Flow cell, flow cell inlet and outlet device and sample analysis system

Abstract

A flow cell (100) comprises a first cover sheet (11), a second cover sheet (12) and at least two spacer layers (13); the first cover sheet (11), the second cover sheet (12) and the two adjacent spacer layers (13) together form a flow path (14); the surface of the first cover sheet (11) close to the second cover sheet (12) forms a detection surface (17); the flow cell (100) further comprises a first opening (15) and a second opening (16) arranged at both ends of the flow path (14); a first sample (a) is injected into the flow path (14) from the first opening (15) and adsorbed on the detection surface (17); a micro droplet (b) is injected into the flow path (14) from the second opening (16); the micro droplet (b) contains a second sample. The flow cell (100) can realize the detection of nucleic acid samples of multi-dimensional sizes, can be reused, and has low cost. A flow cell liquid inlet and outlet device (200) and a sample analysis system (300) are also provided.

Description

Technical Field

[0001] The present application relates to a flow cell for biological sample analysis, a flow cell liquid inlet and outlet device, and a sample analysis system. Background Art

[0002] The core components of gene sequencing technology and droplet digital PCR (Polymerase Chain Reaction) technology are micro-flow cells. As an important carrier of the sample analysis system, the micro-flow cell is mainly used to disperse nucleic acid samples, prepare micro-droplets, and provide a place for the amplification and analysis of nucleic acid samples.

[0003] The current sample analysis system has the following defects: the functional space utilization rate of the microfluidic pool is not saturated, especially the functional division and utilization of the gap area in the microfluidic pool is insufficient, which limits the application of the current sample analysis system in emerging multi-omics or through-omics research. In addition, the current microfluidic pool has a high development cost, but it is a disposable consumable and cannot be reused, resulting in great waste. Summary of the invention

[0004] In order to solve the above deficiencies of the prior art, it is necessary to propose a flow cell, a flow cell liquid inlet and outlet device, and a sample analysis system.

[0005] In a first aspect, a flow cell is provided, comprising a first cover sheet; a second cover sheet arranged opposite to the first cover sheet; at least two spacer layers arranged in parallel between the first cover sheet and the second cover sheet, the two sides of each spacer layer being in contact with the first cover sheet and the second cover sheet respectively, the first cover sheet, the second cover sheet and the two adjacent spacer layers jointly forming a flow path, the surface of the first cover sheet close to the second cover sheet forming a detection surface; a first opening arranged at one end of the flow path, the first opening being used to inject a first sample into the flow path, the first sample being adsorbed on the detection surface; and a second opening arranged at the other end of the flow path, the second opening being used to inject microdroplets into the flow path, the microdroplets containing the second sample.

[0006] In an embodiment of the present application, the hydraulic radius of the first opening is smaller than the hydraulic radius of the second opening.

[0007] In the implementation manner of the present application, the detection surface is also provided on a surface of the second cover sheet close to the first cover sheet.

[0008] In the implementation manner of the present application, the hydraulic radius of the first opening is 0.075mm-0.75mm.

[0009] In the implementation manner of the present application, the hydraulic radius of the second opening is 0.02mm-0.2mm.

[0010] In the implementation manner of the present application, the spacer layer is an adhesive layer.

[0011] In the embodiment of the present application, the first opening and the second opening are both provided on the first cover sheet or the second cover sheet.

[0012] In a second aspect, a flow cell liquid inlet and outlet device is provided, comprising the flow cell as described above, a first coupling device and a second coupling device, wherein the first coupling device is connected to the first opening, and the second coupling device is connected to the second opening.

[0013] In an embodiment of the present application, the second joining device includes a second joining portion, the second joining portion includes a first joining port and a second joining port, the second joining port is connected to the second opening, the first joining port is arranged away from the second opening, and the dimension of the first joining port along the extension direction perpendicular to the flow path is smaller than the dimension of the second joining port along the extension direction perpendicular to the flow path.

[0014] In the embodiment of the present application, the second joining device further includes a second interface block and a second sealing member, the second interface block is communicated with the first joining port, and the second sealing member is sleeved on the second joining portion.

[0015] In the embodiment of the present application, 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 is communicated with the first joint.

[0016] In an embodiment of the present application, 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.

[0017] In an embodiment of the present application, the first joining device includes a first joining portion, a first interface block and a first sealing assembly, one end of the first joining portion is connected to the first opening, and the other end is connected to the first interface block, and the first sealing assembly is sleeved on the first joining portion.

[0018] In an embodiment of the present application, 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 is in communication with the first joint portion.

[0019] In a third aspect, a sample analysis system is provided, comprising: a flow cell liquid inlet and outlet device, wherein the flow cell liquid inlet and outlet device is the flow cell liquid inlet and outlet device as described above.

[0020] The first sample dispensing device is used to inject the first sample into the flow pool through the first coupling device.

[0021] The second sample dispensing device is used to form micro-droplets from the second sample and inject the micro-droplets into the flow pool through the second coupling device.

[0022] and an optical imaging unit for acquiring an image of the flow cell.

[0023] In an embodiment of the present application, the second sample dispensing device includes a micro-droplet generating device.

[0024] In the implementation manner of the present application, the optical imaging unit includes a first lens assembly and a second lens assembly.

[0025] The first lens assembly is used for imaging the first sample.

[0026] The second lens assembly is used for imaging the micro-droplets.

[0027] The focal length of the first lens assembly is smaller than the focal length of the second lens assembly, and the field of view diameter of the first lens assembly is smaller than the field of view diameter of the second lens assembly.

[0028] In the implementation manner of the present application, the focal length of the first lens assembly is 1-2 μm, and the field of view diameter is 1-2 mm; the focal length of the second lens assembly is 1-2 mm, and the field of view diameter ranges from 5-10 mm.

[0029] In the implementation manner of the present application, the magnification of the first lens assembly is greater than the magnification of the second lens assembly.

[0030] In the implementation manner of the present application, the optical imaging unit includes a third lens assembly and a compensation mirror assembly.

[0031] In an embodiment of the present application, the sample analysis system further includes a cleaning unit, and the cleaning unit is connected to the flow pool.

[0032] The flow cell of the present application can arrange different sample arrays in the same flow system by constructing flow paths and detection surfaces in the flow path, significantly increasing the types of identifiable contents in the flow cell, and can be used for the detection and analysis of nucleic acid samples of known and unknown types, while taking into account the cost-effectiveness of both types of analysis. The sample analysis system uses the above-mentioned flow cell in combination with a unique optical imaging unit to achieve analysis and detection of nucleic acid samples of multi-dimensional sizes. The detection content is richer, which can meet low-demand detection as well as high-demand detection, and can obtain sequence information and dynamic concentration information of nucleic acid samples. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 It is a schematic diagram of a flow cell provided in one embodiment of the present application.

[0035] Figure 2 It is a schematic diagram of adding samples to a flow cell provided in one embodiment of the present application.

[0036] Figure 3 It is a schematic diagram of a flow cell provided in another embodiment of the present application.

[0037] Figure 4 yes Figure 3 A cross-sectional view of the flow cell along IV-IV is provided.

[0038] Figure 5 It is a cross-sectional view of a flow cell provided in one embodiment of the present application.

[0039] Figure 6 It is a cross-sectional view of a flow cell provided in another embodiment of the present application.

[0040] Fig. 7A and Figure 7B It is a schematic diagram of a flow cell provided in one embodiment of the present application having a detection surface.

[0041] Fig. 8A and Figure 8B It is a schematic diagram of two detection surfaces in a flow cell provided in one embodiment of the present application.

[0042] Fig. 9 It is a schematic diagram of a liquid inlet and outlet device of a flow cell provided in one embodiment of the present application.

[0043] Fig.10 It is a program architecture diagram of a sample analysis system provided in one embodiment of the present application.

[0044] Fig.11A It is a schematic diagram of an optical imaging unit provided in one embodiment of the present application imaging a first sample.

[0045] Fig. 11B This is the intention of the optical imaging unit provided in one embodiment of the present application to image micro-droplets.

[0046] Fig. 12A It is a schematic diagram of an optical imaging unit provided in another embodiment of the present application imaging a first sample.

[0047] Fig. 12B It is a schematic diagram of imaging micro-droplets by an optical imaging unit provided in another embodiment of the present application.

[0048] Fig.13 a is an imaging image of micro-droplets of uneven size in a flow cell provided in the present application.

[0049] Fig.13 b is Fig.13 a Provided image of droplet imaging at the edge of the flow cell.

[0050] Fig.13 c is Fig.13 a provides an image of some droplets in the flow cell that are not counted in the statistics.

[0051] Fig.13 d is Fig.13 a Normal distribution diagram of the statistical results of fluorescence intensity and radius of microdroplets in the flow cell.

[0052] Fig.14 This is an imaging image of micro-droplets of uniform size in a flow cell provided by the present application.

[0053] Fig.15 This is an imaging image of the first sample in the flow cell provided by the present application.

[0054] The following specific implementation methods will further illustrate the present application in conjunction with the above-mentioned drawings.

[0055] Main component symbols

[0056] Flow cell 100 Second liquid inlet channel 2221

[0057] First cover plate 11 Second liquid outlet channel 2222

[0058] Second cover plate 12 Second control valve group 2223

[0059] Spacer layer 13 Second common channel 2224

[0060] Flow path 14 Second liquid inlet valve 2225

[0061] Detection surface 17 Second liquid outlet valve 2226

[0062] Active area 171 Second sealing component 223

[0063] Separation area 172 Sample analysis system 300

[0064] First opening 15 Sample processing unit 31

[0065] Second opening 16 Optical imaging unit 32

[0066] Flow cell inlet and outlet device 200 First lens assembly 321

[0067] First joint device 21 Second lens assembly 322

[0068] First joint part 211 Third lens assembly 323

[0069] First interface block 212 Compensation mirror assembly 324

[0070] First liquid inlet channel 2121 Sample storage device 33

[0071] First liquid outlet channel 2122 Sample distribution device 34

[0072] First control valve group 2123 First sample distribution device 341

[0073] First public channel 2124 Second sample distribution device 342

[0074] First inlet valve 2125 Upstream pump 35

[0075] First liquid outlet valve 2126 Downstream pump 36

[0076] First sealing component 213 Micro droplet generating device 37

[0077] Second bonding device 22 Cleaning unit 38

[0078] Second joint part 221 First sample a

[0079] First joint 2211 micro droplet b

[0080] Second joint port 2212 extension direction c of the flow path

[0081] Second interface block 222 DETAILED DESCRIPTION

[0082] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0083] It should be noted that when a component is referred to as being "fixed to" or "mounted on" another component, it may be directly on the other component or there may be a central 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 central component at the same time. The term "and / or" as used herein includes all and any combinations of one or more of the relevant listed items.

[0084] See also Figure 1 to Figure 2 In one embodiment of the present application, a flow cell 100 is provided, which is used to carry nucleic acid samples. The flow cell 100 includes a first cover sheet 11, a second cover sheet 12 and at least two spacer layers 13. The first cover sheet 11 and the second cover sheet 12 are spaced apart and arranged opposite to each other. Two adjacent spacer layers 13 are substantially parallel and spaced apart between the first cover sheet 11 and the second cover sheet 12, and both sides of each spacer layer 13 are in contact with the first cover sheet 11 and the second cover sheet 12, respectively, so that 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 two adjacent spacer layers 13. Multiple flow paths 14 can be formed between the first cover sheet 11 and the second cover sheet 12, wherein the aspect ratio of the flow path 14 is greater than or equal to 1.

[0085] In this embodiment, the first cover sheet 11 can be a silicon sheet, inorganic glass, organic glass or transparent plastic, etc. The thickness of the first cover sheet 11 is about 0.75 mm.

[0086] In this embodiment, the second cover sheet 12 can be transparent inorganic glass, transparent organic glass or transparent plastic, etc. The thickness of the second cover sheet 12 is 0.15 mm-0.3 mm.

[0087] The spacer layer 13 is used to form a flow path 14 between the first cover sheet 11 and the second cover sheet 12. Specifically, flow paths 14 of different sizes can be designed by changing the thickness of the spacer layer 13. In this embodiment, the spacer layer 13 is an adhesive layer, which can be curable glue or double-sided tape.

[0088] Please refer to Figure 4 In this embodiment, the first cover sheet 11 forms a detection surface 17 on the surface close to the second cover sheet 12. It is understood that when there is only one detection surface 17, the detection surface 17 can also be set on the surface of the second cover sheet 12 close to the first cover sheet 11. It is also understood that, please refer to Figure 4 Two detection surfaces 17 may be provided, and the two detection surfaces 17 are respectively located on two opposite surfaces of the first cover sheet 11 and the second cover sheet 12 . At this time, the area between the two detection surfaces 17 forms a flow path 14 .

[0089] For details, please refer to Figure 5In this embodiment, the surface of the first cover sheet 11 close to the second cover sheet 12 is formed into a detection surface 17 through a specific treatment. It is understood that, please refer to Figure 6 Alternatively, the two opposite surfaces of the first cover sheet 11 and the second cover sheet 12 may be processed simultaneously to form two detection surfaces 17 , and a flow path 14 is formed between the two detection surfaces 17 .

[0090] In this embodiment, the first sample a is a nano-spherical nucleic acid molecule formed by entanglement and is adsorbed on the detection surface 17 . The detection surface 17 includes an active area 171 and a separation area 172 other than the active area 171 .

[0091] In this embodiment, the detection surface 17 includes a plurality of active areas 171, and the plurality of active areas 171 are arranged in an array. The active areas 171 include chemically active groups such as amino, carboxyl, and hydroxylation for binding to the target or the molecule to be tested in the nucleic acid molecule. The first sample a is a nanosphere formed by the spatial folding and winding of the nucleic acid molecule, with a diameter of about 10-500nm. The ends of the nucleic acid nanospheres have exposed chemical groups and are fixed to the active areas 171 of the detection surface 17 by chemical bonds. The unknown sequence information in these nucleic acid molecules is measured by binding to the fluorescent substance added into the flow path 14.

[0092] In this embodiment, the active area 171 can be circular, polygonal or other shapes, and the active areas 171 of these shapes are distributed in an array on the detection surface 17. The active groups are distributed on the surface of the active areas 171 of the above shapes, so that the nanospheres of the first sample a are arranged in an array on the detection surface 17.

[0093] In a specific detection process, the flow path 14 may be used alone, or in combination with the flow path 14 and the detection surface 17, thereby achieving the purpose of multi-dimensional nucleic acid sample array detection.

[0094] Please refer to Figure 3 , Figure 4 , 7A to 8B In this embodiment, a first opening 15 and a second opening 16 are formed at both ends of each flow path 14. The first opening 15 is used to inject 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 a micro droplet b into the flow path 14, wherein the micro droplet b contains the second sample.

[0095] In this embodiment, the first sample a is a nucleic acid molecule, and the amplified nucleic acid molecule is entangled into a nanoball structure to form the first sample a. Specifically, the diameter of the nanoball structure of the first sample a is in the range of 10nm-500nm. After the first sample a is injected into the flow path 14 through the first opening 15, the nanoball will be adsorbed on the detection surface 17, and then the detection reagent (for example, a reagent for sequencing, that is, the above-mentioned fluorescent substance) is injected into the flow path 14 through the first opening 15 or the second opening 16. Through suction, the detection reagent is fully reacted with the nanoballs on the detection surface 17. After the reaction is completed, the detection reagent is discharged from the flow path 14 through the first opening 15 or the second opening 16. At this time, the first sample a is still adsorbed on the detection surface 17.

[0096] In this embodiment, the micro-droplet b is a micron-sized droplet (the droplet diameter is in the range of 10μm-50μm) coated with a second sample and related reagents. One droplet constitutes a small reaction system. After the micro-droplet b is injected into the flow path 14 from the second opening 16, the micro-droplet b will react in the flow path 14. After the reaction is completed, there is no need to discharge the micro-droplet b from the flow path 14.

[0097] It is understandable that please refer to 7A to 8B , the diameter of the nanosphere of the first sample a is much smaller than the diameter of the microdroplet b.

[0098] Please refer to Figure 3 In this embodiment, 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 technical term in hydraulics, which refers to the ratio of the flow area of ​​a water conveyance section to the side length of the water conveyance pipe in contact with the water body (i.e., the wetted perimeter). It is related to the shape of the section and is often used to calculate the water conveyance capacity of the channel tunnel. The expression is: R = A / χ, where A is the water-passing section area, and χ is the wetted perimeter, i.e., the length of the boundary wetted by the water flow on the water-passing section. The first opening 15 and the second opening 16 can be circular, semicircular, rectangular, fan-shaped, or other shaped openings, where the hydraulic radius of a circular or semicircular opening is R = A / χ = r 2 / d. The hydraulic radius of the rectangular opening R = A / χ = w*H / 2(w+H), w and H are the width and height of the flow cell flow path 14, and the width of the flow path 14 is defined as the dimension perpendicular to the extension direction c of the flow path. Taking the first opening 15 as a circular or semicircular opening as an example, the radius r is between 0.3mm-3mm, then the hydraulic radius of the first opening 15 is 0.075mm-0.75mm, wherein the nanospheres of the first sample a enter the flow path 14 through the first opening 15 by pressure; taking the second opening 16 as a rectangular opening 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 hydraulic radius of the second opening 16 is 0.02mm-0.2mm. The micro droplet b is driven by pressure to enter the flow path 14 through the second opening 16.

[0099] Please refer to Figure 4 In this embodiment, the first opening 15 and the second opening 16 are both formed on the second cover plate 12 .

[0100] Please refer to Fig. 7A When there is only one detection surface 17, at this time, the first sample a is arranged in an array on the detection surface 17, and the micro droplets b can be droplets of uniform size arranged in an array in the flow path 14. It can be understood that, please refer to Figure 7B The micro droplets b may also be droplets of uneven sizes arranged in the flow path 14 .

[0101] Please refer to Fig. 8A When there are two detection surfaces 17, the first sample a is arranged in an array on the two detection surfaces 17, and the micro droplets b can be droplets of uniform size arranged in an array in the flow path 14. It can be understood that, please refer to Figure 8B The micro droplets b may also be droplets of uneven sizes arranged in the flow path 14 .

[0102] In this embodiment, a large number of micron-sized microdroplets b are distributed in an array in the flow path 14, and the microdroplets b have two forms. One type of microdroplet b contains nucleic acid molecules, and the diameter of the microdroplet is about 50μm. A large number of microdroplets b are closely arranged on the flow path 14. Another type of microdroplet b is a magnetic microsphere with nucleic acid molecules labeled with fluorescent substances bound to the surface. The particle size of the microspheres is about 20μm. They are closely arranged with each other and controlled by a magnetic force control device on the outside of the flow path 14. They are arranged in an array in the space of the flow path 14. The microdroplets b can be gel microbeads, mesoporous SiO2 microspheres or oil-in-water microdroplets, etc. The microdroplets b are dripped drop by drop at the position of the second opening 16 through the flared pipette tip and enter the flow path 14 under pressure. By controlling the thickness of the spacer layer 13, most of the microdroplets b are distributed in a single layer. Please refer to Figure 1 Here, the micro-droplets b added to the flow path 14 are micro-droplets of uneven size. The droplet loading is performed by self-priming to ensure the integrity of the droplet. Figure 2 , the micro droplets b in the flow path 14 are incubated by temperature cycling to produce fluorescent droplets. By analyzing the ratio of the number of luminescent droplets in the droplets to the total number of droplets, 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 / μL.

[0103] See also Fig. 9An embodiment of the present application provides a flow pool liquid inlet and outlet device 200, including the flow pool 100 as described above and a first coupling device 21 and a second coupling device 22 respectively arranged at both ends of the flow path 14, wherein the first coupling device 21 is connected to the first opening 15, and the second coupling device 22 is connected to the second opening 16.

[0104] The internal fluid channels of the first engaging device 21 and the second engaging device 22 are both typical one-to-two channels, and the on-off of the fluid is controlled by a valve, and the two channels are divided, one for controlling the fluid inlet flow path 14, and the other for controlling the fluid outlet flow path 14. Specifically, the first engaging device 21 and the second engaging device 22 can be composed of a two-position three-way solenoid valve and a manifold block, or a one-way valve and a manifold block.

[0105] The first joining device 21 includes a first joining portion 211 , a first interface block 212 and a first sealing assembly 213 . One end of the first joining portion 211 is connected to the first opening 15 , and the other end is connected to the first interface block 212 . The first sealing assembly 213 is sleeved on the first joining portion 211 .

[0106] Please refer to 7A to 8B In this embodiment, 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, and the first common channel 2124 is connected to the first joint 211. The first control valve group 2123 controls the on-off of the first liquid inlet channel 2121 and the first liquid outlet channel 2122, so that the first sample a enters the flow path 14 from the first opening 15.

[0107] In this embodiment, the second joining device 22 includes a second joining portion 221 , a second interface block 222 and a second sealing assembly 223 . One end of the second joining portion 221 is connected to the second opening 16 , and the other end is connected to the second interface block 222 . The second sealing assembly 223 is sleeved on the second joining portion 221 .

[0108] In this embodiment, the second joint portion 221 includes a first joint opening 2211 and a second joint opening 2212 . The second joint opening 2212 is communicated with the second opening 16 . The first joint opening 2211 is disposed away from the second opening 16 and is communicated with the second interface block 222 .

[0109] Please refer to 7A to 8BIn this embodiment, the dimension of the first joint 2211 along the extension direction c of the vertical flow path is smaller than the dimension of the second joint 2212 along the extension direction c of the vertical flow path. Specifically, the first joint 2211 and the second joint 2212 are both rectangular openings, the overall second joint 221 is funnel-shaped, and the angle between the two opposite side walls of the second joint 221 is less than 45°. The purpose of such a design is to enable micron-sized micro-droplets b to enter the flow path 14 in the flow path 14 individually and to arrange the micro-droplets b in a single layer in the flow path 14, while preventing the micro-droplets b from breaking at the second joint 221.

[0110] In this embodiment, the second interface block 222 includes a second liquid inlet channel 2221 , a second liquid outlet channel 2222 , a second control valve group 2223 , and a second common channel 2224 . The second common channel 2224 is in communication with the first joint 2211 .

[0111] Please refer to 7A to 8B In this embodiment, 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 diameters of the second liquid inlet channel 2221 and the second common channel 2224 are larger than the diameters of the micro-droplets b, so that the micro-droplets b can smoothly enter the flow path 14 without being squeezed. When the reaction is completed, the micro-droplets b can reduce their diameters by squeezing, so that they can 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 adapted to the second joint 221 and the second sealing assembly 223.

[0112] The first control valve group 2123 and the second control valve group 2223 can realize the purpose of one-way liquid inlet and one-way liquid outlet according to the function of dividing one channel into two channels.

[0113] In this embodiment, 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, and 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 outlet channel 2222.

[0114] In this embodiment, the first sealing component 213 and the second sealing component 223 are both heat-resistant, corrosion-resistant and biocompatible elastic parts, and the material includes silicone rubber or fluororubber.

[0115] In this embodiment, the second sealing component 223 is designed to be a funnel with a rectangular cross section according to the structural shape of the second joint portion 221 , and can be sleeved on the outside of the second joint portion 221 .

[0116] In another embodiment, the second sealing component 223 constitutes the second joint portion 221 , and the structural shape of the second sealing component 223 is designed to be a funnel with a rectangular cross-section, which is used for sealing on the one hand and for realizing the function of the second joint portion 221 on the other hand.

[0117] Please combine FIG. 7A to FIG. 9 , the method for the first sample a to enter and exit the flow cell inlet and outlet device 200 is:

[0118] Step S11, open the first liquid inlet valve 2125 on the first bonding device 21 and the second liquid outlet valve 2226 on the second bonding device 22, and at the same time keep the first liquid outlet valve 2126 on the first bonding device 21 and the second liquid inlet valve 2225 on the second bonding device 22 in a closed state, and the first sample a is pumped into the flow path 14 through the first bonding device 21 and adsorbed on the detection surface 17.

[0119] Similarly, when sequencing analysis is performed on the first sample a, the sequencing detection reagent to be added is added into the flow path 14 through the first opening 15 using the same method each time.

[0120] Step S12, open the first liquid inlet valve 2125 on the first joining device 21 and the second liquid outlet valve 2226 on the second joining device 22, and at the same time keep the first liquid outlet valve 2126 on the first joining device 21 and the second liquid inlet valve 2225 on the second joining device 22 in a closed state, and through pressure-driven, repeatedly and cyclically suck the detection reagent to achieve the combination of the detection reagent and the first sample a, thereby achieving the combination of the nucleic acid molecule and the fluorescent substance (i.e., the detection reagent).

[0121] Step S13, after the reaction is completed, if the detection reagent needs to be discharged, the first liquid outlet valve 2126 on the first bonding device 21 and the second liquid inlet valve 2225 on the second bonding device 22 are opened, and the first liquid inlet valve 2125 on the first bonding device 21 and the second liquid outlet valve 2226 on the second bonding device 22 are kept in a closed state, and the detection reagent after the reaction is completed is discharged from the flow path 14 through the first liquid outlet valve 2126 of the first bonding device 21 by pressure driving. It can be understood that the first liquid inlet valve 2125 on the first bonding device 21 and the second liquid outlet valve 2226 on the second bonding device 22 can also be opened, and the first liquid outlet valve 2126 on the first bonding device 21 and the second liquid inlet valve 2225 on the second bonding device 22 are kept closed, so that the detection reagent is discharged from the second liquid outlet valve 2226.

[0122] Please combine FIG. 7A to FIG. 9 The method for the micro droplet b to enter and exit the flow cell inlet and outlet device 200 is:

[0123] The second liquid inlet valve 2225 on the second joining device 22 and the first liquid outlet valve 2126 on the first joining device 21 are opened, and the first liquid inlet valve 2125 on the first joining device 21 and the second liquid outlet valve 2226 on the second joining device 22 are kept in a closed state, and the micro-droplet b is pumped into the flow path 14 through the second joining device 22, and performs an amplification reaction and combines with the fluorescent substance in the flow path 14. The micro-droplet b does not need to be discharged from the flow path 14 after the reaction is completed.

[0124] In an actual use case, the sample pumping process further includes multiple segmented pumping processes.

[0125] In another practical application, the above droplet pumping process also includes multiple segmented pumping and back-pumping processes.

[0126] See also Fig.10 , combined with reference Fig. 9 In an embodiment of the present application, a sample analysis system 300 is also provided, comprising: a sample processing unit 31, the above-mentioned flow cell liquid inlet and outlet device 200, and an optical imaging unit 32, wherein the sample processing unit 31 is connected to the flow cell liquid inlet and outlet device 200, and is used to add samples into the flow cell liquid inlet and outlet device 200, and the flow cell liquid inlet and outlet device 200 is located in the imaging area of ​​the optical imaging unit 32, so that the optical imaging unit 32 can collect images of the flow path 14 in the flow cell liquid inlet and outlet device 200.

[0127] The sample processing unit 31 is used to pre-process the sample, 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 to independently store 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.

[0128] Please refer to 7A to 8B In this embodiment, the sample to be tested is a nucleic acid molecule, mainly including two types, one is a first sample a, and the other is a second sample used to form a microdroplet b, wherein the multiple reagents can be mineral oil, dNTP, fluorescently labeled molecular probes, deoxyribonucleic acid oligomers, enzymes, phosphate buffers and biological enzymes, etc. The above 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. Other reagents are independently packaged in the above independent slots. The storage temperature of the sample storage device 33 is relatively low, at about 4°C.

[0129] See also Fig.10 In this embodiment, the sample distribution device 34 includes a first sample distribution device 341 and a second sample distribution device 342. The first sample distribution device 341 is connected to the first joint device 21, and is used to inject the first sample a and the corresponding detection reagent into the flow path 14 through the first joint device 21 and the first opening 15. After the first sample a enters the flow path 14, the nanospheres of the first sample a are adsorbed on the detection surface 17. During the reaction process, the required detection reagent stored in the sample storage device 33 is injected into the flow path 14 through the first opening 15 each time through the first sample distribution device 341. The second sample distribution device 342 is connected to the second joint device 22, and is used to mix the second sample with the corresponding reagent and form the mixed liquid into micro droplets b, and then inject it into the flow path 14 through the second joint device 22 and the second opening 16.

[0130] See also Fig.10 In this embodiment, the second sample dispensing device 342 further includes a micro-droplet generating device 37, which is used to generate micro-droplets b from the mixed liquid formed by the second sample and the corresponding reagent, and specifically can be a microfluidic droplet generator or an oscillating emulsifier, etc.

[0131] Please refer to FIG. 11A to FIG. 12B The optical imaging unit 32 has two modes. One mode is to use two lens assemblies to respectively image the nucleic acid molecule array on the detection surface 17 and the droplet array in the flow path 14. The other mode is to use one lens assembly and add a lens with adjustable imaging focal plane and field of view to the lens assembly to achieve the purpose of imaging the nucleic acid molecule array on the detection surface 17 and the droplet array in the flow path 14.

[0132] See also Fig.11A and Fig. 11B In the first mode, the optical imaging unit 32 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 322 is used for imaging the micro-droplet b.

[0133] In this embodiment, 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 the field of view diameter of the second lens assembly 322. Specifically, the focal length of the first lens assembly 321 is 1-2 μm, and the field of view diameter is about 1-2 mm; the focal length of the second lens assembly 322 is about 1-2 mm, and the field of view diameter ranges from 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 clearly image the first sample a of the nanosphere structure.

[0134] Accordingly, the image data collected by the first lens assembly 321 and the second lens assembly 322 are stored in their respective planned storage paths. These image data will be used for analysis of specific applications, including early screening or diagnosis of tumors or cancers, detection and analysis of infectious disease pathogens, etc.

[0135] See also Fig. 12A and Fig. 12B In another mode, the optical imaging unit includes a third lens assembly 323, wherein the third lens assembly 323 includes a lens with a fixed focal length, and is used to image the first sample a of nanometer size. When it is necessary to image the micro droplet b of micrometer size, it is achieved by adding a compensation mirror assembly 324 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.

[0136] Please refer to Fig.13 a to Fig.13 d, is an image of an image of a micro-droplet of uneven size (micro-droplet b) in the flow path 14 and a statistical analysis image. The second lens assembly 322 is used to collect images of the optical imaging area in the flow path 14, and the test conditions are: nucleic acid branches labeled with fluorescent dye Cy5, wherein the Cy5 concentration is about 7mM, and oil-in-water micro-droplets are prepared by the oscillation emulsification method after dilution 500 times; the excitation light is a green laser with a wavelength of 532nm and a power of 2-5W, and the focal plane is fixed at 1.5μm; MATLAB performs image statistical analysis on the diameter and number of micro-droplets.

[0137] The results are as follows: Fig.13 a is a top view of the flow cell, which is a thumbnail composed of 60 consecutively taken microscopic fluorescence images with connected fields of view, and shows microdroplets (microdroplets b) with uneven size distribution in the flow path 14. Fig.13 b shows that the detection surface 17 at the edge of the flow path 14 has densely distributed small droplets (first sample a). Fig.13 Some large droplets in flow path 14 in c are not included in the statistics. Fig.13 b Analysis shows that image stitching may affect the droplet roundness difference, thus affecting the statistical accuracy. Fig.13 d is a statistical result diagram of the fluorescence intensity and radius of the micro-droplet b in the flow path 14, from which it can be seen that the two are basically normally distributed.

[0138] Please refer to Fig.14 , which is an image of a uniform-sized micro-droplet (micro-droplet b) in the flow path 14. The second lens assembly 322 is used to collect images of the optical imaging area in the flow path 14, and it can be seen that the micro-droplets b in the flow path 14 are arranged in an array.

[0139] Please refer to Fig.15, which is an image formed by the optical signal of the first sample a (including the nucleic acid library for sequence determination or the specifically entangled DNA nanospheres) collected by the first lens assembly 321. From the figure, it can be seen that the first sample a (DNA nanospheres, with a diameter of about 200 nm) is arranged in an array with a clear outline of the fluorescent image.

[0140] In this embodiment, the sample analysis system further includes a cleaning unit 38 , which is in communication with the flow path 14 and is used to clean the system pipeline and the flow path 14 .

[0141] See also Fig.10 , combined with reference Fig. 9 In actual application, the flow cell 100 is installed on a mounting table (not shown), the first opening 15 of the flow cell 100 is sealed and connected to the first engaging device 21 through the first sealing component 213, and the second opening 16 on the flow cell 100 is sealed and connected to the second engaging device 22 through the second sealing component 223. The first engaging device 21 and the second engaging device 22 are connected to the sample distribution device 34 through a pipeline, and the sample is sucked by the upstream pump 35 (such as a syringe pump). The first engaging device 21 and the second engaging device 22 are connected to the cleaning unit 38 through a pipeline, and the cleaning agent is injected and the waste liquid is discharged by the downstream pump 36 (such as a syringe pump). The sample distribution device 34, the upstream pump 35, the first engaging device 21, the second engaging device 22 and the downstream pump 36 can all be connected to the control device (not shown), and are started or turned on by the control device.

[0142] Please refer to FIG. 7A to FIG. 10 The method of performing sample analysis using the sample analysis system 300 comprises the following steps:

[0143] Step 1: prepare two samples, namely the first sample a and the second sample.

[0144] In this embodiment, the first sample a is a nanosphere having nanometer-sized characteristics. The second sample is used to generate micro droplets b having micrometer-sized characteristics.

[0145] In this embodiment, the first sample a, the second sample and other required reagents are 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, and 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 above independent slots. The storage temperature of the sample storage device 33 is relatively low, at about 4°C. The various reagents can be mineral oil, dNTP, fluorescently labeled molecular probes, deoxyribonucleic acid oligomers, enzymes, phosphate buffers and biological enzymes, etc.

[0146] Step 2: inject the first sample a into the flow path 14 .

[0147] The sealing membrane of the sealed tube containing the first sample a is pierced, and the first sample a is extracted from the sealed tube in the sample storage device 33 by pressure-driven means. The first sample a is pumped into the flow path 14 through the first coupling device 21 by the first sample distribution device 341, and the first sample a is adsorbed on the detection surface 17 and arranged in an array.

[0148] In this embodiment, the first sample a is pumped into the first bonding device 21 by suction of the upstream pump 35 .

[0149] Step three, let it stand for a period of time (eg, 30 minutes), and the nucleic acid nanospheres (ie, the first sample a) are fully adsorbed on the detection surface 17 due to the interaction between the active groups, forming a relatively regularly arranged nano nucleic acid molecule array.

[0150] Step 4: Detect and analyze the first sample a. Specifically, this embodiment is to sequence the nucleic acid molecule. The sequencing method includes:

[0151] First, the first fluorescent substance is pumped into the flow path 14 under pressure, and the first fluorescent substance is combined with the first sample a by repeated suction. After the reaction is completed, the remaining first fluorescent substance is discharged from the flow path 14 through the second opening 16. Similarly, other fluorescent substances are combined with the first sample a. Every time a fluorescent substance is added, the first sample a combined with the fluorescent substance in the nanosphere array will emit a fluorescent signal.

[0152] Then, the fluorescence signal generated by each cycle reaction is imaged and analyzed to obtain the sequence information of the first sample a.

[0153] Specifically, by driving by pressure, the fluorescent material put into the flow path 14 is repeatedly and cyclically extracted to realize the combination of the nucleic acid molecules in the array and the fluorescent material. The fluorescent signals generated by each cycle reaction are imaged and analyzed to obtain the arrangement order of a segment of bases in the first sample a, until the base sequence analysis of the required length is completed. This method can detect the sequence information of unknown nucleic acid molecules and achieve the purpose of sequencing.

[0154] In this embodiment, imaging analysis is achieved by an optical imaging unit 32. Specifically, a high-magnification first lens assembly 321 is used to achieve imaging analysis of nucleic acid nanospheres. The objective lens and lens combination configured therein have a small focal length and field of view diameter, with a focal length of about 1-2 μm and a field of view diameter of about 1-2 mm. Fig.15 Shown is a fluorescent image of a DNA nanosphere (about 200 nm in diameter).

[0155] Step 5: Form the second sample into micro-droplets b, and inject the micro-droplets b into the flow path 14 .

[0156] The sealing film of the sealed tube containing the second sample is pierced, and the second sample is extracted from the sealed tube in the sample storage device 33 by pressure-driven method, and mixed with other reagents in the sample storage device 33 to form a mixed liquid. The mixed liquid containing the second sample is located in the second sample distribution device 342. Under pressure-driven, the mixed liquid containing the second sample is injected into the micro-droplet generating device 37 through the second sample distribution device 342. The second sample generates micro-droplets b in the micro-droplet generating device 37 (such as a microfluidic droplet generator or an oscillating emulsifier). The micro-droplets b are then injected into the flow path 14 through the second bonding device 22, and left to stand for a period of time. A large number of droplets are stably suspended in the flow path 14 to form an array of droplet arrays arranged in an array.

[0157] In this embodiment, the second sample and other reagents are mixed evenly by suction of the upstream pump 35 , the mixed solution is pumped into the micro-droplet generating device 37 by the upstream pump 35 , and then the micro-droplets b are injected into the flow path 14 through the second bonding device 22 by the upstream pump 35 .

[0158] In this embodiment, the microdroplet b is a nucleic acid molecule coating and copolymer (eg, nucleic acid microdroplet) having micron-sized characteristics, wherein the microdroplet b contains raw materials required for nucleic acid molecule amplification.

[0159] Step 6: Analyze the second sample. The droplet array will undergo multiple cycles of amplification reactions in the flow path 14. The product obtained from each cycle of amplification reactions is used to combine with the corresponding fluorescent substance to generate a fluorescent signal. The fluorescent signal generated by each cycle of amplification reactions is imaged and analyzed to obtain the sequence information of the second sample, or the content of a certain base in the second sample with a known sequence.

[0160] Specifically, after standing for a while, when the microdroplet (microdroplet b) is suspended stably and remains relatively still, the circulation convection flow path 14 is incubated at a specific temperature or a specific temperature gradient, so that the nucleic acid molecules in the microdroplet are combined with the fluorescent substance, and after a specific reaction time, the second lens assembly 322 is used to image and count the fluorescence signals of the microdroplet to analyze the flow pool, and the content of a certain base in the second sample nucleic acid molecule can be obtained. This method can achieve qualitative and quantitative analysis of nucleic acid molecules with known sequences.

[0161] In the cleaning step, the cleaning unit 38 is started, and a specific cleaning reagent (such as Tween-20, sodium hydroxide solution) and water are extracted through the cleaning unit 38 to clean the system pipeline. After cleaning, the system can be used for the next detection and analysis. The cleaning step can be performed during the detection of the first sample a, and the system management and the flow path 14 need to be cleaned between adding two different fluorescent substances. In addition, after the detection of the first sample a, if the detection of the micro droplet b is required, the system pipeline and the flow path 14 need to be cleaned.

[0162] In the above steps, the sequence analysis of the nano nucleic acid molecule array (first sample a) is realized through steps 1 to 4, and the detection and analysis of the microdroplet array (microdroplet b) encapsulating the nucleic acid molecules is realized through steps 5 and 6. The two analysis stages are relatively independent. A complete system working logic should include steps 1 to 4, or steps 5 and 6, or steps consistent with step 6, wherein the cleaning step is carried out according to the actual situation as described above.

[0163] The sample analysis system 300 provided in the embodiment of the present application combines gene sequencing technology and droplet PCR technology, and can realize the analysis of a large number of diseases caused by known and unknown pathogens, with high cost performance and high detection efficiency; it realizes the integration of multiple functions, and the operation process is simple, does not require a large amount of nucleic acid samples, 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 demand for laboratory infrastructure such as power supply, desks, computing and storage resources, and supporting or software requirements for equipment management are relatively low.

[0164] The flow cell of the present application constructs flow paths and detection surfaces in the flow path, so that different sample arrays can be arranged in the same flow path system, thereby significantly increasing the types of identifiable contents in the flow cell. Taking the detection and analysis of pathogens or infectious viruses as an example, the present application can be used for the detection and analysis of known and unknown types of pathogens or infectious viruses, while taking into account the cost-effectiveness of both types of analysis.

[0165] 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 sizes. The detection content is richer, and it can meet the detection of low demands as well as high demands. At the same time, it can improve the analysis performance. Taking the droplet PCR application as an example, the present application can significantly increase the number of droplets analyzed and thus improve the detection dynamic range of the target concentration.

[0166] The flow cell of the present application can be reused, thereby improving the utilization rate of the flow cell and reducing the cost of the flow cell.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application and are not intended to limit it. Although the present application has been described in detail with reference to the preferred embodiments, a person of ordinary skill in the art should understand that the technical solution of the present application may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present application.

Claims

1. A sample analysis system, characterized in that: include: A flow cell liquid inlet and outlet device, the flow cell liquid inlet and outlet device comprising: a flow cell, a first coupling device and a second coupling device, the flow cell comprising: first cover sheet; a second cover sheet, arranged opposite to the first cover sheet; At least two spacer layers are arranged in parallel between the first cover sheet and the second cover sheet, two sides of each spacer layer are in contact with the first cover sheet and the second cover sheet respectively, a flow path is formed between the first cover sheet, the second cover sheet and two adjacent spacer layers, and a detection surface is formed on the surface of the first cover sheet close to the second cover sheet; A first opening is provided at one end of the flow path, the first opening is used to inject a nanometer-scale first sample 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 to inject micron-sized droplets into the flow path, wherein the droplets contain a second sample. The hydraulic radius of the first opening is smaller than the hydraulic radius of the second opening, the first engaging device is in communication with the first opening, and the second engaging device is in communication with the second opening; a first sample dispensing device, connected to the first engaging device, and used to inject a first sample into the flow cell through the first engaging device and the first opening; A second sample dispensing device, connected to the second engaging device, the second sample dispensing device is used to form a plurality of micro-droplets from the second sample, and inject the plurality of micro-droplets into the flow pool through the second engaging device and the second opening, wherein the plurality of micro-droplets are arranged in an array in the flow path; and An optical imaging unit is used to acquire an image of the flow cell.

2. The sample analysis system according to claim 1, characterized in that: The detection surface is also arranged on a surface of the second cover sheet close to the first cover sheet.

3. The sample analysis system according to claim 1, wherein: The hydraulic radius of the first opening is 0.075 mm-0.75 mm.

4. The sample analysis system according to claim 1, wherein: The hydraulic radius of the second opening is 0.02 mm-0.2 mm.

5. The sample analysis system according to claim 1, wherein: The spacer layer is an adhesive layer.

6. The sample analysis system according to claim 1, wherein: The first opening and the second opening are both arranged on the first cover sheet or the second cover sheet.

7. The sample analysis system according to claim 1, wherein: The second joining device includes a second joining portion, the second joining portion includes a first joining port and a second joining port, the second joining port is communicated with the second opening, the first joining port is arranged away from the second opening, and the dimension of the first joining port along the extension direction perpendicular to the flow path is smaller than the dimension of the second joining port along the extension direction perpendicular to the flow path.

8. The sample analysis system according to claim 7, characterized in that: The second joint device further comprises a second interface block and a second sealing assembly, wherein the second interface block is communicated with the first joint port, and the second sealing assembly is sleeved on the second joint portion.

9. The sample analysis system according to claim 8, characterized in that: 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 is communicated with the first joint.

10. The sample analysis system according to claim 9, characterized in that: The hydraulic radius of the second liquid inlet channel and the second common channel are both greater than the hydraulic radius of the second liquid outlet channel.

11. The sample analysis system according to claim 7, wherein: 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 is communicated with the first opening, and the other end is communicated with the first interface block. The first sealing assembly is sleeved on the first engaging portion.

12. The sample analysis system according to claim 11, characterized in that: 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 is in communication with the first joint portion.

13. The sample analysis system according to claim 1, wherein: The second sample dispensing device includes a micro-droplet generating device.

14. The sample analysis system according to claim 1, wherein: 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 field of view diameter of the first lens assembly is smaller than the field of view diameter of the second lens assembly.

15. The sample analysis system according to claim 14, characterized in that: The focal length of the first lens assembly is 1-2µm, and the field of view diameter is 1-2 mm; the focal length of the second lens assembly is 1-2 mm, and the field of view diameter ranges from 5-10 mm.

16. The sample analysis system according to claim 14, characterized in that: The magnification of the first lens assembly is greater than the magnification of the second lens assembly.

17. The sample analysis system according to claim 1, wherein: The optical imaging unit includes a third lens assembly and a compensating mirror assembly.

18. The sample analysis system according to claim 1, wherein: The sample analysis system further includes a cleaning unit, which is communicated with the flow pool.

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