WO2019128841A1 - Microcanal en spirale, méthode d'utilisation associée et structure d'installation connectée en série/parallèle - Google Patents

Microcanal en spirale, méthode d'utilisation associée et structure d'installation connectée en série/parallèle 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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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

L'invention concerne un microcanal en spirale, une méthode d'utilisation associée et une structure d'installation connectée en série/parallèle. L'invention permet de résoudre le problème technique dans le domaine de la séparation ou de l'enrichissement de cellules tumorales circulantes dans le sang périphérique d'un patient atteint d'une tumeur maligne au moyen d'un microcanal en spirale, et permet de concevoir une structure de séparateur inertiel en fonction de microcanaux en spirale connectés en série et connectés en parallèle qui appliquent en cascade, de multiples microcanaux en spirale. Par rapport aux techniques de l'état de la technique dans le même domaine, la présente invention présente une efficacité de capture supérieure et un rendement de traitement supérieur concernant la séparation et l'enrichissement de cellules tumorales circulantes dans le sang périphérique, optimise la pureté des cellules tumorales circulantes, réduit au minimum l'endommagement de ces dernières et conserve le phénotype d'origine des cellules tumorales circulantes. Une suspension de cellules contenant des cellules tumorales circulantes de grande pureté obtenues par l'utilisation de l'invention est particulièrement appropriée pour un essai biomédical, une analyse génétique, une culture cellulaire, une préparation de tumeur de xénogreffe ultérieurs et similaires, et présente une valeur d'application pratique large et à grande portée en médecine clinique.
PCT/CN2018/122352 2017-12-25 2018-12-20 Microcanal en spirale, méthode d'utilisation associée et structure d'installation connectée en série/parallèle WO2019128841A1 (fr)

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