Separation device and biological sample preparation system

Abstract

A separation device and a biological sample preparation system for separating a dispersed phase from a two-phase mixture after demulsification of a microdroplet. The separation device includes a substrate and a porous adsorption layer. The substrate includes a first surface and a second surface disposed opposite each other. The first surface has a plurality of well sites recessed therein, the well sites forming openings in the first surface configured to receive the two-phase mixture into the well sites. The porous adsorption layer is disposed within the well sites. The porous adsorption layer is disposed along an axis of the well sites from the first surface. The porous adsorption layer is configured to selectively adsorb a continuous phase of the two-phase mixture to leave the dispersed phase above the porous adsorption layer.

Description

Technical Field

[0001] This application relates to the field of single-cell sorting, and more particularly to a separation device and a biological sample preparation system having the separation device. Background Technology

[0002] Single-cell sorting is a technology that has gradually emerged in recent years. By confining cells within a single droplet, it can effectively increase the efficiency of biochemical reactions and has important application prospects in single-cell sequencing, antibody synthesis, and the design of targeted drugs.

[0003] In related technologies, droplet microfluidics is commonly used for single-cell sorting. Droplet microfluidics is a technique developed in recent years based on biological sample preparation technology to manipulate tiny volumes of liquid. In droplet microfluidics, the formation of microdroplets involves two immiscible liquids, one as the continuous phase (e.g., oil) and the other as the dispersed phase (e.g., water). Under the influence of shear forces, interfacial tension, and viscous forces, the water phase is broken into tiny droplets and dispersed in the oil phase, forming an emulsion containing microdroplets. Then, the microdroplets need to be demulsified to remove the encapsulated oil phase and obtain single-cell microspheres for subsequent biochemical reactions.

[0004] However, after the microdroplets break the emulsion, the oil phase and the aqueous phase coexist. The two phases cannot be separated well by aspirating the aqueous phase with the instrument. The residual oil phase will affect the subsequent biochemical reactions and reduce the accuracy of biochemical detection. Utility Model Content

[0005] In view of this, it is necessary to provide a separation device for effectively separating the dispersed phase and the continuous phase after microdroplet demulsification. Furthermore, it is also necessary to provide a biological sample preparation system incorporating this separation device.

[0006] This application provides a separation apparatus for separating a dispersed phase from a two-phase mixture after microdroplet demulsification. The separation apparatus includes a substrate and a porous adsorption layer. The substrate includes a first surface and a second surface disposed opposite each other. A plurality of pores are recessed on the first surface, the pores forming openings on the first surface, the openings being configured to allow the two-phase mixture to be injected into the pores. The porous adsorption layer is disposed within the pores. Along the axial direction of the pores, the porous adsorption layer is spaced apart from the first surface. The porous adsorption layer is configured to selectively adsorb the continuous phase in the two-phase mixture, so that the dispersed phase remains above the porous adsorption layer.

[0007] Based on the first aspect, in some possible implementations, the porous adsorption layer includes an oleophilic and hydrophobic base layer connected to the inner wall of the pore sites and a micropore array penetrating the base layer along the axial direction.

[0008] Based on the first aspect, in some possible implementations, the base layer is a polyethylene layer, a polypropylene layer, or a polytetrafluoroethylene layer with a thickness of 6 mm or more along the axial direction.

[0009] Based on the first aspect, in some possible implementations, along the axial direction of the pore, the pore includes a first segment, a second segment, and a third segment arranged sequentially, with the first segment being closer to the first surface than the third segment. The porous adsorption layer is disposed in the second segment.

[0010] Based on the first aspect, in some possible implementations, the porous adsorption layer is bonded to the inner wall of the second segment by an adhesive.

[0011] Based on the first aspect, in some possible implementations, the porous adsorption layer is disposed at the bottom of the pore location opposite to the first surface, along the axial direction of the pore location.

[0012] Based on the first aspect, in some possible implementations, the pore size of the micropore array does not exceed 20 μm.

[0013] Based on the first aspect, in some possible implementations, the hydrophobicity of the substrate is less than that of the porous adsorption layer.

[0014] Based on the first aspect, in some possible implementations, the plurality of holes are arranged in an array on the first surface.

[0015] A second aspect of this application provides a biological sample preparation system, including a microdroplet generating device for preparing microdroplets. The biological sample preparation system further includes a separation device as described above, the separation device being used to separate the dispersed phase from the two-phase mixture after the microdroplets have demulsified.

[0016] The separation device of this application separates the demulsification products of microdroplets from the continuous phase by setting a porous adsorption layer in the pores. This reduces the risk of residual continuous phase affecting subsequent biochemical reactions and improves the accuracy of biochemical detection. Moreover, this separation device can reduce the difficulty of separating the continuous and dispersed phases, improve the recovery rate of the dispersed phase, and facilitate the automation of complex single-cell transcriptome library assembly. Attached Figure Description

[0017] Figure 1 This is a modular architecture diagram of a biological sample preparation system provided in one embodiment of this application.

[0018] Figure 2 for Figure 1 A top view of the separation device of the biological sample preparation system shown.

[0019] Figure 3 For some embodiments Figure 2 A cross-sectional view of the separation device shown.

[0020] Figure 4 For some other embodiments Figure 2 A cross-sectional view of the separation device shown.

[0021] Figure 5 for Figure 3 or Figure 4 A top view of the porous adsorption layer of the separation device shown.

[0022] The following detailed description, in conjunction with the accompanying drawings, will further illustrate this application. Detailed Implementation

[0023] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0024] It should be noted that when a component is considered "set on" another component, it can be directly set on the other component or may have an intervening component present; when a component is considered "installed on" another component, it can be directly installed on the other component or may have an intervening component present. The term "and / or" as used herein includes all and any combination of one or more of the related listed items.

[0025] Please see Figure 1 This application provides a biological sample preparation system 1 for sample preparation during gene sequencing. For single-cell samples, before gene sequencing, the following steps are required: First, microdroplet preparation and intradroplet reverse transcription; Second, demulsification, enrichment and purification, sorting of Oligo and cDNA, amplification, purification, and quantification of cDNA using polymerase chain reaction (PCR); Third, PCR amplification, fragment screening, cDNA fragmentation, end repair, ligation of molecular tag adapters, purification, fragment screening, PCR amplification, purification, and quantification of cDNA products; Fourth, rolling circle amplification of the library to obtain nanospheres (DNB), quantification, etc., finally obtaining the gene sequencing library.

[0026] The biosample preparation system 1 includes a microdroplet generating device 200. The microdroplet generating device 200 is used to prepare single-cell suspensions into microdroplets, i.e., to perform the first step of microdroplet preparation. Specifically, cell fluid (such as cell nuclei or protoplasts), magnetic beads, and an oil phase can be loaded into the microdroplet generating device 200. The cell fluid and magnetic beads, as dispersed phases, are broken into tiny droplets under the action of fluid shear force, interfacial tension, viscosity, etc., and dispersed in the oil phase to form an emulsion containing microdroplets. The oil phase, as a continuous phase, serves as a separator. In some embodiments, the microdroplet generating device 200 can be a microfluidic chip.

[0027] The biosample preparation system 1 may also include a biochemical reaction apparatus 300. The biochemical reaction apparatus 300 comprises multiple different modules, dividing the entire gene sequencing sample preparation process into several different sub-processes. Each module of the biochemical reaction apparatus 300 can execute one or more of these sub-processes. For example, the biochemical reaction apparatus 300 may specifically include a PCR instrument, a purification and sorting device, and a temperature-controlled shaking device. The PCR instrument can be used to perform in-droplet reverse transcription (RT) reactions, PCR amplification, and other reaction processes. The purification and sorting device can be used to perform enrichment, purification, and sorting processes. The temperature-controlled shaking device can be used to perform a shaking incubation process; "incubation" refers to creating the necessary temperature conditions for the biochemical reaction of the biological sample through heating, cooling, or insulation.

[0028] Each microdroplet can encapsulate multiple cell beads and index carriers. The cell beads are linked to polyT primers, which are released within the microdroplet. The index carriers are linked to barcode primers, which can be captured by the cell beads. After the RT reaction, the polyT primers released from the cell beads polymerize with mRNA, synthesizing a single-stranded cDNA under the action of RT enzymes. Additionally, the sequence of the index carrier is also captured by the cell beads, generating an oligo library. Next, the microdroplets need to be demulsified, for example by adding PFO. Then, the dispersed phase is recovered, and the cell beads and index carriers are removed from the dispersed phase.

[0029] The biological sample preparation system 1 also includes a separation device 100. The separation device 100 is used to separate the dispersed phase from the two-phase mixture after the microdroplets have broken down.

[0030] Please see Figure 2 and Figure 3The separation device 100 includes a substrate 10, which has a first surface 11 and a second surface 12 disposed opposite to each other. A plurality of holes 20 are recessed on the first surface 11. Each hole 20 has an opening 110 formed on the first surface 11, allowing a pipetting component 40 (such as a pipette tip) to inject a two-phase mixture into the hole 20 through the opening 110. For example... Figure 3 As shown, when the pipetting component 40 picks up the two-phase mixture, the oil phase may be located on the lower side of the pipetting component 40, and the aqueous phase may be located on the upper side of the pipetting component 40. In some embodiments, the substrate 10 may be made of plastics such as polypropylene (PP) or polystyrene (PS), and the substrate 10 may be integrally formed by injection molding.

[0031] The term "hole" should be understood to mean that the substrate 10 itself has these holes, or it can mean that the substrate 10 has test tubes or containers inside, which may be in the form of cup-shaped parts, hole-shaped parts, hollow parts or similar structures and are used to receive liquids.

[0032] The holes may have a circular, hexagonal, square, or other geometric cross-section. The bottom or base of each hole may be flat, U-shaped, V-shaped, frustum-shaped, or any other suitable shape.

[0033] The separation device 100 further includes a porous adsorption layer 30 disposed within the aperture 20. The porous adsorption layer 30 is positioned at a distance from the first surface 11 along the axial direction of the aperture 20. In some embodiments, the aperture 20 includes a first segment 21, a second segment 22, and a third segment 23 sequentially disposed. The first segment 21 is closer to the first surface 11 of the substrate 10 than the third segment 23, and the third segment 23 is closer to the bottom of the aperture 20 than the first segment 21. The first segment 21 may be the upper segment of the aperture 20, the second segment 22 may be the middle segment of the aperture 20, and the third segment 23 may be the lower segment of the aperture 20. Figure 3 As shown, the porous adsorption layer 30 is disposed in the second segment 22. Preferably, the porous adsorption layer 30 fills the entire second segment 22 and is not only disposed on the inner wall of the second segment 22. The first segment 21 is located above the porous adsorption layer 30, and the third segment 23 is located below the porous adsorption layer 30.

[0034] like Figure 4 As shown, in some other embodiments, the porous adsorption layer 30 may also be disposed at the bottom of the pore position 20, such as in the third segment 23.

[0035] Combined with reference Figure 5The porous adsorption layer 30 may include a base layer 31 connected to the inner wall of the pore sites 20 and a micropore array 32 penetrating the base layer 31 along the axial direction of the pore sites 20. The pore size of the micropore array 32 may be micro- or nano-scale; specifically, the pore size (diameter) of the micropore array 32 may be set to no more than 20 μm, preferably 6-15 μm. Therefore, the porous adsorption layer 30 can selectively adsorb the continuous phase in a two-phase mixture, allowing the dispersed phase to remain above the porous adsorption layer 30. In some embodiments, when the continuous phase is an oil phase and the dispersed phase is an aqueous phase, the base layer 31 of the porous adsorption layer 30 may be made of an oleophilic and hydrophobic material, such as a polyethylene (PE) layer, a polypropylene (PP) layer, or a polytetrafluoroethylene (PTFE) layer, or other polymer layers.

[0036] When the two-phase mixture is injected into the pore 20 of the separation device 100 via the pipetting component 40 and allowed to stand for a period of time, the continuous phase in the mixture will be absorbed by the porous adsorption layer 30, while the dispersed phase will remain on the upper part of the porous adsorption layer 30. In particular, when the porous adsorption layer 30 is located in the second section 22 of the pore 20, after the continuous phase in the mixture is absorbed by the porous adsorption layer 30, the excess continuous phase will permeate to the third section 23 of the pore 20 due to its own gravity. At this time, a new pipetting component 40 is used to remove the dispersed phase located above the porous adsorption layer 30, thus completing the separation of the dispersed phase and the continuous phase.

[0037] Furthermore, when the porous adsorption layer 30 is disposed in the second section 22, the porous adsorption layer 30 can be fixed to the inner wall of the second section 22 by adhesive bonding, thereby improving the stability of the porous adsorption layer 30 and reducing the risk that the porous adsorption layer 30 will fall to the bottom of the pore position 20 during transportation or after adsorbing the continuous phase, thus increasing the difficulty of subsequent dispersion phase separation. Furthermore, the adhesive used to bond the porous adsorption layer 30 can also be made of oleophilic and hydrophobic materials, such as polyethylene (PE), polypropylene (PP), or polytetrafluoroethylene (PTFE) and other polymers, thereby avoiding the problem of low dispersed phase recovery efficiency caused by the adhesive adsorbing the dispersed phase.

[0038] In some embodiments, the hydrophobicity of the substrate 10 is less than that of the porous adsorption layer 30, which is more conducive to the aqueous phase of the two-phase mixture loaded into the pore 20 being fully spread in the pore 20 (such as the first segment 21) between the porous adsorption layer 30 and the first surface 11, so that the subsequent pipetting component 40 can pick up the aqueous phase located above the porous adsorption layer 30.

[0039] In some embodiments, the thickness of the base layer 31 of the porous adsorption layer 30 along the axial direction of the pore site 20 is at least 6 mm. This allows the porous adsorption layer 30 to fully adsorb the continuous phase, especially when the porous adsorption layer 30 is disposed at the bottom of the pore site 20, the thickness of the porous adsorption layer 30 is more conducive to achieving the separation of the dispersed phase and the continuous phase.

[0040] like Figure 2 As shown, in some embodiments, a plurality of pores 20 may be arranged in an array on the first surface 11. The arrangement of a plurality of pores 20 is beneficial to improving the separation efficiency of the separation device 100, thereby improving the efficiency of biological sample preparation.

[0041] Therefore, the separation device 100 of this application, by setting a porous adsorption layer 30 in the pore positions 20, achieves the separation of the demulsification product of microdroplets from the continuous phase, thereby reducing the risk of residual continuous phase affecting subsequent biochemical reactions and improving the accuracy of biochemical detection. Moreover, the separation device 100 can also reduce the difficulty of separating the continuous phase and the dispersed phase, improve the recovery rate of the dispersed phase, and facilitate the automation of complex single-cell transcription library assembly.

[0042] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any way. Although the preferred embodiment has been disclosed above, it is not intended to limit this application. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of this application. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the scope of the technical solution of this application.

Claims

1. A separation apparatus for separating a dispersed phase from a two-phase mixture after microdroplet demulsification, characterized in that, The separation device includes: A substrate, the substrate including a first surface and a second surface disposed opposite to each other, a plurality of holes being recessed on the first surface, the holes forming openings on the first surface, the openings being configured to allow the two-phase mixture to be injected into the holes; and A porous adsorption layer is disposed within the pores along the axial direction of the pores. The porous adsorption layer is positioned at a distance from the first surface. The porous adsorption layer is configured to selectively adsorb the continuous phase in the two-phase mixture, so that the dispersed phase remains above the porous adsorption layer.

2. The separation device as described in claim 1, characterized in that, The porous adsorption layer includes an oleophilic and hydrophobic base layer connected to the inner wall of the pores and a micropore array penetrating the base layer along the axial direction.

3. The separation device as described in claim 2, characterized in that, The base layer is a polyethylene layer, a polypropylene layer, or a polytetrafluoroethylene layer with a thickness of 6 mm or more along the axial direction.

4. The separation device as described in claim 1, characterized in that, Along the axial direction of the pore, the pore includes a first segment, a second segment, and a third segment arranged sequentially. The first segment is closer to the first surface than the third segment, and the porous adsorption layer is disposed in the second segment.

5. The separation device as described in claim 4, characterized in that, The porous adsorption layer is bonded to the inner wall of the second section by an adhesive.

6. The separation device as described in claim 1, characterized in that, Along the axial direction of the pore, the porous adsorption layer is disposed at the bottom of the pore opposite to the first surface.

7. The separation device as described in claim 2, characterized in that, The diameter of the micropore array does not exceed 20 μm.

8. The separation device as described in claim 1, characterized in that, The hydrophobicity of the substrate is less than that of the porous adsorption layer.

9. The separation device as described in claim 1, characterized in that, The plurality of holes are arranged in an array on the first surface.

10. A biological sample preparation system, comprising a microdroplet generation device for preparing microdroplets, characterized in that, The biological sample preparation system further includes a separation device as described in any one of claims 1 to 9, the separation device being used to separate the dispersed phase from the two-phase mixture after the microdroplets have demulsified.

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