，a/h<0.5也是限制条件，粒子尺寸过大可能会导致通道容易堵塞。因此，在设计制备时，要根据分离粒子的尺寸来确定微通道的尺寸。Whether the particle can form an inertial focusing flow in a curved arc microchannel is related to the size and radius of curvature of the microchannel, the size and flow velocity of the particle. Previous studies have shown that in a rectangular cross-section channel with a channel width greater than the height, a certain size of particles forming an inertial focusing flow usually needs to satisfy the condition a/h ≥ 0.07, a is the diameter of the particle, and h is the minimum size of the microchannel. The height of the microchannels in an inertial microfluidic chip is typically the smallest size. If the particle size is too small, it may result in the particle not being able to focus inertially within a finite length microchannel. Studies have also shown that a / h < 0.5 is also a limiting condition, too large particle size may cause the channel to be easily blocked. Therefore, in design preparation, the size of the microchannels is determined according to the size of the separated particles.
本发明提供一种从外周血级联或串联式叠加分选富集外周血循环肿瘤细胞的方法，具体包括：待富集的样本溶液从第一个单螺旋形微通道的细胞入口1进样，鞘液从第一个单螺旋形微通道的鞘液入口2和第二个单螺旋形微通道的鞘液入口5进样，在一定的流体流速条件下，在第二个单螺旋形微通道的细胞出口8收集富含靶细胞的流体，而非靶细胞则从第二个单螺旋形微通道的废液出口7收集。The invention provides a method for enriching peripheral blood circulating tumor cells from peripheral blood cascade or tandem superposition, specifically comprising: the sample solution to be enriched is injected from the cell inlet 1 of the first single spiral microchannel, The sheath fluid is injected from the sheath fluid inlet 2 of the first single spiral microchannel and the sheath fluid inlet 5 of the second single spiral microchannel, at a certain fluid flow rate, in the second single spiral microchannel The cell outlet 8 collects the fluid enriched in the target cells, while the non-target cells are collected from the waste liquid outlet 7 of the second single spiral microchannel.
本发明所述的双螺旋形微通道的工作原理如下：在第一个单螺旋形微通道的入口A处，血液样本自细胞入口1进入微通道，鞘液自鞘液入口2进入微通道，由于鞘液入口2的流体流速高于细胞入口1的流体流速，细胞入口1的所有细胞被挤压至第一个单螺旋形微通道的外侧壁，并由此进入后续的微通道，因此，可以说，在0个迪恩周期时，所有细胞的起始位置是第一个单螺旋形微通道的外侧壁。在惯性升力和迪恩曳力的共同作用下，靶细胞由于其尺寸较大，在0.5个迪恩周期时，在第一个单螺旋形微通道的内侧壁形成聚焦流束，并由于其惯性升力和迪恩曳力的作用，靶细胞在第一个单螺旋形微通道后续的微通道中始终保持在其内侧壁的聚焦流；非靶细胞因其尺寸较小，主要受到迪恩曳力 的作用，以迪恩涡流的形式在第一个单螺旋形微通道的外侧壁和内侧壁来回横向迁移，并且，通常是0.5个迪恩周期时位于第一个单螺旋形微通道的内侧壁，而在1个完整的迪恩周期时返回至其初始位置，即第一个单螺旋形微通道的外侧壁。由于第一个单螺旋形微通道的长度与上述迪恩周期存在一定比例，在其出口处，靶细胞从位于第一个单螺旋形微通道内侧壁的细胞出口4流出并直接进入第二个单螺旋形微通道的细胞入口，而非靶细胞流体则从位于第一个单螺旋形微通道外侧壁的废液出口5流出并经压力平衡微通道3收集；由于第一个单螺旋形微通道的细胞出口4是第二个单螺旋形微通道的细胞入口，因此，在第二个单螺旋形微通道中，按照与第一个单螺旋形微通道的相同工作原理，第一个单螺旋形微通道分选富集到的靶细胞在第二个单螺旋形微通道被第2次分选富集，或级联式分选富集，或串联式分选富集，或叠加式分选富集。因此，可以说，通过本发明所述的双螺旋形微通道，血液样本中的靶细胞被级联或串联式地进行了2次叠加式分选富集，同时，由于大部分非靶细胞已经被第一个螺旋形微通道去除，进入第二个单螺旋形微通道的细胞悬液的细胞总数整体上远远低于第二个单螺旋形微通道的细胞悬液的细胞总数，从而由于细胞因其密度的干扰少，其分选富集效果会比单独使用单个螺旋形微通道更好，最终能够得到高纯度的靶细胞。请注意，本实施例的以下部分实际上是在比较本发明所述双螺旋形通道与传统的单螺旋形微通道(如图11所示)的分选富集效果。The working principle of the double helix microchannel of the present invention is as follows: at the inlet A of the first single spiral microchannel, the blood sample enters the microchannel from the cell inlet 1, and the sheath fluid enters the microchannel from the sheath inlet 2, Since the fluid flow rate of the sheath liquid inlet 2 is higher than the fluid flow rate of the cell inlet 1, all cells of the cell inlet 1 are squeezed to the outer side wall of the first single spiral microchannel, and thereby enter the subsequent microchannel, thus, It can be said that at 0 Dean cycles, the starting position of all cells is the outer side wall of the first single spiral microchannel. Under the combined action of inertial lift and Dean drag, the target cells form a focused stream on the inner side wall of the first single spiral microchannel due to its large size at 0.5 Dein cycles, and due to its inertia With the effect of lift and Dean's drag, the target cells always maintain a focused flow on their inner sidewalls in the subsequent microchannels of the first single-helical microchannel; non-target cells are mainly subjected to Dean drag due to their small size. Acting in the form of a Dean vortex that migrates laterally back and forth between the outer and inner sidewalls of the first single helical microchannel, and is typically located on the inner side of the first single helical microchannel at 0.5 Dean cycles And return to its original position, the outer side wall of the first single spiral microchannel, at a complete Dean cycle. Since the length of the first single-helical microchannel is proportional to the above-described Dean cycle, at its exit, the target cells flow out from the cell outlet 4 located on the inner side wall of the first single-spiral microchannel and directly enter the second The cell inlet of the single-spiral microchannel, but not the target cell fluid, flows out of the waste liquid outlet 5 located on the outer side wall of the first single-spiral microchannel and is collected via the pressure-balanced microchannel 3; due to the first single-spiral micro The cell outlet 4 of the channel is the cell inlet of the second single-spiral microchannel, so in the second single-spiral microchannel, the first single is operated according to the same principle as the first single-spiral microchannel Spiral microchannel sorting enriched target cells are enriched by the second sorting in the second single spiral microchannel, or cascaded enrichment, or tandem sorting enrichment, or superposition Sorting and enriching. Therefore, it can be said that, by the double helix microchannel of the present invention, the target cells in the blood sample are subjected to two-stage superposition sorting enrichment in cascade or tandem, and at the same time, since most non-target cells have been Removed by the first helical microchannel, the total number of cells in the cell suspension entering the second single helical microchannel is generally much lower than the total number of cells in the cell suspension of the second single helical microchannel, Cells have less interference due to their density, and their sorting enrichment effect is better than using a single spiral microchannel alone, and finally high-purity target cells can be obtained. Please note that the following part of this embodiment is actually comparing the sorting enrichment effect of the double helix channel of the present invention with a conventional single spiral microchannel (as shown in FIG. 11).
在具体的实验中，螺旋形微通道的宽度l为600μm，曲率半径1.15cm，微通道高度h为130μm。传统的单螺旋形微通道(如图11所示)是在螺旋形平面结构内部设计入口，平面结构外部构建出口，其中，细胞入口和细胞出口的宽度是0.15mm，分别位于螺旋形微通道的外侧壁和内侧壁，鞘液入口和废液出口的宽度是0.45mm，分别位于螺旋形微通道的内侧壁和外侧壁，且细胞入口和废液入口均位于螺旋形微通道平面几何结构的内侧，细胞出口和鞘液出口均位于螺旋形微通道平面几何结构的外侧。本发明所述的双螺旋形微通道的细胞入口1和细胞出口4的宽度是0.15mm，鞘液入口2和废液出口6的宽度是0.45mm； 每个单螺旋形微通道均有细胞入口1、鞘液入口5、细胞出口4和废液出口6，其中，细胞入口1和鞘液入口2汇聚成每个单螺旋形微通道的入口，细胞出口4和废液出口6汇聚成每个单螺旋形微通道的出口B；细胞入口1位于既定单螺旋形微通道入口的外侧壁部分，鞘液入口2位于既定单螺旋形微通道入口的内侧壁部分；细胞出口4位于既定单螺旋形微通道出口的内侧壁部分，废液出口6位于既定单螺旋形微通道出口的外侧壁部分；第一个单螺旋形微通道的细胞出口4与第二个单螺旋形微通道的细胞入口密封连接成第一个分级循环点；由第一个单螺旋形微通道的废液出口6、第二个单螺旋形微通道的鞘液入口5以及第一个分级循环点构成第一个分级循环处；在第一个分级循环处内的第一个单螺旋形微通道的废液出口6设有压力平衡微通道3。考虑到不同流速的变化以及通道长度两个因素对不同尺寸细胞最终所产生的横向位移聚焦位置的不同，最终优化确定最佳参数。In a specific experiment, the spiral microchannel has a width l of 600 μm, a radius of curvature of 1.15 cm, and a microchannel height h of 130 μm. The conventional single-helical microchannel (shown in Figure 11) is designed to be designed inside the spiral planar structure, and the outer surface of the planar structure is constructed. The width of the cell inlet and the cell outlet is 0.15 mm, respectively located in the spiral microchannel. The outer and inner side walls, the sheath liquid inlet and the waste liquid outlet have a width of 0.45 mm, respectively located on the inner side wall and the outer side wall of the spiral microchannel, and the cell inlet and the waste liquid inlet are both located inside the spiral microchannel planar geometry. The cell outlet and the sheath fluid outlet are both outside the planar geometry of the spiral microchannel. The width of the cell inlet 1 and the cell outlet 4 of the double helix microchannel of the present invention is 0.15 mm, and the width of the sheath liquid inlet 2 and the waste liquid outlet 6 is 0.45 mm; each single spiral microchannel has a cell inlet 1. A sheath fluid inlet 5, a cell outlet 4, and a waste liquid outlet 6, wherein the cell inlet 1 and the sheath fluid inlet 2 converge into the inlet of each single spiral microchannel, and the cell outlet 4 and the waste liquid outlet 6 are aggregated into each The outlet B of the single spiral microchannel; the cell inlet 1 is located in the outer side wall portion of the inlet of the predetermined single spiral microchannel, the sheath fluid inlet 2 is located in the inner side wall portion of the inlet of the predetermined single spiral microchannel; the cell outlet 4 is located in the predetermined single spiral The inner side wall portion of the microchannel outlet, the waste liquid outlet 6 is located at the outer side wall portion of the outlet of the predetermined single spiral microchannel; the cell inlet 4 of the first single spiral microchannel and the cell inlet of the second single spiral microchannel are sealed Connected to the first staged circulation point; the first liquid circulation outlet 6 of the first single spiral microchannel, the sheath liquid inlet 5 of the second single spiral microchannel, and the first staged circulation point constitute the first staged cycle At the first The waste outlet of a single spiral microchannel within a hierarchical cycles at 6 is provided with pressure equalization microchannel 3. Considering the difference of different flow rates and the length of the channel, the difference in the lateral displacement focus position of the cells of different sizes is finally determined, and the optimal parameters are finally determined.
两种规格的螺旋形微通道芯片的制作主要通过基本的微细加工技术，采用SU8光刻胶在硅片上进行标准的软光刻技术制作模具，PDMS脱模后对其相应出入口处进行打孔处理，并键合在相应玻片上，为了增加键合的牢固性，放置70℃烤箱中，烘烤30min。The fabrication of two kinds of spiral microchannel chips is mainly through the basic micro-machining technology. The SU8 photoresist is used to make the mold on the silicon wafer by standard soft lithography technology. After the PDMS is demolded, the corresponding entrance and exit are punched. It was processed and bonded on the corresponding slide. In order to increase the firmness of the bond, it was placed in a 70 ° C oven and baked for 30 min.
键合后的螺旋形微通道芯片出入口采用磁力夹具装置以及精密导针的连接方式与进出微管进行紧密连接，并通过注射泵以及流速控制***从而达到进样稳定并可实时监测流速变化的目的。对螺旋形微通道芯片其分离效果测试前，首先采用鞘流液在1个大气压的高压力下对其密封性进行检测，结果无漏液情况，说明芯片键合处及芯片出入口连接较牢固，通过显微镜下观察芯片完整性及气泡排除情况，待气泡完全排除后。利用标准聚苯乙烯彩色微珠对螺旋形微通道分选富集循环肿瘤细胞的最佳参数进行优化。采用3～6μm微球、7～10μm微球、15～18μm微球进行混合分别模拟红细胞、白细胞、循环肿瘤细胞，采用磷酸盐缓冲液对3～6μm、7～10μm、15～18μm的微球分别稀释到1.0*10 7微粒/毫升、1.0*10 6微粒/毫升、1.0*10 2个微粒/毫升，并按照等体积比进行混合。将装 载混合微球的注射泵与装载磷酸盐缓冲液鞘流液的注射泵通过精密导针分别连接在排除气泡后的螺旋形芯片外侧和内侧壁入口，通过流速监测***以及压力泵控制调整流速，分别记录并收集本发明所述的双螺旋形微通道(如图1所示)、传统的单螺旋形微通道(如图11所示)在不同流速下(即：150μl/min、300μl/min、450μl/min、600μl/min、750μl/min、900μl/min)在各出口的微球分选富集情况。细胞出口处微球分离的结果示例情况见图8，因普通照相机无法清楚捕获到单个稀有细胞或颗粒的运动状态，故为了清楚直观的表述稀有细胞或颗粒的运动轨迹，以较小直径的圆点代表3～6μm和7～10μm的稀有细胞或颗粒，以较大直径的圆点代表15～18μm的稀有细胞或颗粒的运动轨迹。细胞出口8和废液出口7处微球分离的显微镜计数观察结果示例情况见图9。图9-a为传统的单螺旋形微通道细胞出口处的显微图片，三种微球均较多，提示较大粒子的分选富集效果较差，纯度低；图9-b为本发明所述双螺旋形微通道细胞出口处的显微图片，几乎均为较大直径的微球，提示较大粒子的分选富集效果非常良好，纯度极高；图9-c为本发明所述双螺旋形微通道废液出口7处的显微图片，可见几乎均为较小直径的微球，几乎不见较大直径的微球，提示较大微粒均被富集到细胞出口，具有良好的回收率。进一步的分析结果显示，当流速在600～750μl/min时，两种螺旋形微通道均具有最佳的分选富集效果；当流体的流速小于600μl/min时，部分尺寸较大颗粒无法聚焦在靠近内侧壁的细胞出口；当流体的流速大于750μl/min时，较大微粒的细胞纯度下降；相关示例结果如图8所示。 The bonded spiral microchannel chip inlet and outlet are tightly connected with the inlet and outlet micro tubes by means of a magnetic clamp device and a precision guide pin connection, and the injection pump and the flow rate control system are used to achieve stable injection and real-time monitoring of flow rate changes. . Before testing the separation effect of the spiral microchannel chip, the sealing fluid was first tested under the high pressure of one atmosphere under the high pressure of the sheath fluid. The result showed no leakage, indicating that the chip bonding and the chip inlet and outlet connections were firm. The integrity of the chip and the elimination of the bubbles were observed under a microscope until the bubbles were completely removed. The optimal parameters for the enrichment of circulating tumor cells by spiral microchannel sorting were optimized using standard polystyrene colored microbeads. Three to 6 μm microspheres, 7 to 10 μm microspheres, and 15 to 18 μm microspheres were mixed to simulate red blood cells, white blood cells, and circulating tumor cells, respectively, and phosphate buffers were used for microspheres of 3 to 6 μm, 7 to 10 μm, and 15 to 18 μm. Dilute to 1.0*10 7 particles/ml, 1.0*10 6 particles/ml, 1.0*10 2 particles/ml, respectively, and mix according to an equal volume ratio. The syringe pump loaded with the mixed microspheres and the syringe pump loaded with the phosphate buffer sheath fluid are respectively connected to the outer side of the spiral chip and the inlet of the inner side wall after the bubble elimination through the precision guide needle, and the flow rate is adjusted by the flow rate monitoring system and the pressure pump control. The double helix microchannels (shown in Figure 1) and the conventional single spiral microchannels (shown in Figure 11) of the present invention were separately recorded and collected at different flow rates (i.e., 150 μl/min, 300 μl/ Min, 450 μl/min, 600 μl/min, 750 μl/min, 900 μl/min) microspheres were sorted and enriched at each outlet. An example of the result of microsphere separation at the cell exit is shown in Fig. 8. Since the ordinary camera cannot clearly capture the motion state of a single rare cell or particle, a smaller diameter circle is expressed for the clear and intuitive expression of the movement trajectory of rare cells or particles. The dots represent rare cells or particles of 3 to 6 μm and 7 to 10 μm, and the dots of larger diameter represent the trajectories of rare cells or particles of 15 to 18 μm. An example of the microscopic count observation of the separation of the microspheres at the cell outlet 8 and the waste liquid outlet 7 is shown in Fig. 9. Figure 9-a is a micrograph of the exit of a conventional single-spiral microchannel cell, with more microspheres, suggesting that the larger particles are poorly sorted and enriched, and the purity is low; Figure 9-b shows The micrographs at the outlet of the double-spiral microchannel cells of the invention are almost all of the larger diameter microspheres, suggesting that the sorting and enrichment effect of the larger particles is very good and the purity is extremely high; FIG. 9-c is the present invention The microscopic picture of the double spiral microchannel waste liquid outlet 7 shows that almost all of the smaller diameter microspheres have almost no larger diameter microspheres, suggesting that larger particles are enriched to the cell outlet, Good recovery rate. Further analysis showed that when the flow rate was between 600 and 750 μl/min, both spiral microchannels had the best sorting and enrichment effect; when the flow rate of the fluid was less than 600 μl/min, some of the larger particles could not be focused. At the cell outlet near the inner wall; when the flow rate of the fluid is greater than 750 μl/min, the cell purity of the larger particles decreases; the relevant example results are shown in FIG.
如表1所示，进一步的对比研究结果表明，无论在何种流速条件下，双螺旋形微通道分选富集的大尺寸颗粒(即：15～18μm)的纯度及重复性均远远优于传统的单螺旋形微通道，二者具有统计学显著性差异。其主要原因在于，传统的单螺旋形微通道中受其自身单个螺旋形微通道的限制，往往使得尺寸较小的颗粒发生横向位移，从而使分选富集到的大尺寸颗粒(即：15～18μm)纯度及实验结果的重复性均受到影响。但是，本发明所述双螺旋形微通道因能够二级分选富集，从而大大提高了包括纯度和重复性在内的检测效能(表1)，二者具有统计学 显著差异。进一步的实际应用结果表明，在相似的实验条件下，当处理8毫升血液样本时，传统的单螺旋形微通道耗时40分钟，而本发明所述双螺旋形微通道仅耗时10分钟，且本发明所述双螺旋形微通道所获得的循环肿瘤细胞的纯度和实验结果的重复性更高(与表1所述结果相近)，二者具有统计学显著差异。上述研究结果提示，相对于传统的单螺旋形微通道，本发明所述双螺旋形微通道更能满足和符合循环肿瘤细胞的临床实际检测需求及后续检测分析的苛刻条件(如循环肿瘤细胞单细胞水平基因检测)。As shown in Table 1, further comparative studies show that the purity and repeatability of large-sized particles (ie, 15-18 μm) enriched by double-helical microchannels are superior in both flow conditions. In the traditional single-spiral microchannels, there is a statistically significant difference between the two. The main reason is that the traditional single spiral microchannel is limited by its own single spiral microchannel, which tends to cause lateral displacement of smaller particles, so that the large size particles are sorted and enriched (ie: 15 The purity of ~18 μm) and the repeatability of the experimental results were all affected. However, the double helix microchannel of the present invention is highly enriched by secondary sorting, thereby greatly improving the detection efficiency including purity and reproducibility (Table 1), which are statistically significant. Further practical application results show that under similar experimental conditions, the conventional single spiral microchannel takes 40 minutes when processing 8 ml of blood sample, while the double helix microchannel of the present invention takes only 10 minutes. Moreover, the purity of the circulating tumor cells obtained by the double helix microchannel of the present invention and the repeatability of the experimental results are higher (similar to those described in Table 1), and the two have statistically significant differences. The above research results suggest that the double helix microchannel of the present invention can meet and meet the clinical actual detection requirements of circulating tumor cells and the harsh conditions for subsequent detection and analysis (such as circulating tumor cell orders) compared with the conventional single spiral microchannels. Cell level gene detection).
表一 不同流速下记录收集到的颗粒纯度Table 1 Recorded particle purity collected at different flow rates
流速Flow rate 双螺旋Double helix 单螺旋Single helix
150μl/min150μl/min 54.56±1.1254.56±1.12 34.58±5.2834.58±5.28
300μl/min300μl/min 76.24±2.3576.24±2.35 42.56±5.8142.56±5.81
450μl/min450μl/min 86.10±2.8486.10±2.84 62.57±6.1262.57±6.12
600μl/min600μl/min 94.12±2.0894.12±2.08 67.60±7.2767.60±7.27
750μl/min750μl/min 99.08±0.4399.08±0.43 70.36±8.7670.36±8.76
900μl/min900μl/min 97.02±2.4797.02±2.47 54.56±5.2154.56±5.21
采用人工培养的HCC827、MCF-7、SW480肿瘤细胞株分别倍比稀释至10 2个细胞/ml，按照1:5的体积比混入利用红细胞裂解液处理后的正常血细胞，采用本发明所述双螺旋形微通道进行分选富集，检测结果利用免疫荧光技术染色涂片的计数结果见表二。结果显示本发明所述双螺旋形微通道对多种肿瘤细胞的回收率均至少达98％以上，且纯度与上述双螺旋形微通道相似，均约为99％。 Artificially cultured HCC827, MCF-7, and SW480 tumor cell lines were diluted to 10 2 cells/ml, respectively, and mixed into normal blood cells treated with erythrocyte lysate according to a volume ratio of 1:5, using the double of the present invention. Spiral microchannels were sorted and enriched. The results of the staining smear using immunofluorescence technique are shown in Table 2. The results show that the double helix microchannels of the present invention have a recovery rate of at least 98% for a plurality of tumor cells, and the purity is similar to that of the above double helix microchannels, and both are about 99%.
表二 不同种类肿瘤细胞采用双螺旋形微通道进行富集后的回收率Table 2 Recovery of different types of tumor cells after enrichment using double helix microchannels
Figure PCTCN2018122352-appb-000001
Figure PCTCN2018122352-appb-000001
Figure PCTCN2018122352-appb-000002
Figure PCTCN2018122352-appb-000002
与传统的单螺旋形微通道相比，本发明所述双螺旋形微通道检测的时间缩短至传统的单螺旋形微通道的1/4，且分选富集到的稀有细胞或颗粒的纯度、实验结果的重复性和回收率等方法学参数均远远优于传统的单螺旋形微通道，说明相对于传统单螺旋形微通道，本发明所述双螺旋形微通道能够更好地满足临床应用的实际需求。Compared with the conventional single-helical microchannel, the double helical microchannel detection time of the invention is shortened to 1/4 of the conventional single spiral microchannel, and the purity of the rare cells or particles enriched by sorting is shortened. The methodological parameters such as the repeatability of the experimental results and the recovery rate are far superior to the conventional single spiral microchannels, indicating that the double helix microchannels of the present invention can better satisfy the conventional single spiral microchannels. The actual needs of clinical applications.
实施例二 Embodiment 2
本实施例与实施例一不同的是，本实施例中采用外加压力泵实现与实施例一中压力平衡微通道近似的效果，即使第二个单螺旋形微通道的细胞入口的液体流动方向和第一个单螺旋形微通道的细胞入口1的液体流动方向一致，且0.7*v2≤v1≤1.3*v2；使第二个单螺旋形微通道的细胞出口8的液体流动方向和第一个单螺旋形微通道的细胞出口4的液体流动方向一致，且0.7*v4≤v3≤1.3*v4，且v1＝v4；使第二个单螺旋形微通道的废液出口7的液体流动方向和第一个单螺旋形微通道的废液出口8的液体流动方向一致，且0.7*v6≤v5≤1.3*v6。所述压力泵设置在第一个单螺旋形微通道的废液出口6处。This embodiment differs from the first embodiment in that an external pressure pump is used in this embodiment to achieve the effect similar to the pressure balance microchannel in the first embodiment, even if the liquid flow direction of the cell inlet of the second single spiral microchannel is The liquid flow direction of the cell inlet 1 of the first single-spiral microchannel is uniform, and 0.7*v2 ≤ v1 ≤ 1.3*v2; the liquid flow direction of the cell outlet 8 of the second single-spiral microchannel is made and the first The liquid flow direction of the cell outlet 4 of the single spiral microchannel is uniform, and 0.7*v4≤v3≤1.3*v4, and v1=v4; the liquid flow direction of the waste liquid outlet 7 of the second single spiral microchannel is The liquid flow direction of the waste liquid outlet 8 of the first single spiral microchannel is uniform, and 0.7*v6 ≤ v5 ≤ 1.3*v6. The pressure pump is disposed at the waste liquid outlet 6 of the first single spiral microchannel.
实施例三 Embodiment 3
本实施例与上述实施例不同的是，本实施例中，整体的螺旋形微通道由4个单螺旋形微通道组成。事实上，本发明所述双通道螺旋形流道可进一步演变成三螺旋形微通道、四螺旋形微通道，或更多级螺旋形微通道。所述多级螺旋形微通道中所包括的单螺旋形微通道的数量至少是2个以上，并且，所述多个单螺旋形微通道的圆心位于同一点，第(M-1)个单螺旋形微通道的细胞出口是第M个单螺旋形微通道的细胞入口，且第(M-1)个单螺旋形微通道的废液出口直接连接了压力平衡通道。所述压力平衡通道的作用是使第(M-1)个单螺旋形微通道的细胞出口或第M个单螺旋形微通道的细胞入口的流速及方向与第 (M-1)个螺旋形微通道的细胞入口及第M个单螺旋形微通道的细胞出口的流速及方向保持相同或极相近，同时，使第M个单螺旋形微通道的废液出口流速及方向与第(M-1)个螺旋形微通道的废液出口的流速及方向保持相同或极相近，从而使所述多级螺旋形微通道中的每个螺旋形微通道给予靶细胞的流体力学特征保持相同或相似。This embodiment differs from the above embodiment in that, in this embodiment, the entire spiral microchannel is composed of four single spiral microchannels. In fact, the two-channel spiral flow channel of the present invention can be further evolved into a triple spiral microchannel, a quadrilateral microchannel, or a plurality of spiral microchannels. The number of single spiral microchannels included in the multi-stage spiral microchannel is at least two, and the center of the plurality of single spiral microchannels is located at the same point, the (M-1)th single The cell outlet of the spiral microchannel is the cell inlet of the Mth single spiral microchannel, and the waste liquid outlet of the (M-1) single spiral microchannel is directly connected to the pressure balance channel. The pressure equalization channel functions to make the flow rate and direction of the cell inlet of the (M-1) single spiral microchannel or the cell inlet of the Mth single spiral microchannel and the (M-1) spiral The flow rate and direction of the cell inlet of the microchannel and the cell outlet of the Mth single spiral microchannel remain the same or very close, and at the same time, the flow rate and direction of the waste liquid outlet of the Mth single spiral microchannel are compared with the first (M- 1) The flow rate and direction of the waste liquid outlet of the spiral microchannel remain the same or very close, so that the hydrodynamic characteristics of each spiral microchannel in the multi-stage spiral microchannel to the target cell remain the same or similar .
本实施例以四螺旋形微通道为例，如图3所示，第一个单螺旋形微通道的细胞出口4是第二个单螺旋形微通道的细胞入口，第二个单螺旋形微通道的细胞出口是第三个单螺旋形微通道的细胞入口，第三个单螺旋形微通道的细胞出口是第四个单螺旋形微通道的细胞入口，并且，第一个、第二个和第三个单螺旋形微通道的废液出口均分别具有对应的压力平衡通道3。In this embodiment, a four-helical microchannel is taken as an example. As shown in FIG. 3, the cell outlet 4 of the first single spiral microchannel is the cell inlet of the second single spiral microchannel, and the second single spiral micro. The cell outlet of the channel is the cell inlet of the third single-spiral microchannel, and the cell outlet of the third single-spiral microchannel is the cell inlet of the fourth single-spiral microchannel, and the first and second And the waste liquid outlet of the third single spiral microchannel has a corresponding pressure equalization channel 3, respectively.
所述鞘液从第一个单螺旋形微通道的鞘液入口2、第二个单螺旋形微通道的鞘液入口5、第三个单螺旋形微通道的鞘液入口15、以及第四个单螺旋形微通道的鞘液入口17导入；废液从第一个单螺旋形微通道的废液出口6、第二个单螺旋形微通道的废液出口7、第三个单螺旋形微通道的废液出口16以及第四个单螺旋形微通道的废液出口19导出；最终在第四个单螺旋形微通道的细胞出口18处收集分选富集后的稀有细胞或颗粒。The sheath fluid is from the sheath fluid inlet of the first single spiral microchannel, the sheath fluid inlet 5 of the second single spiral microchannel, the sheath fluid inlet 15 of the third single spiral microchannel, and the fourth The sheath liquid inlet 17 of the single spiral microchannel is introduced; the waste liquid is discharged from the waste liquid outlet 6 of the first single spiral microchannel, the waste liquid outlet of the second single spiral microchannel, and the third single spiral The waste liquid outlet 16 of the microchannel and the waste liquid outlet 19 of the fourth single spiral microchannel are derived; finally, the sorted and enriched rare cells or particles are collected at the cell outlet 18 of the fourth single spiral microchannel.
实施例四 Embodiment 4
本实施例与实施例三不同的是，本实施例中采用外加压力泵实现与实施例中压力平衡微通道近似的效果。The difference between this embodiment and the third embodiment is that an external pressure pump is used in the embodiment to achieve the effect similar to the pressure balance microchannel in the embodiment.
实施例五 Embodiment 5
本实施例中，采用串联的方式连接两个螺旋形微通道，第一个螺旋形微通道的第二个单螺旋形微通道的细胞出口8与第二个螺旋形微通道的第一个单螺旋形微通道的细胞入口14相连接，形成两个螺旋形微通道的串联安装处，并在每个螺旋形微通道串联安装处的位置上设置压力平衡通道。In this embodiment, two spiral microchannels are connected in series, the cell outlet 8 of the second single spiral microchannel of the first spiral microchannel and the first single of the second spiral microchannel The cell inlets 14 of the helical microchannels are joined to form a series arrangement of two helical microchannels, and a pressure equalization channel is provided at a location where each spiral microchannel is mounted in series.
所述鞘液从第一个螺旋形微通道的第一个单螺旋形微通道的鞘液入口2、第一个螺旋形微通道的第二个单螺旋形微通道的鞘液入口5、第二个螺旋形微通道 的第一个单螺旋形微通道的鞘液入口9、以及第二个螺旋形微通道的第二个单螺旋形微通道的鞘液入口11导入；废液从第一个螺旋形微通道的第一个单螺旋形微通道的废液出口6、第一个螺旋形微通道的第二个单螺旋形微通道的废液出口7、第二个螺旋形微通道的第一个单螺旋形微通道的废液出口10、以及第二个螺旋形微通道的第二个单螺旋形微通道的废液出口13导出。从第二个螺旋形微通道的第二个单螺旋形微通道的细胞出口12收集分选富集后的稀有细胞或颗粒。The sheath fluid is from the sheath fluid inlet of the first single spiral microchannel of the first spiral microchannel, the sheath fluid inlet 5 of the second single spiral microchannel of the first spiral microchannel, The sheath liquid inlet 9 of the first single spiral microchannel of the two spiral microchannels and the sheath liquid inlet 11 of the second single spiral microchannel of the second spiral microchannel are introduced; the waste liquid is from the first a waste liquid outlet 6 of the first single spiral microchannel of the spiral microchannel, a waste liquid outlet 7 of the second single spiral microchannel of the first spiral microchannel, and a second spiral microchannel The waste liquid outlet 10 of the first single spiral microchannel and the waste liquid outlet 13 of the second single spiral microchannel of the second spiral microchannel are led out. The sorted and enriched rare cells or particles are collected from the cell outlet 12 of the second single helical microchannel of the second helical microchannel.
采用本实施例的结构形式也可以对更多个N级螺旋形微通道进行串联设置，与现有的细胞入口和细胞出口分别位于单个螺旋形流道平面几何结构的内侧和外侧相比较(例如：最后一个附图)，本发明可在不使用任何外接微管的条件下直接级联或串联式使用多个N级螺旋形流道，既简化了制作工艺，也使N级螺旋形流道的复杂性降低，提高工作效率与检测结果的准确性。With the configuration of this embodiment, it is also possible to arrange a plurality of N-stage spiral microchannels in series, compared with the existing cell inlet and cell outlet respectively located on the inner side and the outer side of the single spiral flow channel plane geometry (for example : Finally, the present invention can directly or cascadely use a plurality of N-stage spiral flow paths without using any external microtubes, which simplifies the manufacturing process and also makes the N-stage spiral flow path. Reduced complexity, improved work efficiency and accuracy of test results.
实施例六 Embodiment 6
本实施例与实施例五不同的是，本实施例中采用外加压力泵实现与实施例五中压力平衡微通道近似的效果。The difference between this embodiment and the fifth embodiment is that an external pressure pump is used in the embodiment to achieve the effect similar to the pressure balance microchannel in the fifth embodiment.
实施例七Example 7
本实施例中，采用并联的方式连接两个双螺旋形微通道。样本溶液从两个双螺旋形微通道的第一个单螺旋形微通道的细胞入口1进入，稀有细胞或颗粒从两个双螺旋形微通道的第二个单螺旋形微通道的细胞出口12流出。参与并联的双螺旋形微通道的圆心投影重合。可在同一张微流控芯片上同时处理2个、3个或更多个待检测样本，从而提高检测效率的同时有效地降低检测成本和样本的处理时间。In this embodiment, two double spiral microchannels are connected in parallel. The sample solution enters from the cell inlet 1 of the first single helical microchannel of the two double helix microchannels, and the rare cell or particle exits from the cell outlet of the second single helical microchannel of the two double helix microchannels 12 Flow out. The center projections of the double spiral microchannels participating in the parallel are coincident. Two, three or more samples to be detected can be simultaneously processed on the same microfluidic chip, thereby improving the detection efficiency while effectively reducing the detection cost and the processing time of the sample.
实施例八Example eight
本实施例与实施例一不同的是，本方案中的所有出入口(包括：细胞入口和出口、鞘液入口和废液出口)均设置在整体螺旋形微通道的几何形状外侧，废液出口通过压力平衡微流道沿其他微流道的延伸方向流通至整体螺旋形微通道的几何形状外侧，通过对压力平衡微流道的宽度、深度和长度的调整实现对细胞 出口内容物流速及流向的调整。This embodiment differs from the first embodiment in that all the inlets and outlets (including the cell inlet and outlet, the sheath liquid inlet and the waste liquid outlet) in the present embodiment are disposed outside the geometry of the entire spiral microchannel, and the waste liquid outlet passes through. The pressure-balanced microchannel flows to the outside of the geometry of the entire spiral microchannel along the extending direction of the other microchannels, and the flow rate and flow direction of the cell outlet contents are adjusted by adjusting the width, depth and length of the pressure-balanced microchannel. Adjustment.
在具体的实施过程中，将部分出入口(如：图1所示的第二级鞘液入口5和第一级废液废液6)设置在螺旋形微通道内侧的结构设计为芯片的成型和制造带来了很大不便，在芯片的中心穿孔并导出或导入流体和/或细胞的操作也较为复杂，导致相关的芯片夹具的制作难度高，难于观察第一级出口和第二级入***汇处的流体和/或细胞的流动状态。因此在本实施例中将所有出入口(包括：细胞入口和出口、鞘液入口和废液出口)均设置在整体螺旋形微通道的外侧。In a specific implementation process, a part of the inlet and outlet (such as the second-stage sheath liquid inlet 5 and the first-stage waste liquid waste liquid 6 shown in FIG. 1) is disposed inside the spiral microchannel, and the structure is designed as a chip. Manufacturing brings great inconvenience, and the operation of perforating and introducing or introducing fluids and/or cells in the center of the chip is also complicated, which makes the related chip fixture difficult to manufacture, and it is difficult to observe the intersection of the first-level exit and the second-level entrance. The flow state of fluids and/or cells. Therefore, in the present embodiment, all the inlets and outlets (including the cell inlet and outlet, the sheath liquid inlet, and the waste liquid outlet) are disposed outside the entire spiral microchannel.
基于惯性迁移力分选富集循环肿瘤细胞，采用COMSOL Multiphisics多物理量仿真软件，在构建了标准化单螺旋芯片的基础上，进一步改进芯片结构并成功构建标准化的双螺旋形微流道芯片。设计构建的标准化双螺旋芯片其关键目的是通过惯性聚焦使得尺寸较大的靶细胞(CTC)沿着通道的内壁分布聚焦，尺寸较小的血细胞从螺旋通道的外侧避随鞘流液分离。双螺旋芯片较单螺旋芯片相比，能够通过级联的方式更加快速、高效地捕获到血液样品中的CTC，满足临床实际要求，达到对肿瘤患者动态实时监测、选择最佳治疗方案的目的，同时还可以进一步的对肿瘤转移等相关机制进行深入探讨研究。Based on inertial migration force sorting and enrichment of circulating tumor cells, COMSOL Multiphisics multi-physical simulation software was used to build a standardized single-helical chip, further improve the chip structure and successfully construct a standardized double-spiral micro-channel chip. The key objective of the designed and constructed double helix chip is to focus the larger target cells (CTC) along the inner wall of the channel by inertial focusing, and the smaller blood cells avoid the sheath fluid separation from the outer side of the spiral channel. Compared with the single-helical chip, the double-helical chip can capture the CTC in the blood sample more quickly and efficiently by cascading, meet the clinical requirements, and achieve the purpose of dynamic real-time monitoring of the tumor patient and selection of the optimal treatment plan. At the same time, further research on related mechanisms such as tumor metastasis can be conducted.
不同细胞其惯性聚焦的位置受螺旋形微流道的三维尺寸的影响。通过COMSOL Multiphisics软件的仿真模拟计算，确定了能够使CTC(≥15um)与血细胞(3～15um)分离效果最佳的螺旋形微流道芯片三维尺寸，从而迫使样品能够从第一个单螺旋形微通道的细胞入口完全顺利进入螺旋形微通道内并开始其在流道内的横向位移。考虑到不同流速的变化以及长度两个因素对不同尺寸细胞(CTC、RBC、WBC)最终所产生的横向位移聚焦位置的不同，最终优化确定第二个单螺旋形微通道的细胞出口的最佳参数。双通道螺旋芯片在单通道螺旋芯片基础上增加了芯片内流道长度，避免了大流速高通量下因流道过短导致的尺寸较小颗粒横向位移过大造成细胞纯度低的问题。The position of the inertial focus of different cells is affected by the three-dimensional size of the spiral microchannel. Through the simulation calculation of COMSOL Multiphisics software, the three-dimensional size of the spiral micro-channel chip which can separate CTC (≥15um) and blood cells (3~15um) is determined, which forces the sample to be from the first single spiral. The cell inlet of the microchannel completely enters the spiral microchannel and begins its lateral displacement within the flow channel. Considering the difference of different flow rates and the length of the two factors, the difference in the lateral displacement focus position of different size cells (CTC, RBC, WBC) finally determines the optimal cell outlet of the second single spiral microchannel. parameter. The dual-channel spiral chip increases the in-chip flow path length based on the single-channel spiral chip, and avoids the problem of low cell purity caused by excessive lateral displacement of small particles due to short flow path at high flow rate and high flux.
采用COMSOL Multiphysics V5.2a软件，仿真分析在不同宽度的压力平衡通道3的条件下，双螺旋微流道的第一级出入口(包括：细胞入口1、鞘液入口2、 废液出口6、细胞出口4)与第二级出入口(包括：细胞入口4、鞘液入口5、废液出口7、细胞出口8)的体积流量特征及相关比值。模拟条件是细胞入口的初始条件是150ul/min，鞘液入口的初始条件是750ul/min。模拟结果表明，只有当压力平衡通道3的宽度在350um时，第一级细胞出口4的流速才与第一级细胞入口1的流速相当，在此条件下，其流速是144.18ul/min(表三)。该结果表明，压力平衡通道3的宽度对第一级细胞出口4的流速具有重要的调节作用，并且，只有在其适当的宽度条件下，才能确保第一级细胞出口4的流速与第一级细胞入口1的流速相当。同时，进一步加大压力平衡通道3的宽度，会导致第一级细胞出口4呈反方向流动，说明压力平衡通道3提供的平衡压力过小，导致流体的反流。其它结果表明，当压力平衡通道3的宽度维持在250～400um时，第二级废液出口7和细胞出口8的体积流量比值维持在比较恒定的范围，具体结果见表四。进一步模拟分析结果表明，压力平衡通道3的深度和长度同样可以影响第一级细胞出口4的流速。Using COMSOL Multiphysics V5.2a software, simulation analysis of the first stage inlet and outlet of the double helix microchannel under conditions of pressure equalization channel 3 of different widths (including: cell inlet 1, sheath inlet 2, waste outlet 6, cell) The volumetric flow characteristics and related ratios of the outlet 4) and the second-stage inlet and outlet (including: the cell inlet 4, the sheath fluid inlet 5, the waste liquid outlet 7, and the cell outlet 8). The simulation conditions were that the initial condition of the cell inlet was 150 ul/min and the initial condition of the sheath fluid inlet was 750 ul/min. The simulation results show that the flow rate of the first-stage cell outlet 4 is equivalent to the flow rate of the first-stage cell inlet 1 only when the width of the pressure-balanced channel 3 is 350 μm. Under this condition, the flow rate is 144.18 ul/min (Table) three). This result indicates that the width of the pressure-equalizing channel 3 has an important regulating effect on the flow velocity of the first-stage cell outlet 4, and that the flow rate of the first-stage cell outlet 4 and the first-stage can be ensured only under the appropriate width conditions. The flow rate of the cell inlet 1 is comparable. At the same time, further increasing the width of the pressure balance channel 3 causes the first stage cell outlet 4 to flow in the opposite direction, indicating that the equilibrium pressure provided by the pressure balance channel 3 is too small, resulting in backflow of the fluid. Other results show that when the width of the pressure balance channel 3 is maintained at 250-400 um, the volume flow ratio of the second-stage waste liquid outlet 7 and the cell outlet 8 is maintained in a relatively constant range, and the specific results are shown in Table 4. Further simulation analysis shows that the depth and length of the pressure equalization channel 3 can also affect the flow rate of the first stage cell outlet 4.
表三 在不同宽度的压力平衡通道3的条件下，各级入口的体积流量特征Table 3 Volumetric flow characteristics of inlets at various stages under conditions of pressure equalization channel 3 of different widths
Figure PCTCN2018122352-appb-000003
Figure PCTCN2018122352-appb-000003
表四 在不同宽度的压力平衡通道3的条件下，各级出口的体积流量特征Table 4 Volume flow characteristics of the outlets at each level under the condition of pressure equalization channel 3 of different widths
Figure PCTCN2018122352-appb-000004
Figure PCTCN2018122352-appb-000004
螺旋形微流道芯片的制作主要通过基本的微细加工技术，采用SU8光刻胶在硅片上进行标准的软光刻技术制作模具，PDMS脱模后对其相应出入口处进行打孔处理，并键合在相应玻片(45mm X45mm)上，为了增加键合的牢固性，放置70℃烤箱中，烘烤30min。The spiral micro-flow channel chip is mainly made by the basic micro-machining technology, and the standard soft lithography technology is used to fabricate the mold on the silicon wafer by using the SU8 photoresist. After the PDMS is demolded, the corresponding entrance and exit are punched, and Bonded on the corresponding slide (45mm X45mm), in order to increase the firmness of the bond, place it in a 70 °C oven and bake for 30 minutes.
键合后的螺旋形微流道芯片出入口采用Magnetic probe(Corsolution)装置以及Fluidic Connect(Micronit)夹具连接方式与进样管进行紧密连接，并通过LongerPump注射泵以及MCFS压力流速控制***从而达到进样稳定并可实时监测流速变化的目的。对螺旋形微流道芯片其分离效果测试前，首先采用鞘流液在1bar高压力下对其密封性进行检测，结果无漏液情况，说明芯片键合处及芯片出入口连接较牢固，通过显微镜下观察芯片完整性及气泡排除情况，待气泡完全排除后。利用标准聚苯乙烯彩色微珠对螺旋形微流道分选富集CTC的最佳参数进行优化。The bonded spiral microchannel chip inlet and outlet are tightly connected to the sample tube by a magnetic probe (Corsolution) device and a Fluidic Connect (Micronit) clamp connection, and the injection is achieved by a LongerPump syringe pump and a MCFS pressure flow rate control system. Stable and real-time monitoring of flow rate changes. Before testing the separation effect of the spiral micro-channel chip, the sealing fluid was first tested under the high pressure of 1 bar. The result was no leakage, indicating that the chip bonding and the chip inlet and outlet were firmly connected. Observe the integrity of the chip and the bubble elimination, after the bubble is completely removed. The optimal parameters for the enrichment of CTC by spiral microchannel sorting were optimized using standard polystyrene colored microbeads.
采用3～6um微球、7～10um微球、15～18um微球进行混合分别模拟红细胞、白细胞、CTC，其中利用PBS缓冲液对3～6μm、7～10μm、15～18μm的微球分别稀释到1.0×10 9particles/ml、1.0×10 7particles/ml、1.0×10 3particles/ml并按照等体积比进行混合。将装载混合微球的注射泵与装载PBS鞘流液的注射泵通过芯片夹具分别连接在排除气泡后的螺旋形芯片的细胞入口1以及鞘液入口2，通过流速监测***以及压力泵控制调整流速及压力，分别记录并收集单、双螺旋型芯片(实施例一中的单螺旋型芯片与本实施例)在不同流速下(200μl/min、350μl/min、500μl/min、650μl/min、800μl/min、950μl/min)，芯片出口处有色微球分离情况见图23，并通过显微镜计数观察结果见图24。结果显示当流速在650～800μl/min时分离效果最佳。 The cells were mixed with 3-6 μm microspheres, 7-10 μm microspheres and 15-18 μm microspheres to simulate red blood cells, white blood cells and CTC, respectively, and the microspheres of 3-6 μm, 7-10 μm and 15-18 μm were diluted with PBS buffer. To 1.0 × 10 9 particles/ml, 1.0 × 10 7 particles/ml, 1.0 × 10 3 particles/ml, and mixed in an equal volume ratio. The syringe pump loaded with the mixed microspheres and the syringe pump loaded with the PBS sheath fluid are respectively connected to the cell inlet 1 and the sheath liquid inlet 2 of the spiral chip after the bubble elimination by the chip holder, and the flow rate is adjusted by the flow rate monitoring system and the pressure pump control. And pressure, separately recorded and collected single and double spiral type chip (single spiral type chip in the first embodiment and the present embodiment) at different flow rates (200 μl / min, 350 μl / min, 500 μl / min, 650 μl / min, 800 μl /min, 950μl/min), the separation of colored microspheres at the exit of the chip is shown in Fig. 23, and the observation result by microscopic counting is shown in Fig. 24. The results showed that the separation effect was best when the flow rate was 650-800 μl/min.
流速小于650μl/min部分尺寸较大颗粒无法聚焦在靠近细胞出口，当流速大于800μl/min时，CTC分选富集纯度将受到影响如图23所示，圆点多少及大小并不反映颗粒实际的数量多寡与尺寸大小。The flow rate is less than 650μl/min. The larger size of the particles cannot be focused near the cell outlet. When the flow rate is greater than 800μl/min, the purity of CTC sorting enrichment will be affected. As shown in Figure 23, the number and size of the dots do not reflect the actual particle size. The number and size of the.
利用上述研究方法，对双通道螺旋型芯片进行测试，其结果统计如表五， 反映了双通道螺旋型芯片其分选富集纯度大大高于单通道螺旋型芯片。同时由于单螺旋芯片流道长度的限制，当流速大于800μl/min时，往往使得尺寸较小的颗粒在较大惯性迁移力作用下发生横向位移导致分选富集到的CTC细胞纯度受到影响，然而双螺旋芯片增加了流道长度，进行第二级分离从而大大提高检测通量。结果表明，在相同时间内，单通道螺旋型芯片处理能够在20min内处理8ml血液样本，而双通道螺旋型芯片仅需10min，且其高纯度、高通量的富集结果更加满足符合CTC应用于临床及后续检测分析的苛刻条件。Using the above research method, the two-channel spiral type chip was tested. The results are shown in Table 5. It reflects that the purity of sorting and enrichment of the two-channel spiral type chip is much higher than that of the single-channel spiral type chip. At the same time, due to the limitation of the flow length of the single spiral chip, when the flow rate is greater than 800 μl/min, the lateral displacement of the smaller size particles under the action of large inertial migration force tends to affect the purity of the sorted and enriched CTC cells. However, the double-helical chip increases the length of the flow path and performs the second-stage separation to greatly increase the detection flux. The results show that in the same time, single-channel spiral chip processing can process 8ml blood samples in 20min, while the dual-channel spiral chip only takes 10min, and its high-purity, high-throughput enrichment results are more suitable for CTC applications. The harsh conditions for clinical and subsequent testing analysis.
表五 不同流速下记录收集到颗粒的纯度Table 5 Record the purity of collected particles at different flow rates
Figure PCTCN2018122352-appb-000005
Figure PCTCN2018122352-appb-000005
采用人工培养的HCC827、MCF-7、SW480细胞株分别倍比稀释至10 2cells/ml，按照1:5的体积比混入利用红细胞裂解液处理后的正常血细胞，采用本实施例中的芯片进行检测，检测结果利用免疫荧光技术染色涂片计数见表六。结果显示双螺旋微流道芯片对多种肿瘤细胞的回收率达90％以上，图25所示为分选富集到的SW480免疫荧光染色.通过混入肿瘤细胞与捕获到的细胞结果可见其有很好的线性相关，同时该双螺旋芯片捕获对多种肿瘤细胞捕获效率达90％，见图26。 The artificially cultured HCC827, MCF-7, and SW480 cell lines were diluted to 10 2 cells/ml, respectively, and the normal blood cells treated with the red blood cell lysate were mixed in a volume ratio of 1:5, and the chip in the present example was used. Detection, detection results using immunofluorescence technique staining smear counts are shown in Table 6. The results showed that the recovery rate of multiple tumor cells by the double helix microchannel chip was over 90%. Figure 25 shows the SW480 immunofluorescence staining by sorting and enrichment. The results were observed by mixing the tumor cells with the captured cells. Very good linear correlation, while the double helix chip captures 90% of the capture efficiency of multiple tumor cells, see Figure 26.
表六 双螺旋芯片多种肿瘤细胞捕获回收率Table 6 Double spiral chip multiple tumor cell capture recovery
Figure PCTCN2018122352-appb-000006
Figure PCTCN2018122352-appb-000006
Figure PCTCN2018122352-appb-000007
Figure PCTCN2018122352-appb-000007
此外，分别对20例结直肠癌患者样本进行检测，其中利用标准化单螺旋和双螺旋芯片都检测出其中18例患者CTC细胞，但较单螺旋芯片相比，双螺旋型芯片检测时间缩短一半，贴近满足临床高通量的需求。同时，我们设计的双螺旋通道，通过标准化进出样口设计，使其能够配合大部分商品化的芯片夹具精确完成整个血液样品进样出样的全部流程，使其离临床大量标本、低成本、快速检测CTC更进一步，可在10min处理8ml血液样本.其对标准化微球的分选富集纯度达98％以上，从理论上来讲，其分选富集CTC纯度同样达98％以上，但因为肿瘤患者之间差异性较大(如化疗后WBC显著增高)，故其纯度往往是一个变量.但相比单螺旋型芯片，双螺旋型芯片分选富集CTC细胞纯度均远高于单螺旋芯片，其细胞纯度对CTC细胞后续遗传学等生物操控分析至关重要。In addition, 20 samples of colorectal cancer patients were detected. Among them, 18 patients with CTC cells were detected by standardized single-helix and double-helical chips, but the detection time of double-helix chips was shortened by half compared with single-screw chips. Close to meet the needs of clinical high throughput. At the same time, we designed the double spiral channel, through the standardized inlet and outlet sample design, so that it can cooperate with most commercial chip fixtures to accurately complete the entire process of blood sample injection and sampling, making it away from clinical specimens, low cost, Rapid detection of CTC can further process 8ml blood samples in 10min. Its purity of sorting and enrichment of standardized microspheres is over 98%. In theory, the purity of sorting and enrichment CTC is also over 98%, but because The difference between tumor patients is large (such as WBC is significantly increased after chemotherapy), so its purity is often a variable. However, compared with single-helical chip, the purity of CTC cells enriched by double helix chip is much higher than that of single helix. The chip, its cell purity is critical for biological manipulation analysis such as CTC cell follow-up genetics.
实施例九Example nine
本实施例与实施例五在本质上较为相似，但本方案中的所有出入口(包括：细胞入口和出口、鞘液入口和废液出口)均设置在整体螺旋形微通道的几何形状外侧，更易于观测和分析结果，同时，也更易于标准化夹具，从而有利于实现批量化处理和操作。This embodiment is similar in nature to the fifth embodiment, but all the inlets and outlets in the solution (including: cell inlet and outlet, sheath liquid inlet and waste liquid outlet) are disposed outside the geometry of the overall spiral microchannel, and Easily observe and analyze results, and at the same time, it is easier to standardize fixtures, which facilitates batch processing and operation.
实施例十Example ten
本实施例与实施例三在本质上较为相似，但该设计将第一个单螺旋形微通 道的细胞入口、第一个单螺旋形微通道的鞘液入口、第二个单螺旋形微通道的鞘液入口、第一个单螺旋形微通道的废液出口设置在较为接近的位置，并将两个单螺旋形微通道设置以同心的形式交替设置，同时，所有出入口(包括：细胞入口和出口、鞘液入口和废液出口)均设置在整体螺旋形微通道的几何形状外侧，整体结构更为精巧，便于使用。This embodiment is similar in nature to the third embodiment, but the design has the cell inlet of the first single spiral microchannel, the sheath fluid inlet of the first single spiral microchannel, and the second single spiral microchannel. The sheath liquid inlet, the waste liquid outlet of the first single spiral microchannel are disposed at a relatively close position, and the two single spiral microchannels are arranged alternately in a concentric manner, and at the same time, all the inlets and exits (including: cell inlet) And the outlet, the sheath liquid inlet and the waste liquid outlet are all arranged outside the geometry of the overall spiral microchannel, and the overall structure is more compact and easy to use.
实施例十一 Embodiment 11
本实施例由两个实施例八中的双螺旋形微通道采用并联的方式进行连接的结构示意图，采用上述并联的形式可使同一个样本在单位时间内同时在两个双螺旋形微通道进行处理，或者说，可以在一次筛选的时间内实现更多流体的同时处理，可以有效增加筛选效率。相对于实施例八，临床样本的检测时间至少可缩短1倍。本实施例中仅展示两个双螺旋形微通道的并联形式，在实际使用中可以根据需要进行三条、四条及更多条双螺旋形微通道的并联。In this embodiment, the double spiral microchannels in the two embodiments are connected in a parallel manner. The parallel configuration can be used to simultaneously perform the same sample on two double spiral microchannels in a unit time. Processing, or speaking, can simultaneously process more fluids in one screening time, which can effectively increase the screening efficiency. Compared to the eighth embodiment, the detection time of the clinical sample can be shortened by at least one time. In this embodiment, only the parallel form of two double spiral microchannels is shown. In actual use, three, four or more double spiral microchannels can be connected in parallel according to requirements.
实施例十二Example twelve
本实施例与实施例七在本质上较为相似，但该设计将第一个双螺旋形微通道的第一个单螺旋形微通道的细胞入口、第一个单螺旋形微通道的鞘液入口、第二个单螺旋形微通道的鞘液入口、第一个单螺旋形微通道的废液出口设置在较为接近的位置；将第二个双螺旋形微通道中的第一个单螺旋形微通道的细胞入口、第一个单螺旋形微通道的鞘液入口、第二个单螺旋形微通道的鞘液入口、第一个单螺旋形微通道的废液出口设置在较为接近的位置，并将两个单螺旋形微通道设置以同心的形式交替设置，整体结构更为精巧，便于使用。同时，本方案中的所有出入口(包括：细胞入口和出口、鞘液入口和废液出口)均设置在整体螺旋形微通道的几何形状外侧，更易于观测和分析结果，同时，也更易于标准化夹具，从而有利于实现批量化处理和操作。This embodiment is similar in nature to the seventh embodiment, but the design introduces the cell inlet of the first single spiral microchannel of the first double helix microchannel and the sheath fluid inlet of the first single spiral microchannel. The sheath liquid inlet of the second single spiral microchannel, the waste liquid outlet of the first single spiral microchannel is disposed at a relatively close position; and the first single spiral of the second double spiral microchannel The cell inlet of the microchannel, the sheath inlet of the first single spiral microchannel, the sheath inlet of the second single spiral microchannel, and the waste outlet of the first single spiral microchannel are disposed in close proximity And the two single spiral microchannels are arranged alternately in a concentric manner, and the overall structure is more compact and easy to use. At the same time, all the inlets and exits in this scheme (including: cell inlet and outlet, sheath fluid inlet and waste outlet) are placed outside the geometry of the overall spiral microchannel, making it easier to observe and analyze the results, and at the same time easier to standardize. Fixtures, which facilitate batch processing and operation.
实施例十三Example thirteen
本实施例由两个实施例十中的螺旋形微通道(即串联的双螺旋微通道)采用并联的方式进行连接的结构示意图，采用上述并联的形式可使同一个样本在 单位时间内同时在两个实施例十所示螺旋形微通道进行处理，或者说，可以在一次筛选的时间内实现更多流体的同时处理，可以有效增加筛选效率。相对于实施例十，临床样本的检测时间至少可缩短1倍。本实施例既具有通过串联的形式保证采用多级流道多次筛选，提高细胞纯度的优势，也具有通过并联的形式提高筛选效率的优势。本实施例中仅展示两个实施例十所示螺旋形微通道的并联形式，在实际使用中可以根据需要进行三条、四条及更多条实施例十所示螺旋形微通道的并联。In this embodiment, the spiral microchannels (ie, the series double helix microchannels) in the tenth embodiment are connected in parallel, and the parallel sample is used to make the same sample simultaneously in unit time. The spiral microchannels shown in the two embodiments are processed, or more simultaneous treatment of more fluids can be realized in one screening time, which can effectively increase the screening efficiency. Compared to the tenth embodiment, the detection time of the clinical sample can be shortened by at least one time. This embodiment not only has the advantages of ensuring multi-stage flow channel multiple screening through series connection, improving cell purity, but also has the advantage of improving screening efficiency by parallel form. In this embodiment, only the parallel forms of the spiral microchannels shown in the two embodiments are shown. In actual use, three, four, and more parallel spiral microchannels shown in Embodiment 10 can be performed as needed.
实施例十四 Embodiment 14
本实施例中展示基础设计-单螺旋形微通道的另一种实现形式，该种形式中将细胞出口及废液出口均设置在整体螺旋形微通道的外侧。在具体的实施过程中，将细胞出口及废液出口均设置在螺旋形微通道内侧的结构设计(如图11所示)为芯片的成型和制造带来了很大不便，在芯片的中心穿孔并导出或导入流体和/或细胞的操作也较为复杂，导致相关的芯片夹具的制作难度高，难于观察鞘液和细胞入***汇处的流体和/或细胞的流动状态。因此在本实施例中将细胞出口及废液出口均设置在整体螺旋形微通道的外侧，更易于观测和分析，以及标准化夹具和简便化操作。Another embodiment of the basic design-single helical microchannel is shown in this embodiment, in which both the cell outlet and the waste outlet are disposed outside of the overall helical microchannel. In the specific implementation process, the structural design of the cell outlet and the waste liquid outlet disposed inside the spiral microchannel (as shown in FIG. 11) brings great inconvenience to the molding and manufacturing of the chip, and is perforated at the center of the chip. The operation of exporting or introducing fluids and/or cells is also complicated, resulting in difficulty in making related chip holders, and it is difficult to observe the fluid and/or cell flow state at the junction of the sheath fluid and the cell inlet. Therefore, in the present embodiment, the cell outlet and the waste liquid outlet are both disposed outside the entire spiral microchannel, which is easier to observe and analyze, as well as standardized fixtures and simplified operations.
实施例十五Example fifteen
本实施例中展示两个实施例十四中的单螺旋形微通道采用串联的方式进行连接的结构示意图，采用串联的形式可以实现在一次流动过程中多次筛选，可进一步提高筛选细胞的纯度。本实施例中仅展示两个单螺旋形微通道的串联形式，在实际使用中可以根据需要进行三条、四条及更多条单螺旋形微通道的串联。In this embodiment, the schematic diagrams of the single spiral microchannels in the fourteenth embodiment are connected in a series manner, and the serial connection can realize multiple screening in one flow process, which can further improve the purity of the screening cells. . In this embodiment, only the series form of two single spiral microchannels is shown. In actual use, three, four, and more single spiral microchannels can be connected in series as needed.
实施例十六Example sixteen
本实施例中展示实施例十五的另一种表现形式，两个单螺旋形微通道采用同心并列环绕设置，盘曲折叠螺旋绕制成环，采用串联的形式可以实现在一次流动过程中多次筛选，可进一步提高筛选细胞的纯度。本实施例中仅展示两个 单螺旋形微通道的串联形式，在实际使用中可以根据需要进行三条、四条及更多条单螺旋形微通道的串联。Another embodiment of the fifteenth embodiment is shown in the embodiment. The two single spiral microchannels are arranged in a concentric juxtaposed arrangement, and the spiral folding spiral is wound into a ring, which can be realized in a single flow process in a series connection manner. Screening can further improve the purity of the screened cells. In this embodiment, only two series of single spiral microchannels are shown. In actual use, three, four, and more single spiral microchannels can be connected in series as needed.
实施例十七Example seventeen
本实施例中展示两个实施例十四中的单螺旋形微通道采用并联的方式进行连接的结构示意图，采用上述并联的形式可使同一个样本在单位时间内同时在两个实施例十四所示单螺旋形微通道进行处理，或者说，可以在一次筛选的时间内实现更多流体的同时处理，可以有效增加筛选效率。相对于实施例十四，临床样本的检测时间至少可缩短1倍。本实施例中仅展示两个单螺旋形微通道的并联形式，在实际使用中可以根据需要进行三条、四条及更多条单螺旋形微通道的并联。In the embodiment, the schematic diagrams of the single spiral microchannels in the fourteenth embodiment are connected in parallel, and the same sample can be used in the same time in the unit time in two embodiments. The single spiral microchannels are shown to be processed, or more fluids can be processed simultaneously in one screening time, which can effectively increase the screening efficiency. Compared to Example 14, the detection time of clinical samples can be reduced by at least 1 time. In this embodiment, only two parallel forms of single spiral microchannels are shown. In actual use, three, four, and more single spiral microchannels can be connected in parallel according to requirements.
实施例十八Example 18
本实施例中展示实施例十七的另一种表现形式，两个单螺旋形微通道采用同心并列环绕设置，盘曲折叠螺旋绕制成环，采用并联的形式可以在一次筛选时间内实现更多流体的同时处理，可以有效增加筛选效率，本实施例中仅展示两个单螺旋形微通道的并联形式，在实际使用中可以根据需要进行三条、四条及更多条单螺旋形微通道的并联。Another embodiment of the embodiment 17 is shown in the embodiment. The two single spiral microchannels are arranged in a concentric juxtaposition, and the spiral folding spiral is wound into a ring, and the parallel form can realize more in one screening time. The simultaneous treatment of the fluid can effectively increase the screening efficiency. In this embodiment, only the parallel form of two single spiral microchannels is shown. In actual use, three, four or more single spiral microchannels can be connected in parallel according to requirements. .
在本发明的基础设计为单螺旋形微通道，其延伸设计为单螺旋形微通道的串联、并联、混联，以及多螺旋形微通道(本发明说明书中以双螺旋形微通道为例)的串联、并联、混联等多种实现形式，上述表现形式均为本方案的延伸设计，其基本原理均在本说明书中得到阐述，因此上述各种表现形式也均应落入本发明的保护范围之中。The basic design of the present invention is a single spiral microchannel, the extension of which is designed as a series, parallel, hybrid, and multi-spiral microchannel of a single spiral microchannel (for example, a double spiral microchannel in the present specification) The various implementation forms such as series, parallel, hybrid, etc., all of the above expressions are extended designs of the scheme, and the basic principles are all explained in the present specification, so the above various expressions should also fall under the protection of the present invention. In the range.
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to be limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art Modifications or equivalents are intended to be included within the scope of the appended claims.

Claims (84)

  1. 一种螺旋形微通道，用于分选富集稀有细胞或颗粒，其特征在于：A spiral microchannel for sorting and enriching rare cells or particles, characterized by:
    包括N个单螺旋形微通道，单个的单螺旋形微通道包括入口、与入口密封连接的微通道以及与通道远离入口的一端密封连接的出口，所述的入口由细胞入口及鞘液入口汇聚而成，所述的出口由废液出口及细胞出口汇聚而成；Including N single spiral microchannels, the single single spiral microchannel includes an inlet, a microchannel sealingly connected to the inlet, and an outlet sealingly connected to an end of the channel remote from the inlet, the inlet being concentrated by the cell inlet and the sheath inlet The outlet is made up of a waste liquid outlet and a cell outlet;
    第(M-1)个单螺旋形微通道的细胞出口与第M个单螺旋形微通道的细胞入口密封连接成第(M-1)个分级循环点，由第(M-1)个单螺旋形微通道的废液出口、第M个单螺旋形微通道的鞘液入口以及第(M-1)个分级循环点构成第(M-1)个分级循环处；The cell outlet of the (M-1) single helical microchannel is sealingly connected with the cell inlet of the Mth single helical microchannel into the (M-1)th hierarchical cycle point, by the (M-1)th single The waste liquid outlet of the spiral microchannel, the sheath liquid inlet of the Mth single spiral microchannel, and the (M-1)th graded circulation point constitute the (M-1)th classification cycle;
    在第(M-1)个分级循环处内的第(M-1)个单螺旋形微通道的废液出口设有压力平衡模块，第M个单螺旋形微通道的细胞入口的液体流速为v1，第(M-1)个单螺旋形微通道的细胞入口的液体流速为v2；第M个单螺旋形微通道的细胞出口的液体流速为v3，第(M-1)个单螺旋形微通道的细胞出口的液体流速为v4；第M个单螺旋形微通道的废液出口的液体流速为v5，第(M-1)个单螺旋形微通道的废液出口的液体流速为v6；A liquid pressure flow rate is provided at a waste liquid outlet of the (M-1) single spiral microchannel in the (M-1)th classification cycle, and a liquid flow rate of the cell inlet of the Mth single spiral microchannel is V1, the liquid flow rate of the cell inlet of the (M-1) single spiral microchannel is v2; the liquid flow rate of the cell outlet of the Mth single spiral microchannel is v3, the (M-1) single spiral The liquid flow rate of the cell outlet of the microchannel is v4; the liquid flow rate of the waste liquid outlet of the Mth single spiral microchannel is v5, and the liquid flow rate of the waste liquid outlet of the (M-1) single spiral microchannel is v6 ;
    该压力平衡模块用于使第M个单螺旋形微通道的细胞入口的液体流动方向和第(M-1)个单螺旋形微通道的细胞入口的液体流动方向一致，且0.7*v2≤v1≤1.3*v2；使第M个单螺旋形微通道的细胞出口的液体流动方向和第(M-1)个单螺旋形微通道的细胞出口的液体流动方向一致，且0.7*v4≤v3≤1.3*v4，且v1＝v4；使第M个单螺旋形微通道的废液出口的液体流动方向和第(M-1)个单螺旋形微通道的废液出口的液体流动方向一致，且0.7*v6≤v5≤1.3*v6；The pressure balance module is configured to make the liquid flow direction of the cell inlet of the Mth single spiral microchannel coincide with the liquid flow direction of the cell inlet of the (M-1) single spiral microchannel, and 0.7*v2≤v1 ≤1.3*v2; the liquid flow direction of the cell outlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the cell outlet of the (M-1) single spiral microchannel, and 0.7*v4≤v3≤ 1.3*v4, and v1=v4; the liquid flow direction of the waste liquid outlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the waste liquid outlet of the (M-1) single spiral microchannel, and 0.7*v6≤v5≤1.3*v6;
    所述的N个单螺旋形微通道形成互不交叉的螺旋形微通道；The N single spiral microchannels form spiral microchannels that do not intersect each other;
    其中，第一个单螺旋形微通道的细胞入口用于进入含稀有细胞或颗粒的样本液体；第N个单螺旋形微通道的细胞出口用于收集分选富集后的稀有细胞或颗粒；所述鞘液入口用于添加带有渗透压，保证稀有细胞或颗粒完整的稀释液；1＜M≤N，M、N为正整数。Wherein the cell inlet of the first single spiral microchannel is used to enter a sample liquid containing rare cells or particles; the cell outlet of the Nth single spiral microchannel is used to collect rare cells or particles after sorting and enrichment; The sheath fluid inlet is used to add a diluent with osmotic pressure to ensure the integrity of rare cells or particles; 1 < M ≤ N, M, N are positive integers.
  2. 如权利要求1中所述的螺旋形微通道，其特征在于：所述压力平衡模块为 设置在第(M-1)个分级循环处内的第(M-1)个单螺旋形微通道的废液出口处且与该废液出口密封连接的压力平衡微通道；所述压力平衡微通道设有(N-1)个。The spiral microchannel according to claim 1, wherein said pressure balance module is (M-1) single spiral microchannels disposed in the (M-1)th classification cycle a pressure-balanced microchannel at the outlet of the waste liquid and sealedly connected to the waste liquid outlet; the pressure-balanced microchannel is provided with (N-1).
  3. 如权利要求2中所述的螺旋形微通道，其特征在于：所述压力平衡微通道与N个单螺旋形微通道设置在同一平面内。The spiral microchannel according to claim 2, wherein said pressure equalization microchannel is disposed in the same plane as said N single spiral microchannels.
  4. 如权利要求1中所述的螺旋形微通道，其特征在于：所述压力平衡模块为设置在第(M-1)个分级循环处内的第(M-1)个单螺旋形微通道的废液出口处的压力泵。The spiral microchannel according to claim 1, wherein said pressure balance module is (M-1) single spiral microchannels disposed in the (M-1)th classification cycle Pressure pump at the outlet of the waste liquid.
  5. 如权利要求1中所述的螺旋形微通道，其特征在于：第一个单螺旋形微通道的入口及第N个单螺旋形微通道的出口位于螺旋形微通道几何形状的外侧。The spiral microchannel of claim 1 wherein the inlet of the first single helical microchannel and the outlet of the Nth single helical microchannel are located outside of the helical microchannel geometry.
  6. 如权利要求1中所述的螺旋形微通道，其特征在于：所述的螺旋形微通道由N个单螺旋形微通道形成相互平行的环状。The spiral microchannel according to claim 1, wherein said spiral microchannels are formed by N single spiral microchannels forming mutually parallel annular shapes.
  7. 如权利要求6中所述的螺旋形微通道，其特征在于：所述的螺旋形微通道之间的单螺旋形微通道和与之相邻且平行的单螺旋形微通道之间的水平距离为0.1～1000μm。A spiral microchannel as claimed in claim 6 wherein the horizontal distance between said single helical microchannel between said helical microchannels and a single helical microchannel adjacent and parallel thereto It is 0.1 to 1000 μm.
  8. 如权利要求1中所述的螺旋形微通道，其特征在于：单个的单螺旋形微通道的截面为矩形或梯形。The spiral microchannel according to claim 1, wherein the single single spiral microchannel has a rectangular or trapezoidal cross section.
  9. 如权利要求8中所述的螺旋形微通道，其特征在于：所述的单螺旋形微通道的截面为矩形，该矩形的宽度w与高度h的比值w/h＝1.2～10。A spiral microchannel according to claim 8, wherein said single spiral microchannel has a rectangular cross section, and the ratio of the width w to the height h of the rectangle is w/h = 1.2 to 10.
  10. 如权利要求8中所述的螺旋形微通道，其特征在于：所述的单螺旋形微通道的截面为梯形，所述该梯形靠近螺旋形微通道的几何中心的一侧高度低于该梯形远离螺旋形微通道的几何中心的一侧高度。A spiral microchannel according to claim 8, wherein said single spiral microchannel has a trapezoidal cross section, and said trapezoid is closer to a height of one side of a geometric center of said spiral microchannel than said trapezoid Aside from the height of one side of the geometric center of the spiral microchannel.
  11. 如权利要求9中所述的螺旋形微通道，其特征在于：微通道的宽度w为0.1-1000μm，高度为0.1-800μm。The spiral microchannel according to claim 9, wherein the microchannel has a width w of 0.1 to 1000 μm and a height of 0.1 to 800 μm.
  12. 如权利要求1中所述的螺旋形微通道，其特征在于：N＝2，所述的螺旋形微 通道由第一个单螺旋形微通道及第二个单螺旋形微通道组成。The spiral microchannel of claim 1 wherein N = 2, said helical microchannel consisting of a first single helical microchannel and a second single helical microchannel.
  13. 如权利要求1中所述的螺旋形微通道，其特征在于：单个的单螺旋形微通道的入口及该单螺旋形微通道的出口之间的距离为非靶细胞或非靶颗粒流体力学特征的1.7-2.0个迪恩周期，或者，2.7-3.0个迪恩周期，或者，3.7-4.0个迪恩周期，或者，4.7-5.0个迪恩周期，或者，5.7-6.0个迪恩周期，或者，6.7-7.0个迪恩周期，或者，7.7-8.0个迪恩周期，或者，8.7-9.0个迪恩周期，或者，9.7-10.0个迪恩周期。A helical microchannel as claimed in claim 1 wherein the distance between the inlet of the single single helical microchannel and the exit of the single helical microchannel is a non-target or non-target particle hydrodynamic characteristic 1.7-2.0 Dean cycles, or 2.7-3.0 Dean cycles, or 3.7-4.0 Dean cycles, or 4.7-5.0 Dean cycles, or 5.7-6.0 Dean cycles, or , 6.7-7.0 Dean cycles, or 7.7-8.0 Dean cycles, or 8.7-9.0 Dean cycles, or 9.7-10.0 Dean cycles.
  14. 如权利要求1中所述的螺旋形微通道，其特征在于：所述单螺旋形微通道的通道截面宽度最大处宽度为l1，该单螺旋形微通道的细胞入口和/或细胞出口的宽度为l2，0.1*l1≤l2≤0.4*l1；该单螺旋形微通道的鞘液入口和/或废液出口的宽度为l3，0.1*l1≤l3≤0.9*l1。The spiral microchannel according to claim 1, wherein said single spiral microchannel has a channel section width at a maximum width of l1, and a cell inlet and/or a cell outlet width of said single spiral microchannel It is l2, 0.1*l1≤l2≤0.4*l1; the width of the sheath liquid inlet and/or the waste liquid outlet of the single spiral microchannel is l3, 0.1*l1≤l3≤0.9*l1.
  15. 如权利要求1中所述的螺旋形微通道，其特征在于：N＝4，所述的螺旋形微通道由第一个单螺旋形微通道、第二个单螺旋形微通道、第三个单螺旋形微通道、以及第四个单螺旋形微通道组成。A spiral microchannel as claimed in claim 1 wherein N = 4, said helical microchannel consisting of a first single helical microchannel, a second single helical microchannel, and a third The single spiral microchannel and the fourth single spiral microchannel are composed.
  16. 如权利要求1中所述的螺旋形微通道，其特征在于：所述稀有细胞或颗粒的直径为a，所述微通道的高度为h，0.07≤a/h≤0.5。A spiral microchannel according to claim 1, wherein said rare cells or particles have a diameter a, said microchannels having a height h, 0.07 ≤ a / h ≤ 0.5.
  17. 如权利要求1中所述的螺旋形微通道，其特征在于：所述稀有细胞或颗粒为循环肿瘤细胞、循环祖细胞、循环胎儿细胞、细胞囊泡、外泌体或病原体。The spiral microchannel according to claim 1, wherein the rare cells or particles are circulating tumor cells, circulating progenitor cells, circulating fetal cells, cell vesicles, exosomes or pathogens.
  18. 如权利要求1中所述的螺旋形微通道，其特征在于：还包括用于对该螺旋形通道所分选富集到的稀有细胞或颗粒进行鉴定和/或计数的检测单元；所述检测单元设置在第N个单螺旋形微通道的细胞出口处。A spiral microchannel according to claim 1 further comprising: a detection unit for identifying and/or counting rare cells or particles sorted and enriched for said spiral channel; said detecting The unit is placed at the cell outlet of the Nth single helical microchannel.
  19. 如权利要求18中所述的螺旋形微通道，其特征在于：所述检测单元通过特定技术手段进行稀有细胞或颗粒的性状鉴定；所述的特定技术手段选自由生物光学、生物电学、生物声学、生物磁学及其任意组合组成的组。The spiral microchannel according to claim 18, wherein the detecting unit performs the identification of the characteristics of the rare cells or particles by a specific technical means; the specific technical means is selected from the group consisting of bio-optics, bioelectrics, and bioacoustics. , biomagnetism and any combination of the groups.
  20. 如权利要求18中所述的螺旋形微通道，其特征在于：所述检测单元通过对 特定特征的检测进行稀有细胞或颗粒性状的识别，所述的特定特征选自由稀有细胞或颗粒的太赫兹波谱、拉曼光谱、阻抗、差分阻抗及其任意组合组成的组。A spiral microchannel according to claim 18, wherein said detecting unit performs identification of rare cells or particle traits by detection of specific features selected from terahertz by rare cells or particles A group consisting of a spectrum, a Raman spectrum, an impedance, a differential impedance, and any combination thereof.
  21. 如权利要求2中所述的螺旋形微通道，其特征在于：(N-1)个所述的压力平衡微通道与N个单螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整第(M-1)个压力平衡微通道的长度与内径来调整第(M-1)个细胞出口内通过内容物的流速。The spiral microchannel according to claim 2, wherein: (N-1) said pressure-balanced microchannels and said N single-spiral microchannels form spiral microchannels that do not intersect each other, one or The outlet of the plurality of pressure-balanced microchannels is disposed outside the geometry of the spiral microchannel, and the passage of the (M-1) cell outlet is adjusted by adjusting the length and the inner diameter of the (M-1) pressure-balanced microchannel. The flow rate of the object.
  22. 如权利要求21中所述的螺旋形微通道，其特征在于：(N-1)个所述的压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。A spiral microchannel as claimed in claim 21, wherein the outlets of (N-1) said pressure equalization microchannels are disposed outside the geometry of the spiral microchannel.
  23. 如权利要求1中所述的螺旋形微通道，其特征在于：N个所述细胞出口中的一个或多个设置在螺旋形微通道的几何图形外部。A helical microchannel as claimed in claim 1 wherein one or more of the N said cell outlets are disposed outside of the geometry of the helical microchannel.
  24. 如权利要求23中所述的螺旋形微通道，其特征在于：所有细胞出口均设置在螺旋形微通道的几何图形外部。A spiral microchannel as claimed in claim 23, wherein all cell outlets are disposed outside the geometry of the helical microchannel.
  25. 如权利要求1中所述的螺旋形微通道，其特征在于：N个入口中的一个或多个设置在螺旋形微通道的几何图形外部。A spiral microchannel as claimed in claim 1 wherein one or more of the N inlets are disposed outside of the geometry of the spiral microchannel.
  26. 如权利要求25中所述的螺旋形微通道，其特征在于：所有细胞入口均设置在螺旋形微通道的几何图形外部。A helical microchannel as claimed in claim 25, wherein all of the cell inlets are disposed outside of the geometry of the helical microchannel.
  27. 如权利要求25中所述的螺旋形微通道，其特征在于：所有鞘液入口均设置在螺旋形微通道的几何图形外部。A spiral microchannel as claimed in claim 25, wherein all sheath fluid inlets are disposed outside of the geometry of the helical microchannel.
  28. 如权利要求1～27任一项中所述的螺旋形微通道的使用方法，其特征在于，包括以下步骤：A method of using a spiral microchannel according to any one of claims 1 to 27, comprising the steps of:
    S1：将样本液体从第一个单螺旋形微通道的细胞入口导入，将稀释液从第一个单螺旋形微通道的鞘液入口至第N个单螺旋形微通道的鞘液入口导入；S1: introducing the sample liquid from the cell inlet of the first single spiral microchannel, and introducing the diluent from the sheath liquid inlet of the first single spiral microchannel to the sheath liquid inlet of the Nth single spiral microchannel;
    S2：在第N个单螺旋形微通道的细胞出口收集富含稀有细胞或颗粒的流体； 在第一个单螺旋形微通道的废液出口至第N个单螺旋形微通道的废液出口排出废液。S2: collecting a fluid rich in rare cells or particles at the cell outlet of the Nth single spiral microchannel; discharging the waste liquid in the first single spiral microchannel to the waste liquid outlet of the Nth single spiral microchannel Drain the waste liquid.
  29. 如权利要求28中所述的螺旋形微通道的使用方法，其特征在于，步骤S1中采用的样本液体为具有一定红细胞比积的的血液样本，或通过溶血方式去除大量红细胞的血液样本；所述血液样本中的红细胞比积为0％～70％。The method for using a spiral microchannel according to claim 28, wherein the sample liquid used in step S1 is a blood sample having a certain ratio of red blood cells, or a blood sample for removing a large amount of red blood cells by hemolysis; The ratio of red blood cells in the blood sample is 0% to 70%.
  30. 如权利要求28中所述的螺旋形微通道的使用方法，其特征在于，步骤S1中采用的稀释液具有一定渗透压，用于保护血细胞完整性，为磷酸盐缓冲液、培养血清或生理盐水。A method of using a spiral microchannel according to claim 28, wherein the diluent used in step S1 has a certain osmotic pressure for protecting blood cell integrity, and is a phosphate buffer, culture serum or physiological saline. .
  31. 如权利要求28中所述的螺旋形微通道的使用方法，其特征在于，还包括步骤S1.1，A method of using a spiral microchannel according to claim 28, further comprising the step S1.1,
    S1.1：调整压力平衡模块，使所述第M个单螺旋形微通道的细胞入口的液体流动方向和第(M-1)个单螺旋形微通道的细胞入口的液体流动方向一致，且0.7*v2≤v1≤1.3*v2；使第M个单螺旋形微通道的细胞出口的液体流动方向和第(M-1)个单螺旋形微通道的细胞出口的液体流动方向一致，且0.7*v4≤v3≤1.3*v4，且v1＝v4；使第M个单螺旋形微通道的废液出口的液体流动方向和第(M-1)个单螺旋形微通道的废液出口的液体流动方向一致，且0.7*v6≤v5≤1.3*v6。S1.1: adjusting the pressure balance module such that the liquid flow direction of the cell inlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the cell inlet of the (M-1) single spiral microchannel, and 0.7*v2≤v1≤1.3*v2; the liquid flow direction of the cell outlet of the Mth single spiral microchannel is consistent with the liquid flow direction of the cell outlet of the (M-1) single spiral microchannel, and 0.7 *v4 ≤ v3 ≤ 1.3 * v4, and v1 = v4; liquid flow direction of the waste liquid outlet of the Mth single spiral microchannel and liquid of the waste liquid outlet of the (M-1) single spiral microchannel The flow direction is consistent, and 0.7*v6≤v5≤1.3*v6.
  32. 如权利要求1～27中所述的螺旋形微通道的串联安装结构，其特征在于，采用P个螺旋形微通道串联设置的方式，第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的细胞出口与第Q个螺旋形微通道的第一个单螺旋形微通道入口相连接，形成螺旋形微通道串联安装处，1＜Q≤P。The series mounting structure of the spiral microchannels according to any one of claims 1 to 27, wherein the Nth of the (Q-1)th spiral microchannels is arranged in series by P spiral microchannels The cell outlet of the single spiral microchannel is connected to the first single spiral microchannel inlet of the Qth spiral microchannel to form a spiral microchannel series installation, 1 < Q ≤ P.
  33. 如权利要求32中所述的螺旋形微通道的串联安装结构，其特征在于，在螺旋形微通道串联安装处的第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的废液出口处也设有压力平衡模块。A tandem mounting structure for a spiral microchannel according to claim 32, wherein the Nth single spiral microchannel of the (Q-1)th spiral microchannel at the spiral microchannel is mounted in series A pressure balancing module is also provided at the waste liquid outlet.
  34. 如权利要求33中所述的螺旋形微通道的串联安装结构，其特征在于，所述压力平衡模块为与螺旋形微通道串联安装处的第(Q-1)个螺旋形微通道的 第N个单螺旋形微通道的废液出口密封连接的压力平衡微通道。A series mounting structure for a spiral microchannel according to claim 33, wherein said pressure balance module is Nth of the (Q-1)th spiral microchannel installed in series with the spiral microchannel The waste liquid outlet of the single spiral microchannel is sealed to the pressure-balanced microchannel.
  35. 如权利要求34中所述的螺旋形微通道的串联安装结构，其特征在于，所述的压力平衡微通道和与其相匹配的螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。A series arrangement of spiral microchannels as claimed in claim 34, wherein said pressure-balanced microchannels and their matched helical microchannels form helical microchannels that do not intersect each other, one or more The outlets of the pressure equalization microchannels are disposed outside the geometry of the spiral microchannels, and the flow rate through the contents of the cell outlets matched thereto is adjusted by adjusting the length and inner diameter of the pressure equalization microchannels.
  36. 如权利要求35中所述螺旋形微通道的串联安装结构，其特征在于，所有压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。A series mounting structure for spiral microchannels as recited in claim 35, wherein the outlets of all of the pressure equalization microchannels are disposed outside of the geometry of the helical microchannels.
  37. 如权利要求32中所述的螺旋形微通道的串联安装结构，其特征在于：所述细胞出口中的一个或多个设置在螺旋形微通道的几何图形外部。A tandem mounting structure for a helical microchannel as claimed in claim 32, wherein one or more of said cell outlets are disposed outside of the geometry of the helical microchannel.
  38. 如权利要求37中所述的螺旋形微通道的串联安装结构，其特征在于：所有细胞出口均设置在螺旋形微通道的几何图形外部。A tandem mounting structure for a helical microchannel as claimed in claim 37, wherein all of the cell outlets are disposed outside of the geometry of the helical microchannel.
  39. 如权利要求32中所述的螺旋形微通道的串联安装结构，其特征在于：所述入口中的一个或多个设置在螺旋形微通道的几何图形外部。A series mounting structure for spiral microchannels as recited in claim 32, wherein one or more of said inlets are disposed outside of the geometry of the helical microchannels.
  40. 如权利要求39中所述的螺旋形微通道的串联安装结构，其特征在于：所有细胞入口均设置在螺旋形微通道的几何图形外部。A tandem mounting structure for a helical microchannel as recited in claim 39, wherein all of the cell inlets are disposed outside of the geometry of the helical microchannel.
  41. 如权利要求39中所述的螺旋形微通道的串联安装结构，其特征在于：所有鞘液入口均设置在螺旋形微通道的几何图形外部。A series mounting structure for spiral microchannels as recited in claim 39, wherein all of the sheath fluid inlets are disposed outside of the geometry of the helical microchannels.
  42. 如权利要求33中所述的螺旋形微通道的串联安装结构，其特征在于，所述压力平衡模块为设置在螺旋形微通道串联安装处的第(Q-1)个螺旋形微通道的第N个单螺旋形微通道的废液出口处的压力泵。A series mounting structure for a spiral microchannel according to claim 33, wherein said pressure balance module is a first (Q-1) spiral microchannel disposed at a series connection of the spiral microchannels A pressure pump at the waste outlet of the N single spiral microchannels.
  43. 如权利要求32中所述的螺旋形微通道的串联安装结构，其特征在于，N＝2，所述螺旋形微通道为双螺旋形微通道。A series mounting structure of spiral microchannels as claimed in claim 32, wherein N = 2, said spiral microchannels being double helix microchannels.
  44. 如权利要求32中所述的螺旋形微通道的串联安装结构，其特征在于，P＝2，采用2个螺旋形微通道串联设置的方式，第一个螺旋形微通道的第N个单螺旋形微通道的细胞出口与第二个螺旋形微通道的第一个单螺旋形微通道 入口相连接。A series mounting structure for a spiral microchannel according to claim 32, wherein P = 2, wherein the two spiral microchannels are arranged in series, and the Nth single helix of the first spiral microchannel The cell outlet of the microchannel is connected to the first single helical microchannel inlet of the second helical microchannel.
  45. 如权利要求1～27任一项中所述的螺旋形微通道的并联安装结构，其特征在于，采用X个螺旋形微通道并联设置的方式，样本溶液从X个螺旋形微通道的第一个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从X个螺旋形微通道的第N个单螺旋形微通道的细胞出口流出；X＞1。The parallel mounting structure of the spiral microchannels according to any one of claims 1 to 27, characterized in that, by using X spiral microchannels in parallel, the sample solution is first from the X spiral microchannels The cell inlets of the single helical microchannels enter, and the rare cells or particles flow out from the cell outlet of the Nth single helical microchannel of the X helical microchannels; X>1.
  46. 如权利要求45中所述的螺旋形微通道的并联安装结构，其特征在于，将X个螺旋形微通道的第一个单螺旋形微通道的细胞入口相互连通。A parallel mounting structure for a spiral microchannel according to claim 45, wherein the cell inlets of the first single spiral microchannel of the X spiral microchannels are connected to each other.
  47. 如权利要求45中所述的螺旋形微通道的并联安装结构，其特征在于，将X个螺旋形微通道的第N个单螺旋形微通道的细胞出口相互连通。The parallel mounting structure of the spiral microchannels according to claim 45, wherein the cell outlets of the Nth single spiral microchannels of the X spiral microchannels are connected to each other.
  48. 如权利要求45中所述的螺旋形微通道的并联安装结构，其特征在于，所述的X个螺旋形微通道设置在同一平面内。A parallel mounting structure for a spiral microchannel according to claim 45, wherein said X spiral microchannels are disposed in the same plane.
  49. 如权利要求48中所述的螺旋形微通道的并联安装结构，其特征在于，所述的X个螺旋形微通道同心设置在同一平面内。A parallel mounting structure for a spiral microchannel according to claim 48, wherein said X spiral microchannels are concentrically disposed in the same plane.
  50. 如权利要求45中所述的螺旋形微通道的并联安装结构，其特征在于，所述的X个螺旋形微通道同心且沿垂直于单个螺旋形微通道所在平面的方向设置。A parallel mounting structure for a spiral microchannel according to claim 45, wherein said X spiral microchannels are concentric and disposed in a direction perpendicular to a plane in which the individual spiral microchannels are located.
  51. 如权利要求46中所述的螺旋形微通道的并联安装结构，其特征在于，X＝2，采用两个螺旋形微通道并联设置的方式，样本溶液从两个螺旋形微通道的第一个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从两个螺旋形微通道的第N个单螺旋形微通道的细胞出口流出。A parallel mounting structure for a spiral microchannel according to claim 46, wherein X = 2, the two spiral microchannels are arranged in parallel, and the sample solution is from the first of the two spiral microchannels The cell inlet of the single helical microchannel enters and the rare cells or particles flow out of the cell outlet of the Nth single helical microchannel of the two helical microchannels.
  52. 如权利要求46中所述的螺旋形微通道的并联安装结构，其特征在于，N＝2，采用X个双螺旋形微通道并联设置的方式，样本溶液从X个双螺旋形微通道的第一个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从X个双螺旋形微通道的第二个单螺旋形微通道的细胞出口流出。The parallel mounting structure of the spiral microchannels according to claim 46, wherein N=2, using X double spiral microchannels in parallel, the sample solution is from the X double spiral microchannels The cell inlet of a single helical microchannel enters, and rare cells or particles flow out of the cell outlet of the second single helical microchannel of the X double helical microchannels.
  53. 如权利要求45中所述的螺旋形微通道的并联安装结构，其特征在于，在螺旋形微通道中的第X个螺旋形微通道的第N个单螺旋形微通道的废液出口 处设有压力平衡模块。A parallel mounting structure for a spiral microchannel according to claim 45, wherein a waste liquid outlet of the Nth single spiral microchannel of the Xth spiral microchannel in the spiral microchannel is provided There is a pressure balance module.
  54. 如权利要求53中所述的螺旋形微通道的并联安装结构，其特征在于，所述压力平衡模块为压力泵。A parallel mounting structure for a spiral microchannel according to claim 53, wherein said pressure balance module is a pressure pump.
  55. 如权利要求53中所述的螺旋形微通道的并联安装结构，其特征在于，所述压力平衡模块为压力平衡微通道。A parallel mounting structure for a spiral microchannel according to claim 53 wherein said pressure equalization module is a pressure balanced microchannel.
  56. 如权利要求55中所述的螺旋形微通道的并联安装结构，其特征在于，所述的压力平衡微通道和与其相匹配的螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。A parallel mounting structure for a spiral microchannel according to claim 55, wherein said pressure equalizing microchannel and said matching spiral microchannel form a spiral microchannel which does not intersect each other, one or more The outlets of the pressure equalization microchannels are disposed outside the geometry of the spiral microchannels, and the flow rate through the contents of the cell outlets matched thereto is adjusted by adjusting the length and inner diameter of the pressure equalization microchannels.
  57. 如权利要求56中所述螺旋形微通道道的并联安装结构，其特征在于，所有压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。A parallel mounting structure for a spiral microchannel according to claim 56, wherein the outlets of all of the pressure equalizing microchannels are disposed outside the geometry of the spiral microchannel.
  58. 如权利要求45中所述的螺旋形微通道道的并联安装结构，其特征在于：所述细胞出口中的一个或多个设置在螺旋形微通道的几何图形外部。A parallel mounted structure of spiral microchannels as recited in claim 45, wherein one or more of said cell outlets are disposed outside of the geometry of the helical microchannels.
  59. 如权利要求58中所述的螺旋形微通道道的并联安装结构，其特征在于：所有细胞出口均设置在螺旋形微通道的几何图形外部。A parallel mounting structure for a spiral microchannel as described in claim 58 wherein all of the cell outlets are disposed outside of the geometry of the helical microchannel.
  60. 如权利要求45中所述的螺旋形微通道道的并联安装结构，其特征在于：所述入口中的一个或多个设置在螺旋形微通道的几何图形外部。A parallel mounting structure for a spiral microchannel according to claim 45, wherein one or more of said inlets are disposed outside of the geometry of the spiral microchannel.
  61. 如权利要求60中所述的螺旋形微通道道的并联安装结构，其特征在于：所有细胞入口均设置在螺旋形微通道的几何图形外部。A parallel mounting structure for a spiral microchannel according to claim 60, wherein all of the cell inlets are disposed outside the geometry of the spiral microchannel.
  62. 如权利要求60中所述的螺旋形微通道道的并联安装结构，其特征在于：所有鞘液入口均设置在螺旋形微通道的几何图形外部。A parallel mounting structure for spiral microchannels as claimed in claim 60, wherein all of the sheath liquid inlets are disposed outside the geometry of the spiral microchannels.
  63. 一种螺旋形微通道道的组合连接安装结构，其特征在于：包括K个微通道单体，所述微通道单体为权利要求1～27任一项中所述的螺旋形微通道；所述微通道单体采用串联、并联、多次串联、多次并联、或串并联组合的连接形式相互组合，其中K为正整数且K≥2；串联的连接形式为将第(K-1) 个微通道单体的第N个细胞出口与第K个微通道单体的第一个细胞入口密封连接，组成串联式螺旋形微通道；并联的连接形式为将第(K-1)个微通道单体的第一个细胞入口与第K个微通道单体的第一个细胞入口密封连接汇聚组合成统一的细胞入口；将第(K-1)个微通道单体的第一个鞘液入口与第K个微通道单体的第一个鞘液入口密封连接汇聚组合成统一的鞘液入口，组成并联式螺旋形微通道。A combined connection mounting structure for a spiral microchannel, comprising: K microchannel monomers, wherein the microchannel monomer is a spiral microchannel according to any one of claims 1 to 27; The microchannel cells are combined with each other in a series connection, a parallel connection, a multiple series connection, a multiple parallel connection, or a series-parallel combination, wherein K is a positive integer and K≥2; the connection form of the series is the (K-1) The Nth cell outlet of the microchannel monomer is sealingly connected with the first cell inlet of the Kth microchannel monomer to form a series spiral microchannel; the parallel connection form is the (K-1) micro The first cell inlet of the channel monomer is combined with the first cell inlet of the Kth microchannel monomer to form a uniform cell inlet; the first sheath of the (K-1) microchannel monomer The liquid inlet and the first sheath liquid inlet of the Kth microchannel monomer are sealed and combined to form a uniform sheath liquid inlet to form a parallel spiral microchannel.
  64. 如权利要求63中所述的螺旋形微通道道的组合连接安装结构，其特征在于：多次串联的连接形式为将多个串联式螺旋形微通道以串联的连接形式相互组合；或将串联式螺旋形微通道与微通道单体以串联的连接形式相互组合。A combined connection mounting structure for a spiral microchannel according to claim 63, wherein the plurality of series connection forms are a combination of a plurality of series spiral microchannels in series connection; or a series connection The spiral microchannels and the microchannel monomers are combined with each other in a series connection.
  65. 如权利要求63中所述的螺旋形微通道道的组合连接安装结构，其特征在于：并联的连接形式还包括将第(K-1)个微通道单体的第N个细胞出口与第K个微通道单体的第N个细胞出口密封连接汇聚组合成统一的细胞出口；将第(K-1)个微通道单体的第N个废液出口与第K个微通道单体的第N个废液出口密封连接汇聚组合成统一的废液出口。A combined connection mounting structure for a spiral microchannel according to claim 63, wherein the parallel connection form further comprises the Nth cell outlet of the (K-1)th microchannel monomer and the Kth The Nth cell outlet of the microchannel monomer is sealed and aggregated into a unified cell outlet; the Nth waste liquid outlet of the (K-1) microchannel monomer and the Kth microchannel monomer N waste liquid outlets are sealed and combined to form a unified waste liquid outlet.
  66. 如权利要求63中所述的螺旋形微通道道的组合连接安装结构，其特征在于：多次并联的连接形式为将多个并联式螺旋形微通道以并联的连接形式相互组合；或将并联式螺旋形微通道与微通道单体以并联的形式相互组合。A combined connection mounting structure for a spiral microchannel according to claim 63, wherein the plurality of parallel connection forms are a combination of a plurality of parallel spiral microchannels in parallel connection; or The spiral microchannels and the microchannel cells are combined with each other in parallel.
  67. 如权利要求63中所述的螺旋形微通道道的组合连接安装结构，其特征在于：串并联组合的连接形式为将一个或多个并联式螺旋形微通道和/或一个或多个串联式螺旋形微通道和/或一个或多个微通道单体以串联的连接形式或并联的连接形式相互组合，形成混联式螺旋形流道。A combined connection mounting structure for a spiral microchannel according to claim 63, wherein the series-parallel combination is connected in the form of one or more parallel spiral microchannels and/or one or more series The spiral microchannels and/or one or more microchannel cells are combined with each other in a series connection or a parallel connection to form a hybrid spiral flow path.
  68. 一种单螺旋形微通道，其特征在于：包括入口、与入口密封连接的微通道以及与通道远离入口的一端密封连接的出口，所述的入口由细胞入口及鞘液入口汇聚而成，所述的出口由废液出口及细胞出口汇聚而成，其中，细胞入口用于进入含稀有细胞或颗粒的样本液体；细胞出口用于收集分选富集后的稀有细胞或颗粒；鞘液入口用于添加带有渗透压，保证稀有细胞或 颗粒完整的稀释液。A single spiral microchannel characterized by comprising an inlet, a microchannel sealingly connected to the inlet, and an outlet sealingly connected to an end of the passage away from the inlet, the inlet being formed by the cell inlet and the sheath inlet. The outlet is composed of a waste liquid outlet and a cell outlet, wherein the cell inlet is used to enter a sample liquid containing rare cells or particles; the cell outlet is used to collect rare cells or particles after sorting and enrichment; Add a dilution with osmotic pressure to ensure the integrity of rare cells or particles.
  69. 如权利要求68中所述的单螺旋形微通道，其特征在于：所述细胞入口和/或鞘液入口和/或废液出口和/或细胞出口设置在微通道的几何外形外侧。A single helical microchannel as claimed in claim 68, wherein said cell inlet and/or sheath fluid inlet and/or waste outlet and/or cell outlet are disposed outside of the geometry of the microchannel.
  70. 一种单螺旋形微通道的串联安装结构，其特征在于，应用如权利要求68或69中的单螺旋形微通道，采用P个单螺旋形微通道串联设置的方式，第(Q-1)个单螺旋形微通道的细胞出口与第Q个单螺旋形微通道的细胞入口相连接，1＜Q≤P。A series installation structure of single spiral microchannels, characterized in that a single spiral microchannel according to claim 68 or 69 is used, and P single spiral microchannels are arranged in series, (Q-1) The cell outlet of the single helical microchannel is connected to the cell inlet of the Qth single helical microchannel, 1 < Q ≤ P.
  71. 如权利要求70中所述的单螺旋形微通道的串联安装结构，其特征在于，第(Q-1)个单螺旋形微通道的废液出口处设有压力平衡模块。A tandem mounting structure for a single spiral microchannel according to claim 70, wherein a pressure equalization module is provided at the waste liquid outlet of the (Q-1) single spiral microchannel.
  72. 如权利要求71中所述的单螺旋形微通道的串联安装结构，其特征在于，所述压力平衡模块为压力泵或压力平衡微通道。A series mounting structure for a single spiral microchannel according to claim 71, wherein said pressure balance module is a pressure pump or a pressure balanced microchannel.
  73. 如权利要求72中所述的单螺旋形微通道的串联安装结构，其特征在于，所述压力平衡模块为与第(Q-1)单个螺旋形微通道的废液出口密封连接的压力平衡微通道。A tandem mounting structure for a single spiral microchannel according to claim 72, wherein said pressure equalization module is a pressure balanced micro-tight connection with the waste liquid outlet of said (Q-1) single spiral microchannel aisle.
  74. 如权利要求73中所述的单螺旋形微通道的串联安装结构，其特征在于，所述的压力平衡微通道和与其相匹配的所有单螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。A series-mounted structure of single-spiral microchannels as claimed in claim 73, wherein said pressure-balanced microchannels and all of the single-helical microchannels matched thereto form spiral microchannels that do not intersect each other, The outlet of the one or more pressure-balanced microchannels is disposed outside of the geometry of the helical microchannel, and the flow rate through the contents within the cell outlet that matches it is adjusted by adjusting the length and inner diameter of the pressure-balanced microchannel.
  75. 如权利要求74中所述的单螺旋形微通道的串联安装结构，其特征在于，所有压力平衡微通道的出口均设置在螺旋形微通道的几何图形外部。The tandem mounting structure of single spiral microchannels as recited in claim 74, wherein the outlets of all of the pressure equalization microchannels are disposed outside of the geometry of the helical microchannels.
  76. 如权利要求70中所述的单螺旋形微通道的串联安装结构，其特征在于，所述细胞入口和/或鞘液入口和/或废液出口和/或细胞出口设置在螺旋形微通道的几何外形外侧。A tandem mounting structure for a single spiral microchannel according to claim 70, wherein said cell inlet and/or sheath fluid inlet and/or waste liquid outlet and/or cell outlet are disposed in a spiral microchannel The outer side of the geometric shape.
  77. 一种单螺旋形微通道的并联安装结构，其特征在于，应用如权利要求68或69中的单螺旋形微通道，采用X个单螺旋形微通道并联设置的方式，样本 溶液从X个单螺旋形微通道的细胞入口进入，稀有细胞或颗粒从X个单螺旋形微通道的细胞出口流出；X＞1。A parallel installation structure of single spiral microchannels, characterized in that a single spiral microchannel according to claim 68 or 69 is used, and X single spiral microchannels are arranged in parallel, and sample solutions are taken from X single The cell inlet of the spiral microchannel enters, and rare cells or particles flow out from the cell outlet of the X single helical microchannels; X>1.
  78. 如权利要求77中所述的单螺旋形微通道的并联安装结构，其特征在于，将X个单螺旋形微通道的细胞入口和/或细胞出口相互连通。A parallel mounting structure for a single spiral microchannel as claimed in claim 77, wherein the cell inlets and/or cell outlets of the X single helical microchannels are interconnected.
  79. 如权利要求77中所述的单螺旋形微通道的并联安装结构，其特征在于，在第X个单螺旋形微通道的废液出口处设有压力平衡模块；所述压力平衡模块为压力泵或压力平衡微通道。A parallel installation structure of a single spiral microchannel according to claim 77, wherein a pressure balance module is disposed at a waste liquid outlet of the Xth single spiral microchannel; and the pressure balance module is a pressure pump Or pressure balanced microchannels.
  80. 如权利要求79中所述的单螺旋形微通道的并联安装结构，其特征在于，所述的压力平衡微通道和与其相匹配的单螺旋形微通道形成互不交叉的螺旋形微通道，一个或多个压力平衡微通道的出口设置在螺旋形微通道的几何图形外部，通过调整压力平衡微通道的长度与内径来调整与其相匹配的细胞出口内通过内容物的流速。A parallel mounting structure for a single spiral microchannel according to claim 79, wherein said pressure equalizing microchannel and said matching single spiral microchannel form a spiral microchannel which does not intersect each other, one The outlets of the plurality of pressure-balanced microchannels are disposed outside the geometry of the spiral microchannels, and the flow rate through the contents in the cell outlets matched thereto is adjusted by adjusting the length and inner diameter of the pressure-balanced microchannels.
  81. 如权利要求77中所述的单螺旋形微通道的并联安装结构，其特征在于，所有细胞入口和/或鞘液入口和/或废液出口和/或细胞出口设置在微通道的几何外形外侧。A parallel mounting structure for a single spiral microchannel as claimed in claim 77, wherein all cell inlets and/or sheath fluid inlets and/or waste outlets and/or cell outlets are disposed outside the geometry of the microchannels .
  82. 如权利要求79中所述的单螺旋形微通道的并联安装结构，其特征在于：所有细胞入口和/或鞘液入口均设置在螺旋形微通道的几何图形外部。A parallel mounting structure for a single spiral microchannel as recited in claim 79, wherein all of the cell inlets and/or sheath fluid inlets are disposed outside of the geometry of the helical microchannels.
  83. 一种流道芯片，其特征在于：应用如权利要求1～82任一项中所述的螺旋形微通道。A flow path chip characterized by using the spiral microchannel according to any one of claims 1 to 82.
  84. 一种微粒筛选机，其特征在于：应用如权利要求83中所述的流道芯片。A particle screening machine characterized by applying the flow path chip as claimed in claim 83.
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