WO2022047755A1 - 流动池、流动池进出液装置及样本分析系统 - Google Patents

流动池、流动池进出液装置及样本分析系统 Download PDF

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Publication number
WO2022047755A1
WO2022047755A1 PCT/CN2020/113624 CN2020113624W WO2022047755A1 WO 2022047755 A1 WO2022047755 A1 WO 2022047755A1 CN 2020113624 W CN2020113624 W CN 2020113624W WO 2022047755 A1 WO2022047755 A1 WO 2022047755A1
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Prior art keywords
sample
flow cell
opening
cover sheet
joint
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PCT/CN2020/113624
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English (en)
French (fr)
Inventor
崔兴业
邢楚填
夏贇
席阳
Original Assignee
深圳华大智造科技股份有限公司
深圳华大生命科学研究院
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Application filed by 深圳华大智造科技股份有限公司, 深圳华大生命科学研究院 filed Critical 深圳华大智造科技股份有限公司
Priority to CN202080094079.2A priority Critical patent/CN115003786A/zh
Priority to PCT/CN2020/113624 priority patent/WO2022047755A1/zh
Priority to US18/024,337 priority patent/US20230311125A1/en
Priority to JP2022548386A priority patent/JP2023513559A/ja
Priority to EP20951999.0A priority patent/EP4209572A1/en
Publication of WO2022047755A1 publication Critical patent/WO2022047755A1/zh

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • 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
    • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • B01L3/502784—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 droplet or plug flow, e.g. digital microfluidics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Definitions

  • the present application relates to a flow cell for biological sample analysis, a liquid inlet and outlet device for the flow cell, and a sample analysis system.
  • microfluidic cells The core components of gene sequencing technology and droplet digital PCR (Polymerase Chain Reaction, polymerase chain reaction) technology are microfluidic cells.
  • the microfluidic cell is mainly used for dispersing nucleic acid samples, preparing microdroplets, and providing a place for the amplification and analysis of nucleic acid samples.
  • the current sample analysis system has the following defects: the functional space utilization of the microfluidic cell is not saturated, especially the functional division and utilization of the gap region in the microfluidic cell, which limits the current sample analysis system in emerging multi-omics or penetration applications in omics research.
  • the development cost of the current micro flow cell is high, but it is a one-time consumable and cannot be reused, resulting in a great waste.
  • a flow cell comprising a first cover sheet; a second cover sheet arranged opposite to the first cover sheet; and a parallel arrangement between the first cover sheet and the second cover sheet At least two spacer layers, two sides of each spacer layer are respectively in contact with the first cover sheet and the second cover sheet, the first cover sheet, the second cover sheet and the adjacent two
  • a flow path is jointly formed between the spacer layers, and a surface of the first cover sheet close to the second cover sheet forms a detection surface; a first opening is provided at one end of the flow path, and the first opening is used for A first sample is injected into the flow path, and the first sample is adsorbed on the detection surface; and a second opening is provided at the other end of the flow path, and the second opening is used for droplet injection into the flow path, the droplet containing the second sample.
  • the hydraulic radius of the first opening is smaller than the hydraulic radius of the second opening.
  • the detection surface is further provided on the surface of the second cover sheet close to the first cover sheet.
  • the hydraulic radius of the first opening is 0.075mm-0.75mm.
  • the hydraulic radius of the second opening is 0.02mm-0.2mm.
  • the spacer layer is an adhesive layer.
  • both the first opening and the second opening are provided on the first cover sheet or the second cover sheet.
  • a liquid inlet and outlet device for a flow cell comprising the flow cell as described above, a first engagement device and a second engagement device, the first engagement device communicates with the first opening, and the second engagement device A device communicates with the second opening.
  • the second engaging device includes a second engaging portion, the second engaging portion includes a first engaging port and a second engaging port, and the second engaging port communicates with the second opening, so
  • the first joint is disposed away from the second opening, and the size of the first joint along the extending direction perpendicular to the flow path is smaller than the size of the second joint opening along the extending direction perpendicular to the flow path.
  • the second joint device further includes a second interface block and a second seal, the second interface block communicates with the first joint port, and the second seal assembly is sleeved on the the second joint.
  • the second interface block includes a second liquid inlet channel, a second liquid outlet channel, a second control valve group, and a second common channel, and the second common channel communicates with the first joint port .
  • the hydraulic diameters of the second liquid inlet channel and the second common channel are both larger than the hydraulic diameter of the second liquid outlet channel.
  • the first engaging device includes a first engaging portion, a first interface block, and a first sealing assembly, one end of the first engaging portion communicates with the first opening, and the other end communicates with the first opening.
  • An interface block communicates with each other, and the first sealing component is sleeved on the first joint portion.
  • the first interface block includes a first liquid inlet channel, a first liquid outlet channel, a first control valve group, and a first common channel, and the first common channel communicates with the first joint portion .
  • a sample analysis system comprising: a flow cell inflow and outflow device, the flow cell inflow and outflow device is the above-mentioned flow cell inflow and outflow device.
  • a first sample dispensing device for injecting a first sample into the flow cell through the first engaging device.
  • a second sample dispensing device for forming a second sample into droplets, and injecting the droplets into the flow cell through the second engaging device.
  • the second sample distribution device includes a droplet generating device.
  • the optical imaging unit includes a first lens assembly and a second lens assembly.
  • the first lens assembly is used for imaging the first sample.
  • the second lens assembly is used for imaging the micro droplets.
  • the focal length of the first lens assembly is smaller than the focal length of the second lens assembly, and the diameter of the field of view of the first lens assembly is smaller than the diameter of the field of view of the second lens assembly.
  • the focal length of the first lens assembly is 1-2 ⁇ m, and the diameter of the field of view is 1-2 mm; the focal length of the second lens assembly is 1-2 mm, and the diameter of the field of view is 5-10 mm.
  • the magnification of the first lens assembly is greater than the magnification of the second lens assembly.
  • the optical imaging unit includes a third lens assembly and a compensating mirror assembly.
  • the sample analysis system further includes a cleaning unit, and the cleaning unit communicates with the flow cell.
  • sample arrays By constructing a flow path and a detection surface in the flow cell of the present application, different sample arrays can be arranged in the same flow path system, which significantly improves the types of contents that can be identified in the flow cell, and can be used for known types and unknown types. Detection and analysis of various nucleic acid samples, taking into account the cost-effectiveness of both types of analysis.
  • the sample analysis system adopts the above-mentioned flow cell combined with a unique optical imaging unit, which can realize the analysis and detection of nucleic acid samples of multi-dimensional size, and the detection content is more abundant, which can meet the detection of low demand, and can also meet the detection of high demand, and can obtain nucleic acid samples sequence information and dynamic concentration information.
  • the flow cell of the present application can be reused, which improves the utilization rate of the flow cell and reduces the cost of the flow cell.
  • FIG. 1 is a schematic diagram of a flow cell provided by an embodiment of the present application.
  • FIG. 2 is a schematic diagram of adding a sample to a flow cell according to an embodiment of the present application.
  • FIG. 3 is a schematic diagram of a flow cell provided by another embodiment of the present application.
  • FIG. 4 is a cross-sectional view of the flow cell provided in FIG. 3 taken along IV-IV.
  • FIG. 5 is a cross-sectional view of a flow cell provided by an embodiment of the present application.
  • FIG. 6 is a cross-sectional view of a flow cell provided by another embodiment of the present application.
  • FIG. 7A and 7B are schematic diagrams of a detection surface in a flow cell provided by an embodiment of the present application.
  • FIGS. 8A and 8B are schematic diagrams of two detection surfaces in a flow cell provided by an embodiment of the present application.
  • FIG. 9 is a schematic diagram of a liquid inlet and outlet device of a flow cell provided by an embodiment of the present application.
  • FIG. 10 is a program architecture diagram of a sample analysis system provided by an embodiment of the present application.
  • FIG. 11A is a schematic diagram of imaging a first sample by an optical imaging unit provided by an embodiment of the present application.
  • FIG. 11B is a schematic diagram of an optical imaging unit provided in an embodiment of the present application for imaging microdroplets.
  • FIG. 12A is a schematic diagram of imaging a first sample by an optical imaging unit provided by another embodiment of the present application.
  • FIG. 12B is a schematic diagram of imaging microdroplets by an optical imaging unit provided by another embodiment of the present application.
  • Figure 13a is an imaging image of microdroplets of non-uniform size in a flow cell provided by the present application.
  • Figure 13b is an image of droplet imaging at the edge of the flow cell provided in Figure 13a.
  • Figure 13c is an image of a portion of the droplets in the flow cell provided in Figure 13a that are not counted in the statistics.
  • Fig. 13d is a normal distribution diagram of the statistical results of the fluorescence intensity and radius of microdroplets in the flow cell provided in Fig. 13a.
  • Figure 14 is an imaging image of microdroplets of uniform size within the flow cell provided herein.
  • Figure 15 is an imaging image of the first sample in the flow cell provided by the present application.
  • the first cover sheet 11 The second liquid outlet channel 2222
  • the second cover piece 12 The second control valve group 2223
  • the first joint device 21 The second lens assembly 322
  • the first joint 211 The third lens assembly 323
  • the first liquid inlet channel 2121 Sample storage device 33
  • first control valve group 2123 first sample dispensing device 341
  • the first sealing component 213 Micro droplet generating device 37
  • Second joint 221 First sample a
  • a component when referred to as being “fixed on” or “mounted on” another component, it can be directly on the other component or there may also be an intervening component. When a component is considered to be “set on” another component, it may be directly set on the other component or there may be a co-existing centered component.
  • the term “and/or” includes all and any combinations of one or more of the associated listed items.
  • an embodiment of the present application provides a flow cell 100 for carrying nucleic acid samples.
  • the flow cell 100 includes a first cover sheet 11 , a second cover sheet 12 and at least two spacers Layer 13.
  • the first cover sheet 11 is spaced apart from and opposite to the second cover sheet 12 .
  • the two adjacent spacer layers 13 are substantially parallel and disposed between the first cover sheet 11 and the second cover sheet 12 , and two sides of each spacer layer 13 are respectively in contact with the first cover sheet 11 and the second cover sheet 12 , thereby
  • a flow path 14 is formed between the two opposite surfaces of the first cover sheet 11 and the second cover sheet 12 and the adjacent two spacer layers 13 .
  • a plurality of flow paths 14 may be formed between the first cover sheet 11 and the second cover sheet 12 , wherein the length-to-width ratio of the flow paths 14 is greater than or equal to 1.
  • the first cover sheet 11 may be a silicon wafer, inorganic glass, organic glass, transparent plastic, or the like.
  • the thickness of the first cover sheet 11 is about 0.75 mm.
  • the second cover sheet 12 may be transparent inorganic glass, transparent organic glass, or transparent plastic.
  • the thickness of the second cover sheet 12 is 0.15mm-0.3mm.
  • the spacer layer 13 is used to form the flow path 14 between the first cover sheet 11 and the second cover sheet 12 .
  • the flow path 14 of different sizes can be designed by changing the thickness of the spacer layer 13 .
  • the spacer layer 13 is an adhesive layer, which may be curable glue or double-sided tape.
  • a detection surface 17 is formed on the surface of the first cover sheet 11 close to the second cover sheet 12 . It can be understood that, when there is only one detection surface 17 , the detection surface 17 may also be disposed on the surface of the second cover sheet 12 on the side close to the first cover sheet 11 . It can also be understood that, referring to FIG. 4 , two detection surfaces 17 can be provided, and the two detection surfaces 17 are respectively located on the opposite surfaces of the first cover sheet 11 and the second cover sheet 12 . The area between them forms the flow path 14 .
  • the surface of the first cover sheet 11 close to the second cover sheet 12 forms a detection surface 17 through specific processing. It can be understood that, referring to FIG. 6 , the two opposite surfaces of the first cover sheet 11 and the second cover sheet 12 can also be processed separately to form two detection surfaces 17 , and between the two detection surfaces 17 is formed. Flow path 14.
  • the first sample a is a nano-spherical nucleic acid molecule formed by winding, and is adsorbed on the detection surface 17 .
  • the detection surface 17 includes a plurality of active regions 171, and the plurality of active regions 171 are arranged in an array.
  • the active region 171 includes chemically active groups such as amination, carboxylation, hydroxylation, etc., which are used to bind the target in the nucleic acid molecule or the molecule to be detected.
  • the first sample a is a nanosphere formed by spatial folding and winding of nucleic acid molecules, with a diameter of about 10-500 nm. The end of the nucleic acid nanosphere has exposed chemical groups and is fixed on the active area 171 of the detection surface 17 by chemical bonds. , the unknown sequence information in these nucleic acid molecules is determined by binding to the fluorescent substance added into the channel 14 .
  • the active regions 171 may be circular, polygonal or other shapes, and the active regions 171 of these shapes are distributed on the detection surface 17 in an array.
  • the active groups are distributed on the surface of the active region 171 in the above shape, so that the nanospheres of the first sample a are arranged in an array on the detection surface 17 .
  • the flow channel 14 can be used, or it can be used in combination with the flow channel 14 and the detection surface 17, thus achieving the purpose of multi-dimensional nucleic acid sample array detection.
  • both ends of each flow path 14 are formed with a first opening 15 and a second opening 16 .
  • the first opening 15 is used for injecting the first sample a into the flow path 14 , and the first sample a can be adsorbed on the detection surface 17 .
  • the second opening 16 is used to inject the droplet b into the flow path 14, wherein the droplet b contains the second sample.
  • the first sample a is a nucleic acid molecule, and the amplified nucleic acid molecules are wound into a nanosphere structure to form the first sample a.
  • the diameter of the nanosphere structure of the first sample a is 10 nm- Within the range of 500 nm, after the first sample a is injected into the flow channel 14 through the first opening 15, the nanospheres will be adsorbed on the detection surface 17, and then the detection reagent (for example, the reagent for sequencing, that is, the above-mentioned fluorescent substance) is released from the first sample a.
  • An opening 15 or a second opening 16 is injected into the flow path 14, and the detection reagent is fully reacted with the nanospheres on the detection surface 17 by suction. After the reaction is completed, the detection reagent is passed through the first opening 15 or the second opening 16. The flow path 14 is discharged, and the first sample a is still adsorbed on the detection surface 17 at this time.
  • the micro-droplet b is a micron-scale droplet (with a diameter of the droplet in the range of 10 ⁇ m-50 ⁇ m) coated with the second sample and related reagents.
  • One droplet constitutes a small reaction system, and the micro-liquid After the droplet b is injected into the flow channel 14 through the second opening 16 , the droplet b will react in the flow channel 14 , and there is no need to discharge the droplet b out of the flow channel 14 after the reaction is completed.
  • the diameter of the nanospheres of the first sample a is much smaller than the diameter of the microdroplets b.
  • the hydraulic radius of the first opening 15 is smaller than the hydraulic radius of the second opening 16 .
  • the hydraulic radius R is a proper term in hydraulics, which refers to the ratio of the flow area of a certain water delivery section to the side length of the water delivery pipeline (that is, the wet circumference) in contact with the water body.
  • the hydraulic radius of the first opening 15 is 0.075mm-0.75mm, wherein the nanometer of the first sample a is 0.075mm-0.75mm.
  • the ball enters the flow path 14 through the first opening 15 through pressure; taking the second opening 16 as a rectangular port as an example, the width of the flow path 14 is between 5mm-20mm and the height is between 0.05-0.3mm, then the second opening The hydraulic radius of 16 is 0.02mm-0.2mm.
  • the droplets b are driven by pressure and enter the flow path 14 through the second opening 16 .
  • both the first opening 15 and the second opening 16 are formed on the second cover sheet 12 .
  • the first samples a are arranged on the detection surface 17 in an array, and the droplets b can be uniformly sized droplets arranged in an array in the flow path within 14. It can be understood that, referring to FIG. 7B in conjunction with FIG. 7B , the micro-droplets b may also be droplets with non-uniform sizes arranged in the flow path 14 .
  • the first samples a are arranged in an array on the two detection surfaces 17 , and the droplets b can be uniformly sized droplets arranged in an array in the flow path 14 .
  • the micro-droplets b may also be droplets with non-uniform sizes arranged in the flow path 14 .
  • a large number of micron-sized droplets b are distributed in an array in the flow channel 14 , and the droplets b have two forms.
  • a microdroplet b contains nucleic acid molecules, the diameter of the microdroplets is about 50 ⁇ m, and a large number of microdroplets b are closely arranged on the flow path 14 .
  • Another microdroplet b is a magnetic microsphere with nucleic acid molecules labeled with fluorescent substances bound on its surface. The microspheres have a particle size of about 20 ⁇ m, are closely arranged with each other, and are controlled by the magnetic force control device outside the flow path 14 , arranged in an array. in the space of the flow path 14 .
  • the microdroplets b can be gel microbeads, mesoporous SiO 2 microspheres, or water-in-oil microdroplets, and the like.
  • the microdroplet b is dropped drop by drop at the second opening 16 through the flaring tip, and enters the flow path 14 under the driving of pressure.
  • FIG. 1 where the microdroplets b added to the flow path 14 are microdroplets with non-uniform sizes, and the droplets are loaded by self-priming to ensure the integrity of the droplets. Please refer to FIG.
  • the theoretical dynamic detection value of the target nucleic acid molecule can be calculated. Specifically, in this embodiment, the theoretical dynamic detection range of the target nucleic acid molecule can reach: 10 0 -10 6 copies/ ⁇ L.
  • an embodiment of the present application provides a liquid inlet and outlet device 200 for a flow cell, including the flow cell 100 as described above and a first engagement device 21 and a second engagement device 22 respectively provided at both ends of the flow path 14 , wherein, the first engaging device 21 communicates with the first opening 15 , and the second engaging device 22 communicates with the second opening 16 .
  • the internal fluid channels of the first joint device 21 and the second joint device 22 are typical one-to-two channels, and the on-off of the fluid is controlled by a valve.
  • One is used to control the fluid out of the flow path 14 .
  • both the first engagement device 21 and the second engagement device 22 may be composed of a two-position three-way solenoid valve and a manifold block, or a one-way valve and a manifold block.
  • the first engaging device 21 includes a first engaging portion 211 , a first interface block 212 and a first sealing assembly 213 .
  • One end of the first engaging portion 211 is communicated with the first opening 15 , and the other end is communicated with the first interface block 212 .
  • the assembly 213 is sleeved on the first joint portion 211 .
  • the first interface block 212 includes a first liquid inlet channel 2121, a first liquid outlet channel 2122, a first control valve group 2123, and a first common channel 2124.
  • the first common channel 2124 The channel 2124 communicates with the first engaging portion 211 .
  • the on-off circuit of the first liquid inlet channel 2121 and the first liquid outlet channel 2122 is controlled by the first control valve group 2123 , so that the first sample a enters the flow path 14 through the first opening 15 .
  • the second engaging device 22 includes a second engaging portion 221 , a second interface block 222 and a second sealing assembly 223 , one end of the second engaging portion 221 is communicated with the second opening 16 , and the other end is connected with the second interface block 222 communicated, and the second sealing component 223 is sleeved on the second joint portion 221 .
  • the second engaging portion 221 includes a first engaging opening 2211 and a second engaging opening 2212 , the second engaging opening 2212 communicates with the second opening 16 , and the first engaging opening 2211 is disposed away from the second opening 16 and is connected to the second opening 16 .
  • the interface block 222 communicates.
  • the size of the first joint 2211 along the extending direction c of the vertical flow path is smaller than the size of the second joint 2212 along the extending direction c of the vertical flow path.
  • both the first joint opening 2211 and the second joint opening 2212 are rectangular openings
  • the overall second joint portion 221 is funnel-shaped
  • the angle between the opposite side walls of the second joint portion 221 is less than 45°.
  • the purpose of this design is to make the micro-scale droplets b enter the flow channel 14 in the flow channel 14 individually and make the micro-droplets b form a single layer in the flow channel 14, and at the same time avoid the micro-droplets b in the first flow channel 14.
  • the two joints 221 are broken.
  • the second interface block 222 has a second liquid inlet channel 2221 , a second liquid outlet channel 2222 , a second control valve group 2223 and a second common channel 2224 , and the second common channel 2224 communicates with the first joint port 2211 .
  • the hydraulic diameters of the second liquid inlet channel 2221 and the second common channel 2224 are both larger than the hydraulic diameter of the second liquid outlet channel 2222 .
  • the hydraulic diameter of the second liquid inlet channel 2221 and the second common channel 2224 is larger than the diameter of the micro-droplet b, so that the micro-droplet b can smoothly enter the flow path 14 without being broken.
  • the diameter of the microdroplet b can be reduced by extrusion, so as to be smoothly discharged from the second liquid outlet channel 2222 with a small hydraulic diameter.
  • the second common channel 2224 with a larger hydraulic diameter is compatible with the second joint portion 221 and the second sealing component 223 .
  • the first control valve group 2123 and the second control valve group 2223 can achieve the purpose of one-way liquid inlet and one-way liquid outlet according to the function of one-to-two channels.
  • the first control valve group 2123 includes a first liquid inlet valve 2125 and a first liquid outlet valve 2126 .
  • the first liquid inlet valve 2125 is used to control the opening and closing of the first liquid inlet channel 2121
  • the first liquid outlet valve 2126 is used to control the opening and closing of the first liquid outlet channel 2122 .
  • the second control valve group 2223 includes a second liquid inlet valve 2225 and a second liquid outlet valve 2226, wherein the second liquid inlet valve 2225 is used to control the opening and closing of the second liquid inlet channel 2221, and the second liquid outlet valve 2226 is used to control the opening and closing of the second liquid inlet channel 2221. Control the opening and closing of the second liquid outlet channel 2222 .
  • first sealing component 213 and the second sealing component 223 are both heat-resistant, corrosion-resistant and biocompatible elastomer parts, and the materials include silicone rubber or fluorine rubber.
  • the second sealing component 223 is designed into a funnel shape with a rectangular cross section according to the structural shape of the second engaging portion 221 , and can be sleeved on the outside of the second engaging portion 221 .
  • the second sealing component 223 constitutes the second joint portion 221, and the structural shape of the second sealing component 223 is designed as a funnel shape with a rectangular cross section. In order to realize the function of the second joint portion 221 .
  • the method for the first sample a to enter and exit the flow cell entry and exit device 200 is as follows:
  • Step S11 open the first liquid inlet valve 2125 on the first engaging device 21 and the second liquid outlet valve 2226 on the second engaging device 22, and keep the first liquid outlet valve 2126 on the first engaging device 21 and the second liquid outlet valve 2226 on the first engaging device 21 at the same time.
  • the second liquid inlet valve 2225 on the second coupling device 22 is in a closed state, and the first sample a is pumped into the flow path 14 through the first coupling device 21 and adsorbed on the detection surface 17 .
  • the same method is used each time to add the detection reagent for sequencing that needs to be added into the flow channel 14 through the first opening 15 .
  • Step S12 open the first inlet valve 2125 on the first engagement device 21 and the second outlet valve 2226 on the second engagement device 22, and keep the first outlet valve 2126 on the first engagement device 21 and the second outlet valve 2226 on the first engagement device 21 at the same time.
  • the second liquid inlet valve 2225 on the second coupling device 22 is in a closed state, and driven by pressure, the detection reagent is pumped repeatedly and cyclically to realize the combination of the detection reagent and the first sample a, and then realize the nucleic acid molecule and the fluorescent substance (ie, the detection reagent). ) combination.
  • Step S13 after the reaction is completed, the detection reagent needs to be discharged, then open the first liquid outlet valve 2126 on the first engaging device 21 and the second liquid inlet valve 2225 on the second engaging device 22, and keep the first engaging device 21 at the same time.
  • the first liquid inlet valve 2125 and the second liquid outlet valve 2226 on the second joint device 22 are in a closed state. Driven by pressure, the detection reagent after the reaction is completed is discharged from the first liquid outlet valve 2126 of the first joint device 21.
  • Flow path 14
  • first liquid inlet valve 2125 on the first engaging device 21 and the second liquid outlet valve 2226 on the second engaging device 22 can also be opened, and the first liquid outlet valve on the first engaging device 21 can be kept 2126 and the second inlet valve 2225 on the second engaging device 22 are closed, so that the detection reagent is discharged from the second outlet valve 2226.
  • the method for the micro-droplet b to enter and exit the flow cell entry and exit device 200 is as follows:
  • the second liquid outlet valve 2226 on the 22 is in a closed state, and the microdroplet b is pumped into the flow channel 14 in the flow channel 14 through the second coupling device 22, and the amplification reaction and fluorescence are carried out in the flow channel 14. combination of substances. Wherein, it is not necessary to discharge the flow path 14 after the reaction of the microdroplets b is completed.
  • the above-mentioned sample pumping process also includes a process of multiple staged pumping.
  • the above-mentioned droplet pumping process also includes multiple staged pumping and pumping processes.
  • an embodiment of the present application further provides a sample analysis system 300 , including: a sample processing unit 31 , the above-mentioned flow cell inlet and outlet device 200 , and an optical imaging unit 32 , wherein the sample processing unit 31 It is communicated with the flow cell inlet and outlet device 200, and is used for adding samples to the flow cell inlet and outlet device 200.
  • the flow cell inlet and outlet device 200 is located in the imaging area of the optical imaging unit 32, which is convenient for the optical imaging unit 32 to collect the flow cell inlet and outlet device 200. Image of flow path 14 in the middle.
  • the sample processing unit 31 is used to preprocess the samples, wherein the sample processing unit 31 mainly includes a sample storage device 33 and a sample distribution device 34 .
  • the sample storage device 33 is used for independent storage of multiple samples and multiple reagents, and the sample distribution device 34 is used to add multiple samples and multiple reagents into the flow path 14 respectively.
  • the samples to be tested are nucleic acid molecules, which mainly include two types, one is a first sample a, and the other is a second sample for forming droplets b
  • Samples among which various reagents can be mineral oil, dNTPs, fluorescently labeled molecular probes, deoxyribonucleic acid oligomers, enzymes, phosphate buffers and biological enzymes, etc.
  • the above-mentioned reagents can also be added to the microdroplet b according to actual needs.
  • the first sample a and the second sample are distributed and stored independently in a small tube sealed by a sealing film.
  • the small tube is located on the sample storage device 33.
  • the sample storage device 33 is provided with a plurality of independent slots, and other reagents are independently packaged in the sample storage device 33. In the above-mentioned independent slot, the storage temperature of the sample storage device 33 is relatively low, about 4°C.
  • the sample distribution device 34 includes a first sample distribution device 341 and a second sample distribution device 342 .
  • the first sample dispensing device 341 communicates with the first engaging device 21 , and is used for injecting the first sample a and the corresponding detection reagent into the flow path 14 through the first engaging device 21 and the first opening 15 .
  • the nanospheres of the first sample a are adsorbed on the detection surface 17 .
  • all the samples stored in the sample storage device 33 are passed through the first sample distribution device 341 each time during the reaction.
  • the required detection reagent is injected into the flow path 14 through the first opening 15 .
  • the second sample dispensing device 342 is in communication with the second engaging device 22 for mixing the second sample with the corresponding reagent and forming the mixed liquid into droplets b, which are then injected into the flow through the second engaging device 22 and the second opening 16 Inside Road 14.
  • the second sample distributing device 342 further includes a microdroplet generating device 37 for generating microdroplets b from the mixture formed by the second sample and the corresponding reagent, which may be microdroplets b specifically.
  • a microdroplet generating device 37 for generating microdroplets b from the mixture formed by the second sample and the corresponding reagent, which may be microdroplets b specifically.
  • the optical imaging unit 32 has two modes. One mode uses two lens assemblies to image the nucleic acid molecule array on the detection surface 17 and the droplet array in the flow path 14 respectively, and the other One mode is to use a lens assembly, and the purpose of imaging the nucleic acid molecule array on the detection surface 17 and the droplet array in the flow path 14 is realized by adding a lens on the lens assembly that can adjust the imaging focal plane and field of view. .
  • the optical imaging unit 32 in the first mode, includes a first lens assembly 321 and a second lens assembly 322, wherein the first lens assembly 321 is used for imaging the first sample a, and the second lens assembly 321 is used for imaging the first sample a.
  • the lens assembly 322 is used for the imaging of the microdroplet b.
  • the focal length of the first lens assembly 321 is smaller than the focal length of the second lens assembly 322 , and the field of view diameter of the first lens assembly 321 is smaller than that of the second lens assembly 322 .
  • the focal length of the first lens assembly 321 is 1-2 ⁇ m, and the diameter of the field of view is about 1-2 mm; the focal length of the second lens assembly 322 is about 1-2 mm, and the diameter of the field of view is about 5-10 mm.
  • the magnification of the first lens assembly 321 is greater than the magnification of the second lens assembly 322 , so the first lens assembly 321 can be used to image the first sample a of the nanosphere structure clearly.
  • the image data collected by the first lens assembly 321 and the second lens assembly 322 are respectively saved in their respective planned storage paths. These image data will be used for analysis in specific applications, including early screening or diagnosis of tumors or cancers, detection and analysis of infectious disease pathogens, etc.
  • the optical imaging unit 39 in another mode, includes a third lens assembly 323, wherein the third lens assembly 323 includes a fixed focal length lens, which is used for the first sample a in nanoscale size. Imaging.
  • a compensation mirror assembly 324 is added to the third lens assembly 323, and the compensation mirror assembly 324 is used to adjust the imaging focal plane and the field of view.
  • FIG. 13 a to FIG. 13 d are the imaging and statistical analysis images of a micro-droplet (micro-droplet b) with non-uniform size in the flow path 14 .
  • the second lens assembly 322 is used to capture the image of the optical imaging area in the flow path 14, and the test conditions are: the nucleic acid branch marked with the fluorescent dye Cy5, wherein the Cy5 concentration is about 7mM, and the oil prepared by the oscillating emulsification method after being diluted 500 times Water-containing microdroplets; the excitation light is a green laser with a wavelength of 532nm, a power of 2-5W, and the focal plane is fixed at 1.5 ⁇ m; MATLAB performs image statistical analysis on the diameter and number of microdroplets.
  • Figure 13a is a top view of the flow cell, which is a thumbnail image spliced and synthesized from 60 consecutively photographed microscopic fluorescence images that are connected with each other.
  • drop b shows that the detection surface 17 at the edge of the flow path 14 has a dense distribution of small droplets (the first sample a).
  • Figure 14c some large droplets in the flow path 14 are not included in the statistical data.
  • FIG. 13d is a graph showing the statistical results of the fluorescence intensity and radius of the microdroplet b in the flow path 14, and it can be seen from the graph that the two are basically in a normal distribution.
  • FIG. 14 is an imaging image of a microdroplet (microdroplet b) of uniform size in the flow path 14 .
  • the second lens assembly 322 is used to capture an image of the optical imaging area in the flow path 14, and it can be seen that the microdroplets b in the flow path 14 in the figure are arranged in an array.
  • FIG. 15 is an image formed by the optical signal of the first sample a (including the sequenced nucleic acid library or the specific wound DNA nanospheres) collected by the first lens assembly 321 .
  • a fluorescent image of a sample a (DNA nanospheres, about 200 nm in diameter) arranged in an array with clear outlines.
  • the sample analysis system further includes a cleaning unit 38 , and the cleaning unit 38 communicates with the flow path 14 and is used for cleaning the system pipeline and the flow path 14 .
  • the flow cell 100 is installed on a mounting table (not shown), and the first opening 15 of the flow cell 100 and the first engaging device 21 are sealed by the first sealing component 213
  • the second opening 16 on the flow cell 100 is sealedly connected to the second engaging device 22 through the second sealing assembly 223 .
  • the first engaging device 21 and the second engaging device 22 are connected with the sample dispensing device 34 through pipelines, and meanwhile, the suction of the sample is realized through an upstream pump 35 (eg, a syringe pump).
  • an upstream pump 35 eg, a syringe pump
  • the first engaging device 21 and the second engaging device 22 are connected with the cleaning unit 38 through pipelines, and simultaneously, the cleaning agent injection and waste liquid discharge are realized through a downstream pump 36 (eg, a syringe pump).
  • a downstream pump 36 eg, a syringe pump.
  • the sample dispensing device 34 , the upstream pump 35 , the first engagement device 21 , the second engagement device 22 and the downstream pump 36 can all be connected to a control device (not shown) and activated or turned on by the control device.
  • FIG. 7A to FIG. 10 in conjunction with the sample analysis method using the above-mentioned sample analysis system 300, including the following steps:
  • Step 1 prepare two samples, namely the first sample a and the second sample.
  • the first sample a is a nanosphere with nano-sized features.
  • the second sample was used to generate microdroplets b with micron-sized features.
  • the first sample a, the second sample and other required reagents are all stored in the sample storage device 33 .
  • the first sample a and the second sample are distributed and stored independently in a small tube sealed by a sealing film, the small tube is located on the sample storage device 33, and the sample storage device 33 is provided with a plurality of independent slots, and other reagents are independently Packaged in the above-mentioned independent slot, the storage temperature of the sample storage device 33 is relatively low, about 4°C.
  • various reagents can be mineral oil, dNTPs, fluorescently labeled molecular probes, deoxyribonucleic acid oligomerization, enzymes, phosphate buffers and biological enzymes, etc.
  • step 2 the first sample a is injected into the flow channel 14 .
  • the first sample a is pumped into the flow path 14 through the first coupling device 21 , and the first sample a is adsorbed on the detection surface 17 and arranged in an array.
  • the first sample a is pumped into the first engagement device 21 by the suction of the upstream pump 35 .
  • Step 3 stand for a period of time (for example, 30 minutes), and the nucleic acid nanospheres (ie, the first sample a) are fully adsorbed on the detection surface 17 under the interaction between the active groups to form relatively regularly arranged nanonucleic acids.
  • Molecular array for example, 30 minutes
  • the fourth step is to detect and analyze the first sample a.
  • this embodiment is to sequence nucleic acid molecules, and the sequencing method includes:
  • the first fluorescent substance is pumped into the flow path 14, and through repeated suction, the first fluorescent substance is combined with the first sample a, and after the reaction is completed, the residual first fluorescent substance is The fluorescent substance is discharged from the flow path 14 through the second opening 16 .
  • the combination of other fluorescent substances and the first sample a will be carried out.
  • the first sample a combined with the fluorescent substance in the nanosphere array will emit a fluorescent signal.
  • the fluorescent substances put into the flow channel 14 are repeatedly and cyclically extracted to realize the combination of nucleic acid molecules and fluorescent substances in the array, and the fluorescent signal generated by each cyclic reaction is imaged and analyzed to obtain the first The sequence of a segment of bases in a sample a, until the base sequence analysis of the required length is completed, by this method, the sequence information of the unknown nucleic acid molecule can be detected, and the purpose of sequencing can be achieved.
  • the imaging analysis is realized by the optical imaging unit 32 , and specifically, the imaging analysis of the nucleic acid nanospheres is realized by using the first lens assembly 321 with high magnification.
  • the configured objective lens and lens combination focal length and field of view diameter are small, the focal length is about 1-2 ⁇ m, and the field of view diameter is about 1-2 mm. As shown in Figure 15, it is a fluorescence image of a DNA nanosphere (about 200 nm in diameter).
  • step 5 the second sample is formed into microdroplets b, and the microdroplets b are injected into the flow path 14 .
  • the sealing membrane of the sealed small tube containing the second sample is pierced, and the second sample is extracted from the sealed small tube in the sample storage device 33 in a pressure-driven manner, and mixed with other reagents in the sample storage device 33 to form Mixed liquid, the mixed liquid containing the second sample is located in the second sample distribution device 342 , and the mixed liquid containing the second sample is injected into the droplet generating device 37 through the second sample distribution device 342 under pressure driving.
  • the second sample generates droplets b within a droplet generating device 37 (eg, a microfluidic droplet generator or oscillating emulsifier).
  • the microdroplets b are injected into the flow path 14 through the second bonding device 22 and left for a period of time, and a large number of droplets are stably suspended in the flow path 14 to form a droplet array arranged in an array.
  • the second sample and other reagents are uniformly mixed by the suction of the upstream pump 35 , the mixed solution is pumped into the microdroplet generating device 37 by the upstream pump 35 , and the microdroplet b is passed through the second The bonding device 22 is injected into the flow path 14 .
  • the droplet b is a nucleic acid molecule coating and copolymer (eg, nucleic acid droplet) with micron-sized features, wherein the droplet b contains the raw material required for nucleic acid molecule amplification.
  • Step 6 analyze the second sample.
  • the droplet array will undergo multiple cyclic amplification reactions in the flow channel 14, and the products obtained from each cyclic amplification reaction are used to combine with corresponding fluorescent substances to generate fluorescent signals.
  • Image analysis is performed on the fluorescent signal generated by each cycle of the amplification reaction to obtain the sequence information of the second sample, or the content of a certain base in the second sample with known sequence.
  • the second lens assembly 322 is used to image and count the flow cell to analyze the fluorescence signal of the microdroplets, so that the content of a certain base in the second sample nucleic acid molecule can be obtained.
  • Qualitative and quantitative analysis of nucleic acid molecules of known sequence can be achieved by this method.
  • the cleaning unit 38 is activated, and specific cleaning reagents (eg Tween-20, sodium hydroxide solution) and water are extracted from the cleaning unit 38 to clean the system pipeline, and the cleaned system can be subjected to the next detection and analysis.
  • specific cleaning reagents eg Tween-20, sodium hydroxide solution
  • the system management and the flow path 14 need to be cleaned between adding two different fluorescent substances.
  • the system pipeline and the flow channel 14 need to be cleaned.
  • the sequence analysis of the nano-nucleic acid molecule array (the first sample a) is realized through steps 1 to 4, and the detection and analysis of the micro-droplet array (micro-droplet b) encapsulating nucleic acid molecules is realized in steps 5 and 6.
  • the two analysis stages are relatively independent, and a complete system working logic should include steps 1 to 4, or steps 5 and 6, or step 6 with the same steps.
  • the cleaning step is carried out according to the above description and according to the actual situation.
  • the sample analysis system 300 provided by the embodiment of the present application combines the gene sequencing technology and the droplet PCR technology, which can realize the analysis of a large number of diseases caused by known and unknown pathogens, and has high cost performance and high detection efficiency; realizes a variety of functions integrated, the operation process is simple, and there is no need to consume a large number of nucleic acid samples, which reduces the sampling requirements and is conducive to the comprehensive analysis of rare nucleic acid samples; the demand for physical resources is relatively small, and the laboratory power, desk, computing and storage resources and other infrastructure and equipment management supporting or software requirements are relatively low.
  • sample arrays By constructing a flow path and a detection surface in the flow cell of the present application, different sample arrays can be arranged in the same flow path system, which significantly improves the types of contents that can be identified in the flow cell, and can detect pathogenic bacteria or infectious viruses.
  • the present application can be used for the detection and analysis of known and unknown pathogenic bacteria or infectious viruses, and at the same time, the cost-effectiveness of both types of analysis can be taken into account.
  • the sample analysis system adopts the above-mentioned flow cell combined with a unique optical imaging unit, which can realize the analysis and detection of multi-dimensional nucleic acid samples, and the detection content is more abundant, which can meet the detection of low demand and high demand, and can improve the analysis performance at the same time , taking the droplet PCR application as an example, the present application can significantly increase the number of droplets analyzed, thereby improving the detection dynamic range of the target concentration.
  • the flow cell of the present application can be reused, which improves the utilization rate of the flow cell and reduces the cost of the flow cell.

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Abstract

一种流动池（100），包括第一盖片（11）；与第一盖片（11）相对设置的第二盖片（12）；并列设于第一盖片（11）与第二盖片（12）之间的至少两间隔层（13），第一盖片（11）、第二盖片（12）和相邻两间隔层（13）之间共同形成一流路（14），第一盖片（11）靠近第二盖片（12）的表面形成一检测面（17）；设于流路（14）的一端的第一开口（15），第一开口（15）用于将第一样本（a）注入流路（14），第一样本（a）吸附于检测面（17）；以及设于流路（14）的另一端的第二开口（16），第二开口（16）用于将微液滴（b）注入流路（14），微液滴（b）包含第二样本。流动池（100）可实现多维尺寸核酸样本的检测，可重复利用，成本低。另提供一种流动池进出液装置（200）及样本分析系统（300）。

Description

流动池、流动池进出液装置及样本分析系统 技术领域
本申请涉及生物样本分析用流动池、流动池进出液装置及样本分析系统。
背景技术
基因测序技术和液滴数字PCR(Polymerase Chain Reaction,聚合酶链式反应)技术中的核心部件均为微流动池。微流动池作为样本分析系统的重要载体主要用于核酸样本的分散，制备微液滴，为核酸样本的扩增和分析提供场所。
目前的样本分析系统存在以下缺陷：微流动池的功能性空间利用率不饱和，特别是微流动池中间隙区域的功能划分和利用不足，限制了当前样本分析系统在新兴的多组学或贯穿组学等研究中的应用。另外，目前的微流动池的开发成本高，但却为一次性消耗品，不能重复利用，造成了极大的浪费。
发明内容
为了解决现有技术以上不足之处，有必要提出一种流动池、流动池进出液装置及样本分析系统。
第一方面，提供一种流动池，包括第一盖片；与所述第一盖片相对设置的第二盖片；并列设于所述第一盖片与所述第二盖片之间的至少两间隔层，每一所述间隔层的两侧分别与所述第一盖片和所述第二盖片接触，所述第一盖片、所述第二盖片和相邻两所述间隔层之间共同形成一流路，所述第一盖片靠近所述第二盖片的表面形成一检测面；设于所述流路的一端的第一开口，所述第一开口用于 将第一样本注入所述流路，所述第一样本吸附于所述检测面；以及设于所述流路的另一端的第二开口，所述第二开口用于将微液滴注入所述流路，所述微液滴包含第二样本。
本申请实施方式中，所述第一开口的水力半径小于所述第二开口的水力半径。
本申请实施方式中，所述检测面还设于所述第二盖片靠近所述第一盖片的表面。
本申请实施方式中，所述第一开口的水力半径为0.075mm-0.75mm。
本申请实施方式中，所述第二开口的水力半径为0.02mm-0.2mm。
本申请实施方式中，所述间隔层为胶粘层。
本申请实施方式中，所述第一开口和所述第二开口均设于所述第一盖片或所述第二盖片上。
第二方面，提供一种流动池进出液装置，包括如上所述的流动池、第一接合装置和第二接合装置，所述第一接合装置与所述第一开口连通，所述第二接合装置与所述第二开口连通。
本申请实施方式中，所述第二接合装置包括第二接合部，所述第二接合部包括第一接合口和第二接合口，所述第二接合口与所述第二开口连通，所述第一接合口远离所述第二开口设置，所述第一接合口沿垂直所述流路的延伸方向的尺寸小于所述第二接合口沿垂直所述流路的延伸方向的尺寸。
本申请实施方式中，所述第二接合装置还包括第二接口块和第二密封件，所述第二接口块与所述第一接合口连通，所述第二密封组件套设于所述第二接合部。
本申请实施方式中，所述第二接口块包括第二进液通道、第二出液通道、第二控制阀组以及第二公共通道，所述第二公共通道与所述第一接合口连通。
本申请实施方式中，所述第二进液通道和所述第二公共通道的水力直径均大于所述第二出液通道的水力直径。
本申请实施方式中，所述第一接合装置包括第一接合部、第一接口块以及第一密封组件，所述第一接合部的一端与所述第一开口连通，另一端与所述第一接口块连通，所述第一密封组件套设于所述第一接合部。
本申请实施方式中，所述第一接口块包括第一进液通道、第一出液通道、第一控制阀组以及第一公共通道，所述第一公共通道与所述第一接合部连通。
第三方面，提供一种样本分析系统，包括：流动池进出液装置，所述流动池进出液装置为如上所述的流动池进出液装置。
第一样本分发装置，用于将第一样本通过所述第一接合装置注入所述流动池。
第二样本分发装置，用于将第二样本形成微液滴，并将所述微液滴通过所述第二接合装置注入所述流动池。
以及光学成像单元，用于获取所述流动池的图像。
本申请实施方式中，所述第二样本分发装置包括微液滴发生装置。
本申请实施方式中，所述光学成像单元包括第一镜头组件和第二镜头组件。
所述第一镜头组件用于所述第一样本的成像。
所述第二镜头组件用于所述微液滴的成像。
所述第一镜头组件的焦距小于所述第二镜头组件的焦距，所述第一镜头组件的视场直径小于所述第二镜头组件的视场直径。
本申请实施方式中，所述第一镜头组件的焦距为1-2μm，视场直径为1-2mm；所述第二镜头组件的焦距为1-2mm，视场直径范围为5-10mm。
本申请实施方式中，所述第一镜头组件的放大倍率大于所述第二镜 头组件的放大倍率。
本申请实施方式中，所述光学成像单元包括第三镜头组件以及补偿镜组件。
本申请实施方式中，所述样本分析系统还包括清洗单元，所述清洗单元与所述流动池连通。
本申请流动池通过在流路中构建流路和检测面，能够使不同的样本阵列排布于同一流路系统中，显著提高流动池内可识别的内容物种类，能用于已知种类和未知种类核酸样本的检测分析，并同时兼顾两类分析的成本效益。样本分析系统采用上述的流动池结合独特的光学成像单元，可以实现多维尺寸核酸样本的分析检测，检测内容更加丰富，能满足低需求的检测，同时也能满足高需求的检测，能得到核酸样本的序列信息和动态浓度信息。本申请的流动池可以重复利用，提高了流动池的利用率，同时降低了流动池的成本。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本申请一实施方式提供的流动池的示意图。
图2是本申请一实施方式提供的流动池添加样本的示意图。
图3是本申请另一实施方式提供的流动池的示意图。
图4是图3提供的流动池沿IV-IV的剖视图。
图5是本申请一实施方式提供的流动池的剖视图。
图6是本申请另一实施方式提供的流动池的剖视图。
图7A与图7B是本申请一实施方式提供的流动池内有一个检测面的示意图。
图8A与图8B是本申请一实施方式提供的流动池内有两个检测面的示意图。
图9是本申请一实施方式提供的流动池进出液装置的示意图。
图10是本申请一种实施方式提供的样本分析系统的程序架构图。
图11A是本申请一实施方式提供的光学成像单元对第一样本进行成像的示意图。
图11B是本申请一实施方式提供的光学成像单元对微液滴进行成像的意图。
图12A是本申请另一实施方式提供的光学成像单元对第一样本进行成像的示意图。
图12B是本申请另一实施方式提供的光学成像单元对微液滴进行成像的示意图。
图13a是本申请提供的流动池内尺寸不均一的微液滴的成像图像。
图13b是图13a提供的流动池边缘液滴成像的图像。
图13c是图13a提供的流动池内部分微液滴未被计入统计数据的图像。
图13d是图13a提供的流动池中微液滴荧光强度和半径统计结果正态分布图。
图14是本申请提供的流动池内尺寸均一的微液滴的成像图像。
图15是本申请提供的流动池内第一样本的成像图像。
如下具体实施方式将结合上述附图进一步说明本申请。
主要元件符号说明
流动池         100       第二进液通道    2221
第一盖片       11        第二出液通道    2222
第二盖片       12        第二控制阀组    2223
间隔层         13        第二公共通道    2224
流路              14    第二进液阀        2225
检测面            17    第二出液阀        2226
活性区域          171   第二密封组件      223
分隔区域          172   样本分析系统      300
第一开口          15    样本处理单元      31
第二开口          16    光学成像单元      32、39
流动池进出液装置  200   第一镜头组件      321
第一接合装置      21    第二镜头组件      322
第一接合部        211   第三镜头组件      323
第一接口块        212   补偿镜组件        324
第一进液通道      2121  样本存储装置      33
第一出液通道      2122  样本分发装置      34
第一控制阀组      2123  第一样本分发装置  341
第一公共通道      2124  第二样本分发装置  342
第一进液阀        2125  上游泵            35
第一出液阀        2126  下游泵            36
第一密封组件      213   微液滴发生装置    37
第二接合装置      22    清洗单元          38
第二接合部        221   第一样本          a
第一接合口        2211  微液滴            b
第二接合口        2212  流路的延伸方向    c
第二接口块        222
具体实施方式
以下将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实 施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
需要说明的是，当组件被称为“固定于”、“安装于”另一个组件，它可以直接在另一个组件上或者也可以存在居中的组件。当一个组件被认为是“设置于”另一个组件，它可以是直接设置在另一个组件上或者可能同时存在居中组件。本文所使用的术语“及/或”包括一个或多个相关的所列项目的所有的和任意的组合。
请参阅图1至图2，本申请一实施方式提供了一种流动池100，该流动池100用于承载核酸样本，流动池100包括第一盖片11、第二盖片12及至少两间隔层13。该第一盖片11与第二盖片12间隔且相对设置。相邻两间隔层13大致平行且间隔设置于第一盖片11与第二盖片12之间，每一间隔层13的两侧分别与第一盖片11和第二盖片12接触，从而使得第一盖片11、第二盖片12相对的两表面与相邻两间隔层13之间形成一条流路14。第一盖片11和第二盖片12之间可以形成多条流路14，其中流路14的长宽比大于或等于1。
本实施方式中，第一盖片11可以是硅片、无机玻璃、有机玻璃或透明塑料等。第一盖片11的厚度为0.75mm左右。
本实施方式中，第二盖片12可以是透明无机玻璃、透明有机玻璃或透明塑料等。第二盖片12的厚度为0.15mm-0.3mm。
间隔层13用于在第一盖片11和第二盖片12之间形成流路14，具体可以通过改变间隔层13的厚度来设计不同尺寸的流路14。本实施方式中，间隔层13为胶粘层，具体可以为可固化的胶水或双面胶等。
请结合参阅图4，本实施方式中，第一盖片11靠近所述第二盖片12的表面形成一检测面17。可以理解的是，只有一个检测面17时，检测面17还可以设置于第二盖片12靠近第一盖片11一侧的表面。还可以理解的是，请结合参阅图4，可以设置两检测面17，两个检测面17 分别位于第一盖片11和第二盖片12相对的两表面，此时，两检测面17之间的区域便形成了流路14。
具体地，请结合参阅图5，本实施方式中，所述第一盖片11靠近所述第二盖片12的表面通过特定的处理形成了检测面17。可以理解的是，请结合参阅图6，也可以同时在第一盖片11和第二盖片12相对的两个面分别做处理，形成两个检测面17，两检测面17之间便形成流路14。
本实施方式中，第一样本a为缠绕形成的纳米球状的核酸分子，被吸附在检测面17上，检测面17包括活性区域171和除活性区域171之外的分隔区域172。
本实施方式中，检测面17包括多个活性区域171，多个活性区域171呈阵列排布。活性区域171上包括用于结合核酸分子内目标物或待测分子的氨基化、羧基化、羟基化等化学活性基团。第一样本a是核酸分子通过空间折叠缠绕而形成的纳米球，直径约10-500nm，核酸纳米球的末端具有暴露的化学基团，并通过化学键被固定在检测面17的活性区域171上，这些核酸分子中未知的序列信息通过结合添加进流路14的荧光物质被测定。
本实施方式中，活性区域171可以是圆形、多边形或其它形状，这些形状的活性区域171呈阵列分布在检测面17上。活性基团分布在以上形状活性区域171的表面，从而使第一样本a的纳米球在检测面17上呈阵列排布。
在具体检测过程中可以只用流路14，也可以结合流路14和检测面17使用，因此实现了多维尺寸核酸样本阵列检测的目的。
请结合参阅图3、图4、图7A至图8B，本实施方式中，每一流路14的两端形成有第一开口15和第二开口16。其中第一开口15用于将第一样本a注入流路14内，第一样本a能够吸附在检测面17上。第二开口16用于将微液滴b注入流路14内，其中微液滴b中包含有第二 样本。
本实施方式中，第一样本a为核酸分子，经过扩增的核酸分子缠绕成纳米球的结构从而形成第一样本a，具体地，第一样本a的纳米球结构直径在10nm-500nm的范围内，第一样本a由第一开口15注入流路14内后，纳米球会吸附在检测面17上，再将检测试剂(例如测序用的试剂，即上述荧光物质)由第一开口15或第二开口16注入流路14内，通过抽吸，使检测试剂与检测面17上的纳米球充分反应，反应完成后，再将检测试剂通过第一开口15或者第二开口16排出流路14，此时第一样本a还依旧吸附在检测面17上。
本实施方式中，微液滴b是包覆有第二样本和相关试剂的微米级液滴(液滴直径在10μm-50μm范围内)，一个液滴构成了一个小的反应体系，将微液滴b由第二开口16注入流路14后，微液滴b将在流路14内进行反应，反应完成后无需将微液滴b排出流路14。
可以理解的是，请结合参阅图7A至图8B，第一样本a的纳米球的直径远小于微液滴b的直径。
请结合参阅图3，本实施方式中，第一开口15的水力半径小于第二开口16的水力半径。其中，水力半径R是水力学中的专有名词，指某输水断面的过流面积与水体接触的输水管道边长(即湿周)之比，与断面形状有关，常用于计算渠道隧道的输水能力，表达式为：R＝A/χ，其中A为过水断面面积，χ为湿周，即过水断面上水流所湿润的边界长度。第一开口15和第二开口16可以是圆形口、半圆形口、矩形口、扇形口或其他形状的开口，其中，圆形口或半圆形口的水力半径R＝A/χ＝r 2/d。矩形开口的水力半径R＝A/χ＝w*H/2(w+H)，w和H为流动池流路14的宽和高，这里定义流路14的宽为垂直流路的延伸方向c的尺寸。以第一开口15为圆形口或半圆形口为例，半径r在0.3mm-3mm之间，则第一开口15的水力半径为0.075mm-0.75mm，其中第一样本a的纳米球通过压力由第一开口15进入流路14内；以第二开口16为矩 形口为例，流路14的宽在5mm-20mm之间，高度在0.05-0.3mm之间，则第二开口16的水力半径为0.02mm-0.2mm。其中微液滴b通过压力驱动由第二开口16进入流路14内。
请结合参阅图4，本实施方式中，第一开口15和第二开口16均形成于第二盖片12上。
请结合参阅图7A，当只有一检测面17时，此时，第一样本a呈阵列排布于检测面17上，微液滴b可以是尺寸均一的液滴呈阵列排布在流路14内。可以理解的是，请结合参阅图7B，微液滴b也可以是尺寸不均一的液滴排布在流路14内。
请结合参阅图8A，当有两个检测面17时，此时，第一样本a呈阵列排布于两个检测面17上，微液滴b可以是尺寸均一的液滴呈阵列排布在流路14内。可以理解的是，请结合参阅图8B，微液滴b也可以是尺寸不均一的液滴排布在流路14内。
本实施方式中，流路14中阵列分布着大量微米级微液滴b，微液滴b有两种形式。一种微液滴b内包含有核酸分子，微液滴直径约50μm，大量微液滴b紧密排列在流路14上。另一种微液滴b为表面结合有标记了荧光物质的核酸分子的磁性微球，微球粒径约20μm，彼此紧密排列，并通过流路14外侧的磁力控制装置控制，呈阵列排布于流路14的空间中。微液滴b可以是凝胶微珠、介孔SiO 2微球或油包水微液滴等。微液滴b通过扩口吸头被逐滴的滴加在第二开口16的部位，在压力驱动下进入流路14。通过控制间隔层13的厚度，使多数微液滴b呈单层分布。请结合参阅图1，这里加入流路14的微液滴b为尺寸不均一的微液滴，通过自吸的方式进行液滴加载，可以保证液滴的完整性。请结合参阅图2，流路14中的微液滴b通过温度循环孵育，产生了具有荧光的液滴。通过分析液滴中发光的液滴数目和总的液滴数目的比例，可以计算出目标核酸分子的理论动态检测值。具体地，本实施方式中，目标核酸分子的理论动态检测范围可以达到：10 0-10 6copies/μL。
请参阅图9，本申请一实施方式提供了一种流动池进出液装置200，包括如上所述的流动池100以及分别设于流路14两端的第一接合装置21和第二接合装置22，其中，第一接合装置21与第一开口15连通，第二接合装置22与第二开口16连通。
第一接合装置21和第二接合装置22的内部流体通道均为典型的一分二通道，通过阀门来控制流体的通断，分出的二通道，一条用于控制流体进流路14，另一条用于控制流体出流路14。具体地，第一接合装置21和第二接合装置22均可以由两位三通电磁阀和歧管块组成，或者由单通阀和歧管块组成。
第一接合装置21包括第一接合部211、第一接口块212以及第一密封组件213，第一接合部211一端与第一开口15连通，另一端与第一接口块212连通，第一密封组件213套设于第一接合部211上。
请结合参阅图7A至图8B，本实施方式中，第一接口块212包括第一进液通道2121、第一出液通道2122、第一控制阀组2123以及第一公共通道2124，第一公共通道2124与第一接合部211连通。通过第一控制阀组2123控制第一进液通道2121和第一出液通道2122的通断路，实现第一样本a由第一开口15进入流路14内。
本实施方式中，第二接合装置22包括第二接合部221、第二接口块222和第二密封组件223，第二接合部221一端与第二开口16连通，另一端与第二接口块222连通，第二密封组件223套设于第二接合部221上。
本实施方式中，第二接合部221包括第一接合口2211和第二接合口2212，第二接合口2212与第二开口16连通，第一接合口2211远离第二开口16设置并与第二接口块222连通。
请结合参阅图7A至图8B，本实施方式中，第一接合口2211沿垂直流路的延伸方向c的尺寸小于第二接合口2212沿垂直流路的延伸方向c的尺寸。具体地，第一接合口2211和第二接合口2212均是矩形口， 整体第二接合部221呈漏斗状，第二接合部221相对的两侧壁之间的夹角小于45°。如此设计的目的是，能够使微米级的微液滴b单个进入流路14内的流路14中并使微液滴b在流路14中排成单层，同时避免微液滴b在第二接合部221处破裂。
本实施方式中，第二接口块222第二进液通道2221、第二出液通道2222、第二控制阀组2223以及第二公共通道2224，第二公共通道2224与第一接合口2211连通。
请结合参阅图7A至图8B，本实施方式中，第二进液通道2221和第二公共通道2224的水力直径均大于第二出液通道2222的水力直径。其中第二进液通道2221和第二公共通道2224的水力直径要大于微液滴b的直径，这样可以保证微液滴b顺利进入流路14不被挤破。而当反应完成后的微液滴b可以通过挤破的方式减小直径，从而顺利从水力直径小的第二出液通道2222排出。其中，水力直径较大的第二公共通道2224与第二接合部221以及第二密封组件223相适应。
第一控制阀组2123和第二控制阀组2223按照一分二通道的功能，可以实现单向进液和单向出液的目的。
本实施方式中，第一控制阀组2123包括第一进液阀2125和第一出液阀2126。其中，第一进液阀2125用于控制第一进液通道2121的开合，第一出液阀2126用于控制第一出液通道2122的开合。第二控制阀组2223包括第二进液阀2225和第二出液阀2226，其中，第二进液阀2225用于控制第二进液通道2221的开合，第二出液阀2226用于控制第二出液通道2222的开合。
本实施方式中，第一密封组件213和第二密封组件223均为耐热、耐腐蚀和具有生物兼容性的弹性体件，材质包括硅橡胶或氟橡胶等。
本实施方式中，第二密封组件223根据第二接合部221的结构形状被设计成横截面为矩形的漏斗状，可以套在第二接合部221的外侧。
在另一实施方式中，第二密封组件223构成了第二接合部221，第 二密封组件223的结构形状被设计成横截面为矩形的漏斗状，一方面用于密封作用，另一方面用于实现第二接合部221的功能。
请结合图7A至图9，第一样本a进出流动池进出液装置200的方法为：
步骤S11，打开第一接合装置21上的第一进液阀2125和第二接合装置22上的第二出液阀2226，并同时保持第一接合装置21上的第一出液阀2126和第二接合装置22上的第二进液阀2225处于关闭状态，第一样本a经过第一接合装置21被泵送至流路14内，并吸附在检测面17上。
同理，对第一样本a进行测序分析时，每次采用同样的方法将需要添加的测序用检测试剂通过第一开口15加入流路14内。
步骤S12，打开第一接合装置21上的第一进液阀2125和第二接合装置22上的第二出液阀2226，并同时保持第一接合装置21上的第一出液阀2126和第二接合装置22上的第二进液阀2225处于关闭状态，通过压力驱动，反复循环地抽吸检测试剂实现检测试剂与第一样本a的结合，进而实现核酸分子与荧光物质(即检测试剂)的结合。
步骤S13，反应完成后，需要排出检测试剂，则打开第一接合装置21上的第一出液阀2126和第二接合装置22上的第二进液阀2225，并同时保持第一接合装置21上的第一进液阀2125和第二接合装置22上的第二出液阀2226处于关闭状态，通过压力驱动，将反应完成的检测试剂由第一接合装置21的第一出液阀2126排出流路14。可以理解的是，也可以打开第一接合装置21上的第一进液阀2125和第二接合装置22上的第二出液阀2226，并保持第一接合装置21上的第一出液阀2126和第二接合装置22上的第二进液阀2225关闭，使检测试剂从第二出液阀2226处排出。
请结合图7A至图9，微液滴b进出流动池进出液装置200的方法为：
打开第二接合装置22上的第二进液阀2225和第一接合装置21上的第一出液阀2126，并同时保持第一接合装置21上的第一进液阀2125和第二接合装置22上的第二出液阀2226处于关闭状态，微液滴b经过第二接合装置22被泵送至流路14内的流路14内，并在流路14内进行扩增反应以及与荧光物质的结合。其中，微液滴b反应完成后无需排出流路14。
一种实际使用情况中上述样本泵送过程还包含有多次分段泵送的过程。
另一种实际使用情况中上述液滴泵送过程还包含有多次分段泵送和回抽的过程。
请参阅图10，结合参阅图9，本申请实施方式中还提供一种样本分析系统300，包括：样本处理单元31、上述流动池进出液装置200以及光学成像单元32，其中，样本处理单元31与流动池进出液装置200连通，用于向流动池进出液装置200内加样，流动池进出液装置200位于光学成像单元32的成像区域内，便于光学成像单元32采集流动池进出液装置200中流路14的图像。
样本处理单元31用于对样本进行预处理，其中，样本处理单元31主要包括样本存储装置33和样本分发装置34。其中，样本存储装置33用于多种样本及多种试剂的独立存储，样本分发装置34用于将多种样本和多种试剂分别加入流路14内。
请结合参阅图7A至图8B，本实施方式中，待测的样本为核酸分子，主要包括两种，一种是第一样本a，另一种是用于形成微液滴b的第二样本，其中多种试剂可以是矿物油，dNTP，荧光标记的分子探针，脱氧核糖核酸寡聚，酶、磷酸盐缓冲液和生物酶等。其中微液滴b中还可以根据实际需要加入上述试剂。第一样本a和第二样本分布独立存储在被密封膜封住的小管内，小管位于样本存储装置33上，样本存储装置33上设置有多个独立的槽位，其他试剂被独立封装在上述独立的槽位 内，样本存储装置33的储存温度比较低，在4℃左右。
请参阅图10，本实施方式中，样本分发装置34包括第一样本分发装置341和第二样本分发装置342。第一样本分发装置341与第一接合装置21连通，用于将第一样本a和相应的检测试剂通过第一接合装置21与第一开口15注入流路14内。第一样本a进入流路14后，第一样本a的纳米球吸附在检测面17上，反应过程中，每次通过第一样本分发装置341将存储在样本存储装置33内的所需要的检测试剂通过第一开口15注入流路14内。第二样本分发装置342与第二接合装置22连通，用于将第二样本与相应的试剂进行混合并将混合液形成微液滴b，再通过第二接合装置22与第二开口16注入流路14内。
请参阅图10，本实施方式中，第二样本分发装置342还包括一微液滴发生装置37，用于将第二样本和相应的试剂形成的混合液生成微液滴b，具体可以是微流控液滴生成器或震荡乳化器等。
请结合参阅图11A至图12B，光学成像单元32有两种模式，一种模式是利用两种镜头组件分别对检测面17上的核酸分子阵列和流路14内的液滴阵列进行成像，另一种模式是采用一个镜头组件，通过在镜头组件上增加可调节成像焦面和视场范围的镜头来实现对检测面17上的核酸分子阵列和流路14内的液滴阵列进行成像的目的。
请参阅图11A与图11B，第一种模式中，光学成像单元32包括第一镜头组件321和第二镜头组件322，其中，第一镜头组件321用于第一样本a的成像，第二镜头组件322用于微液滴b的成像。
本实施方式中，第一镜头组件321的焦距小于第二镜头组件322的焦距，第一镜头组件321的视场直径小于第二镜头组件322的视场直径。具体地，第一镜头组件321的焦距为1-2μm，视场直径约1-2mm；第二镜头组件322的焦距约1-2mm，视场直径范围约5-10mm。第一镜头组件321的放大倍率大于第二镜头组件322的放大倍率，因此第一镜头组件321可以用来对纳米球结构的第一样本a进行清晰的成像。
相应地，第一镜头组件321和第二镜头组件322采集的图像数据分别保存在各自规划的存储路径中。这些图像数据将用于具体应用方面的分析，包括肿瘤或癌症的早期筛查或诊断，传染病病原的检测分析等。
请参阅图12A与图12B，另一种模式中，光学成像单元39包括第三镜头组件323，其中第三镜头组件323包括一固定焦距的镜头，用于对纳米级尺寸的第一样本a进行成像。当需要对微米级尺寸的微液滴b进行成像时，通过在第三镜头组件323上增加一补偿镜组件324来实现，补偿镜组件324用于调节成像焦面和视场范围。
请结合参阅图13a至图13d，为一种尺寸不均一的微液滴(微液滴b)在流路14中的成像及统计分析图像。其中，采用第二镜头组件322对流路14中的光学成像区域进行图像采集，测试条件为：标记有荧光染料Cy5的核酸分支，其中Cy5浓度约7mM，稀释500倍后采用震荡乳化法制备的油包水微液滴；激发光为波长532nm的，功率为2-5W的绿色激光，焦面固定为1.5μm；MATLAB对微液滴直径和数目进行图像统计分析。
结果如下：图13a为流动池俯视图，是由连续拍摄的60幅视野相接的显微荧光图拼接合成的缩略图，图中显示流路14中具有尺寸分布不均一的微液滴(微液滴b)。图14b显示流路14边缘的检测面17有密集小液滴(第一样本a)分布。图14c中流路14中的部分大液滴未被计入统计数据，对比图13b分析，可能是图像拼接影响了液滴圆度差，从而影响了统计准确度。图13d为流路14中微液滴b的荧光强度和半径统计结果图，从图中可看出二者基本呈正态分布。
请结合参阅图14，为一种尺寸均一的微液滴(微液滴b)在流路14中的成像图像。其中，采用第二镜头组件322对流路14中的光学成像区域进行图像采集，可以看出图中流路14中的呈阵列排布的微液滴b。
请结合参阅图15，为采用第一镜头组件321采集的第一样本a(包 括序列测定的核酸文库或特定缠绕的DNA纳米球)的光学信号所成的图像，从图中可看到第一样本a(DNA纳米球，直径约200nm)呈阵列排布且轮廓清晰的荧光图像。
本实施方式中，样本分析系统还包括清洗单元38，清洗单元38与流路14连通，用于对系统管路及流路14进行清洗。
请参阅图10，结合参阅图9，实际应用中，流动池100被安装于安装台(图未示)上，流动池100的第一开口15与第一接合装置21通过第一密封组件213密封连接，流动池100上的第二开口16与第二接合装置22通过第二密封组件223密封连接。第一接合装置21以及第二接合装置22与样本分发装置34通过管路连接，同时通过上游泵35(如注射泵)实现样本的抽吸。第一接合装置21以及第二接合装置22与清洗单元38通过管路连接，同时通过下游泵36(如注射泵)实现清洗剂注入和废液排出。样本分发装置34，上游泵35、第一接合装置21、第二接合装置22以及下游泵36均可连接至控制装置(图未示)，被控制装置启动或导通。
请结合参阅图7A至图10，采用上述样本分析系统300进行样本分析的方法，包括以下步骤：
步骤一，准备两份样本，分别为第一样本a和第二样本。
本实施方式中，第一样本a为具有纳米尺寸特征的纳米球。第二样本用于生成具有微米尺寸特征的微液滴b。
本实施方式中，第一样本a、第二样本以及其他所需要的多种试剂都被存储在样本存储装置33内。其中，第一样本a和第二样本分布独立存储在被密封膜封住的小管内，小管位于样本存储装置33上，样本存储装置33上设置有多个独立的槽位，其他试剂被独立封装在上述独立的槽位内，样本存储装置33的储存温度比较低，在4℃左右。其中，多种试剂可以是矿物油，dNTP，荧光标记的分子探针，脱氧核糖核酸寡聚，酶、磷酸盐缓冲液和生物酶等。
步骤二，将第一样本a注入流路14内。
将装有第一样本a的密封小管的密封膜刺穿，采用压力驱动的方式将第一样本a从样本存储装置33中的密封小管中抽取出，通过第一样本分发装置341将第一样本a经过第一接合装置21泵入流路14内，第一样本a吸附在检测面17上并呈阵列排布。
本实施方式中，通过上游泵35的抽吸将第一样本a泵入第一接合装置21。
步骤三，静置一段时间(例如30分钟)，在活性基团之间的相互作用下核酸纳米球(即第一样本a)充分的吸附在检测面17上，形成较为规则排列的纳米核酸分子阵列。
步骤四，对第一样本a进行检测分析，具体地，本实施方式为对核酸分子进行测序，测序方法包括：
首先，在压力驱动下，将第一种荧光物质泵如流路14内，并通过反复抽吸，使第一种荧光物质与第一样本a进行结合，反应完成后将残余的第一种荧光物质通过第二开口16排出流路14。同理再进行其他荧光物质与第一样本a的结合，每投入一种荧光物质，纳米球阵列中与这种荧光物质结合的第一样本a便会发出荧光信号。
然后，对每一个循环反应产生的荧光信号进行成像分析，得到第一样本a的序列信息。
具体地，通过压力驱动，反复循环地抽取流路14中投入的荧光物质，来实现阵列中核酸分子与荧光物质的结合，对每一个循环反应所产生的荧光信号进行成像分析，即可获得第一样本a中一段碱基的排列顺序，直至完成需要长度的碱基顺序分析，通过这种方法可以检测未知核酸分子的序列信息，实现测序的目的。
本实施方式中，成像分析是通过光学成像单元32实现，具体的，采用高放大倍率的第一镜头组件321来实现核酸纳米球的成像分析。其所配置的物镜和透镜组合焦距和视场直径较小，焦距约1-2μm，视场 直径约1-2mm。如图15所示，为一种DNA纳米球(直径约200nm)的荧光图像。
步骤五，将第二样本形成微液滴b，并将微液滴b注入流路14内。
将装有第二样本的密封小管的密封膜刺穿，采用压力驱动的方式将第二样本从样本存储装置33中的密封小管中抽取出，并与样本存储装置33中的其他试剂进行混合形成混合液，含有第二样本的混合液位于第二样本分发装置342内，在压力驱动下，通过第二样本分发装置342将含有第二样本的混合液注入微液滴发生装置37。第二样本在微液滴发生装置37(例如微流控液滴生成器或震荡乳化器)内产生微液滴b。再将微液滴b经第二接合装置22注入流路14内，静置一段时间，大量液滴稳定悬浮在流路14内形成阵列排布的液滴阵列。
本实施方式中，通过上游泵35的抽吸将第二样本和其他试剂混合均匀，通过上游泵35将混合液泵入微液滴发生装置37，再通过上游泵35将微液滴b经第二接合装置22注入流路14内。
本实施方式中，微液滴b是具有微米尺寸特征的核酸分子包覆物及共聚物(例如核酸微液滴)，其中，微液滴b内包含了核酸分子扩增所需要的原料物质。
步骤六，对第二样本进行分析。液滴阵列在流路14内将发生多个循环扩增反应，每一循环扩增反应得到的产物用于与相应的荧光物质结合产生荧光信号。对每一循环扩增反应产生的荧光信号进行成像分析，得到第二样本的序列信息，或者已知序列的第二样本中某个碱基的含量。
具体地，静置片刻，待微液滴(微液滴b)悬浮稳定并保持相对静止时，维持特定温度或特定温度梯度的循环对流路14进行孵育，使得微液滴中的核酸分子与荧光物质相结合，通过特定反应时间，通过第二镜头组件322对流动池进行成像计数分析微液滴的荧光信号，可得出第二样本核酸分子中某一碱基的含量。通过这种方法可以实现已知序列核 酸分子的定性和定量分析。
清洗步骤，启动清洗单元38，通过清洗单元38抽取特定的清洗试剂(例如吐温-20，氢氧化钠溶液)和水对系统管路进行清洗，清洗后的系统即可进行下次检测分析。其中清洗步骤可以在第一样本a检测过程中，添加两种不同荧光物质之间需要对系统管理和流路14进行清洗。另外，在进行完第一样本a的检测，如需进行微液滴b的检测时，需要对系统管路和流路14进行清洗。
上述步骤中通过步骤一到步骤四实现了纳米核酸分子阵列(第一样本a)的序列分析，步骤五和步骤六实现了包裹核酸分子的微液滴阵列(微液滴b)的检测分析，两个分析阶段具有相对独立性，一个完整的系统工作逻辑应包括步骤一到步骤四，或者步骤五和步骤六，或者步骤一致步骤六，其中清洗步骤的进行按如上表述根据实际情况进行。
本申请实施方式提供的样本分析系统300结合了基因测序技术和液滴PCR技术，可以实现对大量已知和未知病原引起的疾病的分析，且性价比较高，检测效率高；实现了多种功能的整合，操作流程简单，无需消耗大量的核酸样本，降低了采样需求，有利于珍稀核酸样本的全面分析；对物理资源的需求相对较少，对实验室的电源、桌台、计算以及存储资源等基础设施以及设备管理的配套或软件需求相对较低。
本申请流动池通过在流路中构建流路和检测面，能够使不同的样本阵列排布于同一流路系统中，显著提高流动池内可识别的内容物种类，以病原菌或传染性病毒的检测分析为例，本申请能用于已知种类和未知种类病原菌或传染性病毒的检测分析，并同时兼顾两类分析的成本效益。
样本分析系统采用上述具有流动池结合独特的光学成像单元，可以实现多维尺寸核酸样本的分析检测，检测内容更加丰富，能满足低需求的检测，也能满足高需求的检测，同时能提升分析性能，以液滴PCR应用为例，本申请能显著提升所分析的液滴的数目进而提升目标物浓度 的检测动态范围。
本申请的流动池可以重复利用，提高了流动池的利用率，同时降低了流动池的成本。
最后应说明的是，以上实施例仅用以说明本申请的技术方案而非限制，尽管参照较佳实施例对本申请进行了详细说明，本领域的普通技术人员应当理解，可以对本申请的技术方案进行修改或等同替换，而不脱离本申请技术方案的精神和范围。

Claims (21)

  1. 一种流动池，其特征在于，包括：
    第一盖片；
    第二盖片，与所述第一盖片相对设置；
    至少两间隔层，并列设于所述第一盖片与所述第二盖片之间，每一所述间隔层的两侧分别与所述第一盖片和所述第二盖片接触，所述第一盖片、所述第二盖片和相邻两所述间隔层之间共同形成一流路，所述第一盖片靠近所述第二盖片的表面形成一检测面；
    第一开口，设于所述流路的一端，所述第一开口用于将第一样本注入所述流路，所述第一样本吸附于所述检测面；以及
    第二开口，设于所述流路的另一端，所述第二开口用于将微液滴注入所述流路，所述微液滴包含第二样本。
  2. 如权利要求1所述的流动池，其特征在于，所述第一开口的水力半径小于所述第二开口的水力半径。
  3. 如权利要求1所述的流动池，其特征在于，所述检测面还设于所述第二盖片靠近所述第一盖片的表面。
  4. 如权利要求2所述的流动池，其特征在于，所述第一开口的水力半径为0.075mm-0.75mm。
  5. 如权利要求2所述的流动池，其特征在于，所述第二开口的水力半径为0.02mm-0.2mm。
  6. 如权利要求1所述的流动池，其特征在于，所述间隔层为胶粘层。
  7. 如权利要求1所述的流动池，其特征在于，所述第一开口和所述第二开口均设于所述第一盖片或所述第二盖片上。
  8. 一种流动池进出液装置，其特征在于，包括权利要求1-7任一项所述的流动池、第一接合装置和第二接合装置，所述第一接合装置与所述第一开口连通，所述第二接合装置与所述第二开口连通。
  9. 如权利要求8所述的流动池进出液装置，其特征在于，所述第二接 合装置包括第二接合部，所述第二接合部包括第一接合口和第二接合口，所述第二接合口与所述第二开口连通，所述第一接合口远离所述第二开口设置，所述第一接合口沿垂直所述流路的延伸方向的尺寸小于所述第二接合口沿垂直所述流路的延伸方向的尺寸。
  10. 如权利要求9所述的流动池进出液装置，其特征在于，所述第二接合装置还包括第二接口块和第二密封件，所述第二接口块与所述第一接合口连通，所述第二密封组件套设于所述第二接合部。
  11. 如权利要求10所述的流动池进出液装置，其特征在于，所述第二接口块包括第二进液通道、第二出液通道、第二控制阀组以及第二公共通道，所述第二公共通道与所述第一接合口连通。
  12. 如权利要求11所述的流动池进出液装置，其特征在于，所述第二进液通道和所述第二公共通道的水力半径均大于所述第二出液通道的水力半径。
  13. 如权利要求8所述的流动池进出液装置，其特征在于，所述第一接合装置包括第一接合部、第一接口块以及第一密封组件，所述第一接合部的一端与所述第一开口连通，另一端与所述第一接口块连通，所述第一密封组件套设于所述第一接合部。
  14. 如权利要求13所述的流动池进出液装置，其特征在于，所述第一接口块包括第一进液通道、第一出液通道、第一控制阀组以及第一公共通道，所述第一公共通道与所述第一接合部连通。
  15. 一种样本分析系统，其特征在于，包括：
    流动池进出液装置，所述流动池进出液装置为如权利要求8-14任一项所述的流动池进出液装置；
    第一样本分发装置，用于将第一样本通过所述第一接合装置注入所述流动池；
    第二样本分发装置，用于将第二样本形成微液滴，并将所述微液滴通过所述第二接合装置注入所述流动池；以及
    光学成像单元，用于获取所述流动池的图像。
  16. 如权利要求15所述的样本分析系统，其特征在于，所述第二样本分发装置包括微液滴发生装置。
  17. 如权利要求15所述的样本分析系统，其特征在于，所述光学成像单元包括第一镜头组件和第二镜头组件；
    所述第一镜头组件用于所述第一样本的成像；
    所述第二镜头组件用于所述微液滴的成像；
    所述第一镜头组件的焦距小于所述第二镜头组件的焦距，所述第一镜头组件的视场直径小于所述第二镜头组件的视场直径。
  18. 如权利要求17所述的样本分析系统，其特征在于，所述第一镜头组件的焦距为1-2μm，视场直径为1-2mm；所述第二镜头组件的焦距为1-2mm，视场直径范围为5-10mm。
  19. 如权利要求17所述的样本分析系统，其特征在于，所述第一镜头组件的放大倍率大于所述第二镜头组件的放大倍率。
  20. 如权利要求15所述的样本分析系统，其特征在于，所述光学成像单元包括第三镜头组件以及补偿镜组件。
  21. 如权利要求15所述的样本分析系统，其特征在于，所述样本分析系统还包括清洗单元，所述清洗单元与所述流动池连通。
PCT/CN2020/113624 2020-09-04 2020-09-04 流动池、流动池进出液装置及样本分析系统 WO2022047755A1 (zh)

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