Chip flow channel structure, nanopore sequencing device and film forming method

Abstract

The present application provides a chip flow channel structure, a nanopore sequencing device and a film forming method. Along the flowing direction of the introduced fluid, the chip flow channel structure includes: a first transition region, a chip inlet region, a chip region, a chip outlet region and a second transition region arranged in sequence; the first transition region is arranged at an angle with the chip inlet region, and the second transition region is arranged at an angle with the chip outlet region; the chip region is formed with sunken sequencing holes; the chip inlet region and the chip outlet region respectively form an angle greater than 90° with the chip region. The present invention can ensure that during the replacement film forming process, the interface of the introduced fluid sweeps across the sequencing holes in the chip region translationally to achieve effective replacement, thereby ensuring the consistency of the film forming positions in each sequencing hole, avoiding film forming differences caused by different flow sequences and flow rates when the introduced fluid flows through each sequencing hole, and further enabling uniform flow and effective replacement of multiphase fluids within a relatively wide flow rate range.

Description

Technical Field

[0001] The present application relates to the field of biological detection technologies, and in particular, to a chip flow channel structure, a nanopore sequencing device, and a film forming method. Background Art

[0002] Nanopore detection technology has advantages such as single-molecule resolution, high throughput, parallelization, diversification, and automation, and has wide applications in fields such as protein detection, gene sequencing, and nanoparticle characterization. For example, when performing gene sequencing, nanopores are embedded in the thin film of the sequencing unit of a gene sequencing device. The nanopore is the only communication channel between electrolyte solutions (polar solvents) located on both sides of the thin film separated by the thin film. A potential difference is applied across the thin film so that an ionic current flows through the nanopore. When a single-stranded deoxyribonucleic acid molecule passes through the nanopore, the effective cross-sectional area of the nanopore is reduced due to the base blocking the nanopore, resulting in a decrease in the ionic current. Or, due to differences in the interactions between different bases and the nanopore, specific reactions occur, causing changes in the resistance of the nanopore, and further causing changes in the current flowing through the nanopore. By detecting and analyzing the current changes, the base sequence can be inferred.

[0003] The nanopore sequencing chip includes a pore array, and a single pore in the array is an independent sequencing unit. The arrangement quantity and preparation success rate of the thin films formed in the pore array on a single nanopore sequencing chip are crucial for the detection throughput and accuracy. Existing replacement film forming methods include: repeatedly introducing different fluids into the flow channel 1' provided with the pore array. The fluids include an oil phase, an aqueous phase, and a gas. The fluid introduced this time (for example, the oil phase) can replace a part of the fluid introduced last time (for example, the aqueous phase) in the pore 2'. In this way, through multiple replacements, a thin film 3' as shown in Figure 1 is formed in the pore 2'.

[0004] However, when the existing flow channel 1' is replaced, as shown in Figure 2 , at the portions where the two-phase fluid interface 6' contacts the side walls of the flow channel 1' on both sides in the flow channel width direction, three-phase contact lines 7' are formed. The existing flow channel 1' has a mutation turning structure 8' as shown in Figure 3 (for example, a vertical turn). In this way, the forward slip of the three-phase contact line 7' is hindered, making it difficult for the two-phase fluid interface 6' to maintain uniform translation in the flow channel 1', resulting in an incomplete replacement situation as shown in Figure 4 in the pore 2' located in the middle part. The lower interface position of the thin film 3' is relatively high, and the formed film is unstable and prone to rupture after film formation; while the lower interface position of the thin film 3' in the pore 2' located in the two side parts is relatively low, affecting the implementation of subsequent replacement processes, and thus a film as shown in Figure 1The thin film 3' shown; in addition, the structure of the existing flow channel 1' allows a relatively small range of fluid displacement speeds. For a relatively fast displacement process (such as a flow rate greater than 1000 μL / min), the difference between the flow speeds in the middle part and the two side parts will increase significantly, and even make the oil-water two-phase fluid interface 6' present a water droplet-like shape as shown in Figure 3 , resulting in the failure of the preparation of the thin film 3'. SUMMARY OF THE INVENTION

[0005] In view of this, the purpose of the present application is to provide a chip flow channel structure, a nanopore sequencing device and a film forming method, so as to solve the problem that it is difficult to achieve uniform flow of multiphase fluids with significantly different physical properties (such as density, viscosity, polarity) on a nanopore sequencing chip, resulting in a low film forming rate.

[0006] According to a first aspect of the present invention, there is provided a chip flow channel structure, wherein, along the flow direction of the introduced substance in the chip flow channel structure, the chip flow channel structure includes:

[0007] A first transition zone, a chip inlet zone, a chip zone, a chip outlet zone and a second transition zone arranged in sequence;

[0008] The first transition zone is arranged at an angle with the chip inlet zone, and the second transition zone is arranged at an angle with the chip outlet zone;

[0009] The chip zone is formed with sunken sequencing holes;

[0010] The chip inlet zone and the chip outlet zone respectively form an angle greater than 90° with the chip zone.

[0011] Preferably, the first transition zone, the second transition zone and the chip zone are parallel to each other.

[0012] Preferably, along the first direction, the second direction and the third direction, the chip flow channel structure is an axisymmetric structure;

[0013] The first direction, the second direction and the third direction are perpendicular to each other in pairs, and the first direction is the same as the flow direction of the introduced substance.

[0014] Preferably, the range of the set angle is 110° to 160°.

[0015] Preferably, the chip zone is located at the bottom of the chip flow channel structure and is horizontally arranged;

[0016] A plurality of the sequencing holes are arranged at the bottom of the chip zone.

[0017] Preferably, a plurality of the sequencing holes are distributed in an array;

[0018] The velocity of the introduced material flowing through each of the sequencing holes in each row along the direction perpendicular to the flow direction in the array is the same.

[0019] Preferably, the introduced materials introduced twice in succession are not miscible, so that a contact part is formed between the introduced material introduced at the (N + 1)-th time and the introduced material introduced at the N-th time. The contact part translates within the chip flow channel structure, so that the contact part is formed into a plane-like shape perpendicular to the flow direction of the introduced material.

[0020] Preferably, the introduced material is a gas or the introduced material is a liquid that is a polar solvent or a non-polar solvent.

[0021] Preferably, along the flow direction of the introduced material, the first transition region includes a first straight-through region and a gradually expanding region that are sequentially connected. The gradually expanding region is formed into a flared structure communicating with the chip inlet region;

[0022] Along the flow direction of the introduced material, the second transition region includes a gradually contracting region and a second straight-through region that are sequentially connected. The gradually contracting region is formed into a constricted structure communicating with the chip outlet region.

[0023] Preferably, the gradually expanding region and the first straight-through region are coplanar, and the gradually contracting region and the second straight-through region are coplanar.

[0024] Preferably, along the flow direction of the introduced material, the first transition region includes a first straight-through region, and the chip inlet region is formed into a flared structure;

[0025] Along the flow direction of the introduced material, the second transition region includes a second straight-through region, and the chip outlet region is formed into a constricted structure.

[0026] Preferably, the chip flow channel structure further includes:

[0027] An inlet part, arranged upstream of the first transition region. The inlet part is arranged at an angle with the first transition region. The channel for the introduced material to flow from the inlet part to the chip region is formed into a stepped structure extending downward;

[0028] An outlet part, arranged downstream of the second transition region. The outlet part is arranged at an angle with the second transition region. The channel for the introduced material to flow from the chip region to the outlet part is formed into a stepped structure extending upward.

[0029] Preferably, along the flow direction of the introduced material, the starting end of the chip outlet region is formed into an outlet intersection part communicating with the chip region;

[0030] The ratio of the equivalent diameter of the outlet intersection part to the equivalent diameter of the chip region is not greater than 3;

[0031] The formula for the equivalent diameter D is as follows:

[0032]

[0033] Wherein, A is the cross-sectional area of the chip flow channel structure;

[0034] P is the cross-sectional perimeter of the chip flow channel structure;

[0035] h is the height dimension of A;

[0036] w is the width dimension of A;

[0037] The cross-section is a plane perpendicular to the flow direction of the introduced substance.

[0038] Preferably, along the flow direction of the introduced substance, the end of the chip inlet region is formed as an inlet intersection part communicating with the chip region;

[0039] The ratio of the equivalent diameter of the inlet intersection part to the equivalent diameter of the chip region is not greater than 3.

[0040] Preferably, the ratio of the equivalent diameter of the chip inlet region to the equivalent diameter of the chip region is not greater than 3.

[0041] Preferably, the ratio of the equivalent diameter of the chip outlet region to the equivalent diameter of the chip region is not greater than 3.

[0042] Preferably, along the flow direction of the introduced substance, the connection line between the end of the upper surface of the chip region and the end of the bottom wall of the chip flow channel structure on the same side forms the maximum height dimension h of the inlet intersection part max1 ; or forms the maximum height dimension h of the outlet intersection part max2 .

[0043] Preferably, along the flow direction of the introduced substance, the end of the inlet intersection part is formed as a first transition section extending vertically downward; the beginning of the outlet intersection part is formed as a second transition section extending vertically upward.

[0044] Preferably, the ratio not being greater than 3 is not achieved by increasing the equivalent diameter of the chip region.

[0045] The second aspect of the present invention provides a nanopore sequencing device, which includes the chip flow channel structure according to any one of the above technical solutions, and:

[0046] A housing assembly, including a base and an upper cover assembled with each other, and the first transition region, the chip inlet region, the chip outlet region and the second transition region are formed on the upper cover;

[0047] A sequencing chip is disposed above the base. The sequencing chip and the upper cover enclose the chip area, and the sequencing holes are disposed on the surface of the sequencing chip.

[0048] A sealing gasket is clamped between the sequencing chip and the upper cover.

[0049] Preferably, the upper cover includes a first upper cover and a second upper cover which are buckled up and down.

[0050] The first upper cover is formed with a protruding portion extending towards the second upper cover, and the second upper cover is formed with a flow channel hole corresponding to the protruding portion for the protruding portion to extend into.

[0051] A part of the bottom of the first upper cover and the top of the second upper cover enclose the first transition area and the second transition area.

[0052] The two side walls in the length direction of the protruding portion and the two side walls in the length direction of the flow channel hole respectively enclose the chip inlet area and the chip outlet area.

[0053] The sequencing chip and the bottom wall of the protruding portion enclose the chip area.

[0054] Preferably, the upper cover is formed of a transparent material.

[0055] The third aspect of the present invention provides a film forming method, wherein the film forming method is applied to the nanopore sequencing device described in any of the above technical solutions, and the film forming method includes the following steps:

[0056] Pretreatment: Coating a non-polar solvent on the side wall of the sequencing hole.

[0057] Replacement film forming: Sequentially introducing different introduced substances into the chip flow channel structure. The introduced substance introduced for the (N + 1)-th time can replace part of the introduced substance introduced for the N-th time in the sequencing hole, or can replace the introduced substance introduced for the N-th time in the sequencing hole and part of the introduced substance introduced for the (N - 1)-th time, so as to form an amphiphilic molecular film in the sequencing hole.

[0058] Preferably, the circumferential side wall of the sequencing hole is formed into a toothed structure, and the non-polar solvent in the pretreatment step is coated in the toothed structure.

[0059] Preferably, the side wall of the sequencing hole is formed into a stepped structure to divide the sequencing hole into upper and lower layers; the amphiphilic molecular film is formed between the upper and lower layers of the sequencing hole.

[0060] Preferably, the replacement film forming step includes:

[0061] S10. The introduced substance is a first polar solvent. The first polar solvent enters from the inlet part of the chip flow channel structure and flows out from the outlet part of the chip flow channel structure, so that the first polar solvent fills the sequencing pores.

[0062] S20. The introduced substance is a non-polar solvent. The non-polar solvent enters the inlet part at a first flow rate. When the non-polar solvent flows through the sequencing pores, it can displace part of the first polar solvent in the sequencing pores, and the displaced first polar solvent is discharged from the outlet part.

[0063] S30. The introduced substance is a gas. The gas enters the inlet part at a second flow rate. When the gas flows through the sequencing pores, it can displace part of the non-polar solvent in the sequencing pores, and the displaced non-polar solvent is discharged from the outlet part.

[0064] S40. The introduced substance is a second polar solvent. The second polar solvent enters from the inlet part. When the second polar solvent flows through the sequencing pores, it can displace the gas and part of the non-polar solvent in the sequencing pores, and the displaced gas and non-polar solvent are discharged from the outlet part.

[0065] Preferably, in step S20, the junction between the first polar solvent and the non-polar solvent forms the lower surface of the amphiphilic molecular film.

[0066] In step S40, the junction between the non-polar solvent and the second polar solvent forms the upper surface of the amphiphilic molecular film.

[0067] According to the chip flow channel structure, nanopore sequencing device and film-forming method of the present invention, it can ensure that during the replacement and film-forming process, the interface of the introduced substance sweeps across the sequencing pores in the chip area translationally to achieve effective replacement, thereby ensuring the consistency of the film-forming positions in each sequencing pore, avoiding film-forming differences caused by different flow sequences and flow rates when the introduced substance flows through each sequencing pore, and further enabling uniform flow and effective replacement of multiphase fluids within a relatively wide flow rate range.

[0068] To make the above objects, features and advantages of the present application more obvious and understandable, the following specifically gives preferred embodiments and, in conjunction with the accompanying drawings, makes a detailed description as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and thus should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0070] Figure 1 It is a schematic diagram of the film formation position required for detection;

[0071] Figure 2 It is a schematic diagram of the two-phase fluid interface when the fluid flows through the flow channel in the prior art;

[0072] Figure 3 It is a schematic diagram of the two-phase fluid interface when the fluid flows through the sudden turning structure in the prior art;

[0073] Figure 4 It is a schematic diagram of the high position of the lower interface of the film caused by the ineffective replacement of the water phase by the oil phase in the prior art;

[0074] Figure 5 It is a top view schematic diagram when the first straight-through area and the gradually expanding area are coplanarly arranged in the chip flow channel structure provided in Embodiment 1 of the present invention;

[0075] Figure 6 It is Figure 5 The structural schematic diagram cut along the plane formed by A-A in;

[0076] Figure 7 It is a flowchart of the non-polar solvent replacing the polar solvent process in the chip flow channel structure provided in Embodiment 1 of the present invention;

[0077] Figure 8 It is a structural schematic diagram when the first straight-through area and the gradually expanding area are arranged at an angle in the chip flow channel structure provided in Embodiment 1 of the present invention;

[0078] Figure 9 It is Figure 8 The structural schematic diagram in the top view state;

[0079] Figure 10 It is Figure 9 The structural schematic diagram cut along the plane formed by B-B in;

[0080] Figure 11 It is a fluid simulation schematic diagram of the chip flow channel structure provided by the present invention;

[0081] Figure 12 It is the appearance of Figure 11 The flowchart of the gas replacing the non-polar solvent process in the case;

[0082] Figure 13 is Figure 12 The schematic cross-sectional view of the C-C formed surface in process e;

[0083] Figure 14 is Figure 12 The schematic cross-sectional view of the D-D formed surface in process f;

[0084] Figure 15 is Figure 12 The schematic cross-sectional view of the E-E formed surface in process g;

[0085] Figure 16 The top view schematic diagram of the chip flow channel structure provided by the second embodiment of the present invention;

[0086] Figure 17 is Figure 16 The schematic cross-sectional view of the F-F formed surface in;

[0087] Figure 18 The schematic diagram of the structure of the outlet intersection part in the chip flow channel structure provided by the second embodiment of the present invention;

[0088] Figure 19 is Figure 18 The schematic diagram of the position of the maximum height dimension of the outlet intersection part in;

[0089] Figure 20 The schematic diagram of the position of the maximum height dimension of the inlet intersection part in the chip flow channel structure provided by the second embodiment of the present invention;

[0090] Figure 21 The flow chart of the process of gas replacing non-polar solvent in the chip flow channel structure provided by the second embodiment of the present invention;

[0091] Figure 22 The schematic diagram of the structure of the chip flow channel structure provided by the third embodiment of the present invention;

[0092] Figure 23 is Figure 22 The schematic cross-sectional view of the G-G formed surface in;

[0093] Figure 24 The schematic diagram of the structure of the outlet intersection part in the chip flow channel structure provided by the third embodiment of the present invention;

[0094] Figure 25 is Figure 24 The schematic diagram of the position of the maximum height dimension of the outlet intersection part in;

[0095] Figure 26 The schematic diagram of the position of the maximum height dimension of the inlet intersection part in the chip flow channel structure provided by the third embodiment of the present invention;

[0096] Figure 27 It is a schematic structural diagram of increasing the equivalent ratio diameter of the chip area by increasing the height dimension of the chip area;

[0097] Figure 28 It is a schematic diagram of the film-forming morphology of the sequencing holes in the chip flow channel structure provided in Embodiment 3 of the present invention;

[0098] Figure 29 It is a schematic assembly structure diagram of the nanopore sequencing device provided in the embodiment of the present invention;

[0099] Figure 30 It is an exploded view of the structure of the nanopore sequencing device provided in the embodiment of the present invention;

[0100] Figure 31 It is a schematic structural diagram corresponding to Embodiment 2 of the chip flow channel structure of the nanopore sequencing device provided in the embodiment of the present invention;

[0101] Figure 32 It is Figure 31 A schematic enlarged view of the structure at H in;

[0102] Figure 33 It is a schematic structural diagram corresponding to Embodiment 3 of the chip flow channel structure of the nanopore sequencing device provided in the embodiment of the present invention;

[0103] Figure 34 It is Figure 33 A schematic enlarged view of the structure at I in;

[0104] Figure 35 It is a schematic structural diagram of the nanopore sequencing device provided in the embodiment of the present invention when the side wall of the through hole on the sealing gasket is inclined;

[0105] Figure 36 It is a schematic diagram of the pretreatment process in the film-forming method provided in the embodiment of the present invention;

[0106] Figure 37 It is a schematic diagram of the sequencing hole structure in the film-forming method provided in the embodiment of the present invention;

[0107] Figure 38 It is a schematic diagram of the replacement film-forming process in the film-forming method provided in the embodiment of the present invention.

[0108] Icons: 1'-Flow channel; 2'-Hole; 3'-Film; 4'-Oil phase; 5'-Water phase; 6'-Two-phase fluid interface; 7'-Three-phase contact line; 8'-Sudden turning structure; 1-Polar solvent; 2-Non-polar solvent; 3-Gas; 4-Amphiphilic molecular film; 5-Included angle; 6-Contact part; 10-First transition zone; 11-First straight-through zone; 12-Tapered expansion zone; 20-Chip inlet zone; 30-Inlet intersection; 31-First transition section; 40-Chip zone; 41-Sequencing hole; 42-Upper part; 43-Lower part; 50-Outlet intersection; 51-Second transition section; 60-Chip outlet zone; 70-Second transition zone; 71-Second straight-through zone; 72-Tapered contraction zone; 80-Inlet part; 90-Outlet part; 100-Base; 200-Upper cover; 201-First upper cover; 2011-Protrusion; 202-Second upper cover; 2021-Flow channel hole; 300-Sequencing chip; 400-Sealing gasket; 401-Through hole; 500-Printed board; D1-First direction; D2-Second direction; D3-Third direction. Detailed implementation mode

[0109] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments.

[0110] The components of the embodiments of the present invention usually described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention.

[0111] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0112] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0113] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", and "coupling" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0114] According to a first aspect of the present invention, a chip flow channel structure is provided, which includes a first transition region 10, a chip inlet region 20, a chip region 40, a chip outlet region 60, and a second transition region 70 that are sequentially arranged along the flow direction of the introduced substance in the chip flow channel structure.

[0115] Hereinafter, the specific structure of the chip flow channel structure according to the present invention will be described in detail.

[0116] Embodiment 1

[0117] In this embodiment, as Figures 5 to 7 shown, multiple introduced substances are introduced into the chip flow channel structure, and the introduced substances introduced adjacent times are different; the introduced substance introduced at the (N + 1)-th time can displace a part of the introduced substance introduced at the N-th time in the sequencing pore 41, or can displace a part of the introduced substances introduced at the N-th and (N - 1)-th times in the sequencing pore 41, and remain in the chip flow channel structure, so as to realize the formation of a film by replacement (this film is the amphiphilic molecular film 4 described below).

[0118] In this embodiment, the introduced substance is a gas 3, such as an inert gas such as nitrogen or air, or the introduced substance is a liquid that is a polar solvent 1 or a non-polar solvent 2, where the polar solvent 1 can be an aqueous solution, and the non-polar solvent 2 is an oil phase containing amphiphilic molecules such as phospholipids and / or block polymers.

[0119] In this embodiment, as Figures 5 to 7 shown, the chip region 40 is formed with a recessed sequencing pore 41 (for example, refer to Figure 28 ), the chip region 40 is located in the middle position of the chip flow channel structure, and the process of forming a film by replacement is carried out in the chip region 40.

[0120] In this embodiment, as Figure 6 shown, the chip inlet region 20 and the chip outlet region 60 respectively form an angle 5 greater than 90° with the chip region 40, so as to avoid the occurrence of a sudden turning structure at the connection part between the chip inlet region 20 and the chip region 40 and at the connection part between the chip outlet region 60 and the chip region 40, making the connection smooth, thereby ensuring that the introduced substance can translate in the chip region 40 to achieve effective replacement.

[0121] Furthermore, in a preferred embodiment, asFigure 6 As shown, the setting ranges of the included angle 5 between the chip inlet area 20 and the chip area 40 and the included angle 5 between the chip outlet area 60 and the chip area 40 are both 110° to 160°; preferably, the included angle 5 can be set to 150°, so that the transition between the chip inlet area 20 and the chip area 40 and between the chip outlet area 60 and the chip area 40 in the chip flow channel structure is gentle, and the replacement effect is better.

[0122] In addition, in this embodiment, as Figures 5 to 7 and Figure 28 shown, the chip area 40 is preferably arranged horizontally to ensure that the introduced substance can move uniformly in translation in the chip area 40. A plurality of sequencing holes 41 are arranged at the bottom of the chip area 40, so that the introduced substance can flow towards the sequencing holes 41 during the process of sweeping through the chip area 40, and can fill the sequencing holes 41 during the first introduction, or replace a part of the introduced substance introduced last time in the sequencing holes 41 to achieve replacement.

[0123] Specifically, the plurality of sequencing holes 41 are distributed in an array, for example, the plurality of sequencing holes 41 are distributed in a rectangular array; the speed of the introduced substance flowing through each sequencing hole 41 in each row along the direction perpendicular to the flow direction (i.e., the third direction D3 as described below) in the array is the same, so as to improve the film-forming replacement effect; in a preferred embodiment, the speed of the introduced substance flowing through all the sequencing holes 41 is the same, so as to further ensure the consistency of the film-forming positions in each sequencing hole 41, thereby improving the sequencing accuracy.

[0124] More specifically, as Figure 7 shown, the introduced substances introduced twice in succession are not miscible, so that a contact part 6 is formed between the introduced substance introduced for the (N + 1)-th time and the introduced substance introduced for the N-th time. For example, the introduced substance introduced for the N-th time is a polar solvent 1 (aqueous solution), and the introduced substance introduced for the (N + 1)-th time is a non-polar solvent 2 (oil) containing amphiphilic molecules. The contact part 6 translates in the chip flow channel structure, so that the contact part 6 is formed into a plane-like shape perpendicular to the flow direction of the introduced substance, thereby ensuring that the introduced substance moves uniformly in translation in the chip area 40.

[0125] It should be noted that since the introduced substance is a fluid, and a three-phase contact line is formed between the two introduced fluids and the side wall of the chip flow channel structure, the actual contact part 6 cannot be formed into a flat and straight-extending surface, but a curved surface shape approaching a plane, that is, formed into the plane-like shape as described above, that is to say, the contact part 6 is similar to a plane, but as Figure 3 compared with Figure 7 shown, the flatness of the contact part 6 in this embodiment is significantly better than the flatness of the two-phase fluid interface formed in the flow channel with a mutation turning structure in the prior art.

[0126] In this embodiment, as Figure 5 andFigure 6 As shown, along the flow direction of the introduced fluid, the chip inlet area 20 and the chip outlet area 60 are located at both ends of the chip area 40, and the chip area 40 is located at the bottom of the chip flow channel structure; the first transition area 10 is arranged at an angle with the chip inlet area 20. Along the flow direction of the introduced fluid, the introduced fluid flowing from the first transition area 10 to the chip inlet area 20 has a downward flow trend, so as to direct the introduced fluid to the chip area 40 at the bottom; the second transition area 70 is arranged at an angle with the chip outlet area 60. Along the flow direction of the introduced fluid, the introduced fluid flowing from the chip outlet area 60 to the second transition area 70 has an upward flow trend, so that the introduced fluid introduced for the (N + 1)-th time ejects the introduced fluid of a part of the N-th introduction from the chip flow channel structure, or ejects all the introduced fluid of the N-th introduction and a part of the introduced fluid of the (N - 1)-th introduction from the chip flow channel structure. In the latter case, N is greater than or equal to 2, avoiding the acceleration of the flow rate of the introduced fluid when flowing out of the chip flow channel structure, so as to ensure the uniform translation of the introduced fluid in the chip area 40 and make the film replacement effect meet the sequencing requirements.

[0127] In a preferred embodiment, as Figure 5 and Figure 6 shown, the angles between the first transition area 10 and the chip inlet area 20 and between the second transition area 70 and the chip outlet area 60 are both greater than 90°, so as to further avoid the dead zone of right-angle transition in the chip flow channel structure, improve the reliability of the contact part 6 forming a quasi-plane in the chip inlet area 20 or the chip outlet area 60, ensure the uniform translation of the contact part 6, and thus realize the effective replacement of the introduced fluid.

[0128] In addition, in this embodiment, as Figure 5 and Figure 6 shown, the first transition area 10, the second transition area 70 and the chip area 40 are parallel to each other, that is, the first transition area 10 and the second transition area 70 are also horizontally arranged, so that the introduced fluid smoothly transitions in the first transition area 10 or the second transition area 70. In a preferred embodiment, the first transition area 10 and the second transition area 70 are coplanar, so as to improve the success rate of film replacement.

[0129] In this embodiment, as Figures 5 to 7 shown, along the first direction D1, the second direction D2 and the third direction D3, the chip flow channel structure is an axisymmetric structure, so that the flow channel structures through which the introduced fluid flows before entering the chip area 40 and after flowing out of the chip area 40 are mirror-symmetrical, so as to reduce the influence of the shape of the flow channel structure on the film replacement effect, and thus further ensure effective replacement; among them, the first direction D1, the second direction D2 and the third direction D3 are perpendicular to each other in pairs, and the first direction D1 is the same as the flow direction of the introduced fluid.

[0130] In this embodiment, as Figure 5 and Figure 6As shown, the chip flow channel structure further includes an inlet portion 80 disposed upstream of the first transition region 10 and an outlet portion 90 disposed downstream of the second transition region 70. The inlet portion 80 is used to connect to a container filled with a feed-in substance, and the outlet portion 90 is used to connect to a suction pump. Specifically, the inlet portion 80 is disposed at an angle with respect to the first transition region 10. For example, the inlet portion 80 is vertically disposed to facilitate multiple disassembly and assembly of the inlet portion 80 with different containers filled with the feed-in substance. The channel through which the feed-in substance flows from the inlet portion 80 to the chip region 40 is formed as a downward-extending stepped structure to facilitate the transport of the feed-in substance to the chip region 40. The outlet portion 90 is disposed at an angle with respect to the second transition region 70. For example, the outlet portion 90 is vertically disposed, such that the channel through which the feed-in substance flows from the chip region 40 to the outlet portion 90 is formed as an upward-extending stepped structure to prevent the flow rate of the feed-in substance from accelerating when flowing out of the chip flow channel structure, thereby enhancing the effectiveness of film replacement.

[0131] In this embodiment, as Figure 5 and Figure 6 shown, the inlet portion 80 and the outlet portion 90 are generally disposed as circular tubular structures with relatively small radial dimensions to facilitate the connection of the chip flow channel structure with the container filled with the feed-in substance and the suction pump. The chip region 40 provided with the array of sequencing holes 41 is formed as an elongated rectangular structure in a cross-section perpendicular to the flow direction (i.e., the plane formed by the second direction D2 and the third direction D3).

[0132] To enable a smooth transition of the feed-in substance during the process of flowing from the circular tubular flow channel structure with relatively small radial dimensions to the elongated rectangular flow channel structure, in this embodiment, as Figure 5 and Figure 6 shown, along the flow direction of the feed-in substance, the first transition region 10 includes a first straight-through region 11 and a gradually expanding region 12 that are connected in sequence. Among them, the first straight-through region 11 is disposed at an angle with respect to the inlet portion 80, and the first straight-through region 11 is formed as a straight pipe structure with a certain length for realizing the turning connection between the inlet portion 80 and the first transition region 10. The gradually expanding region 12 is formed as a flared structure connected to the chip inlet region 20. Specifically, along the flow direction of the feed-in substance, the width dimension of the gradually expanding region 12 in the third direction D3 gradually increases from the upstream to the downstream of the flow direction, such that the two side walls of the gradually expanding region 12 in the third direction D3 are formed as inclined surfaces, thus avoiding the generation of a dead zone structure due to a sudden increase in size. The size of the inlet end portion of the gradually expanding region 12 is the same as the size of the end portion of the first straight-through region 11, and the size of the outlet end portion of the gradually expanding region 12 in the third direction D3 is the same as the size of the chip region 40 in the third direction D3. In this way, the chip inlet region 20 disposed between the gradually expanding region 12 and the chip region 40 is formed as a constant-width structure along the flow direction of the feed-in substance to ensure that the contact portion 6 before the feed-in substance enters the chip region 40 can translate at a uniform speed, thereby ensuring the translation effect of the contact portion 6 within the chip region 40.

[0133] The second transition region 70 is similar to the first transition region 10 in terms of the setting principle. Specifically, along the flow direction of the inflow, the second transition region 70 includes a gradually shrinking region 72 and a second straight-through region 71 that are connected in sequence. Among them, the second straight-through region 71 is arranged at an angle with the outlet portion 90. The second straight-through region 71 is formed into a straight pipe structure with a certain length for realizing the turning connection between the outlet portion 90 and the second transition region 70. Along the flow direction of the inflow, the gradually shrinking region 72 is formed into a reduced-port structure communicating with the chip outlet region 60. Specifically, along the flow direction of the inflow, the dimension of the gradually shrinking region 72 in the third direction D3 gradually decreases from the upstream to the downstream of the flow direction, so that the two side walls of the gradually shrinking region 72 in the third direction D3 are formed into inclined surfaces, thus avoiding the generation of a dead zone structure due to a sudden decrease in size. The dimension of the outlet end of the gradually shrinking region 72 is the same as the dimension of the end of the second straight-through region 71, and the dimension of the inlet end of the gradually shrinking region 72 in the third direction D3 is the same as the dimension of the chip region 40 in the third direction D3. In this way, the chip outlet region 60 arranged between the gradually shrinking region 72 and the chip region 40 is formed into an equal-width structure along the flow direction of the inflow, so as to ensure that the contact portion 6 can translate uniformly after flowing out of the chip region 40, and avoid the acceleration of the flow rate of the inflow when flowing out of the chip flow channel structure, thereby further ensuring the translation effect of the contact portion 6 in the chip region 40.

[0134] In an alternative embodiment, as Figure 5 and Figure 6 shown, the gradually expanding region 12 and the first straight-through region 11 are coplanar, so that the overall first transition region 10 is arranged at an angle with the chip inlet region 20. The gradually shrinking region 72 and the second straight-through region 71 are coplanar, so that the overall second transition region 70 is arranged at an angle with the chip outlet region 60. In this way, the translation effect of the contact portion 6 in the chip inlet region 20 and the chip outlet region 60 is ensured, thereby further ensuring the translation effect of the contact portion 6 in the chip region 40.

[0135] In another alternative embodiment, as Figures 8 to 10As shown, the gradually expanding area 12 is arranged at an angle with the first straight-through area 11, and the gradually expanding area 12 is coplanar with the chip inlet area 20, so that a part of the first transition area 10 is arranged at an angle with the chip inlet area 20; along the flow direction of the inflow, the chip inlet area 20 is formed into a flared structure connected to the gradually expanding area 12. Specifically, along the flow direction of the inflow, the dimensions of the end of the gradually expanding area 12 and the start of the chip inlet area 20 in the third direction D3 are the same, so that the dimensions of the gradually expanding area 12 and the chip inlet area 20 in the third direction D3 gradually increase from the upstream to the downstream of the flow direction; the gradually contracting area 72 is arranged at an angle with the second straight-through area 71, and the gradually contracting area 72 is coplanar with the chip outlet area 60, so that a part of the second transition area 70 is arranged at an angle with the chip outlet area 60; along the flow direction of the inflow, the chip outlet area 60 is formed into a constricted structure connected to the gradually contracting area 72. Specifically, along the flow direction of the inflow, the dimensions of the end of the chip outlet area 60 and the start of the gradually contracting area 72 in the third direction D3 are the same, so that the dimensions of the gradually contracting area 72 and the chip outlet area 60 in the third direction D3 gradually increase from the upstream to the downstream of the flow direction; thus, the flow channel structures of the gradually expanding area 12, the chip inlet area 20, the gradually contracting area 72, and the chip outlet area 60 are integrated, so that while the flow channel structure gradually widens or gradually contracts, it can also smoothly transition from the higher inlet part 80 to the chip area 40 in the form of a slope in the second direction D2, thereby also ensuring the translation effect of the contact part 6 in the chip area 40.

[0136] In one such as Figure 5 and Figure 6In the illustrated embodiment, the dimensions of the chip flow channel structure are as follows: The length dimension of the first straight-through region 11 in the first direction D1 is 6 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 is 1.2 mm; the second straight-through region 71 may have the same dimensions as the first straight-through region 11; the length dimension of the gradually expanding region 12 in the first direction D1 is 5.3 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 gradually expands from 1.2 mm to 7 mm; the length dimension of the gradually shrinking region 72 in the first direction D1 is 5.3 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 gradually shrinks from 7 mm to 1.2 mm; the length dimension of the chip inlet region 20 in the inclined direction forming an angle of 5 with the chip region 40 is 6 mm, the thickness dimension in the direction perpendicular to both the inclined direction and the third direction D3 is 0.6 mm, and the width dimension in the third direction D3 is 7 mm. It should be noted that the length direction of the chip inlet region 20 is the same as the flow direction of the introduced substance in the chip inlet region 20, and the height (or thickness) is the distance between the top wall and the bottom wall of the chip inlet region 20 in the direction perpendicular to the top wall and the bottom wall of the chip inlet region 20; the chip outlet region 60 has the same dimensions as the chip inlet region 20; the length dimension of the chip region 40 in the first direction D1 is 18 mm, the height dimension in the second direction D2 is 0.4 mm, and the width dimension in the third direction D3 is 7 mm.

[0137] However, the dimensions of the above chip flow channel structure are only an optional embodiment, and the dimensions of each region can be adjusted according to the requirements of film formation or sequencing.

[0138] Example Two

[0139] Example Two is a further improvement based on the chip flow channel structure in Example One. The structure disclosed in Example One is applied to Example Two and will not be repeated here.

[0140] During the process of replacing the film formation, the introduced substance in the (N + 1)-th introduction is gas 3, and the introduced substance in the N-th introduction is the oil phase of non-polar solvent 2. During the process of gas 3 replacing the oil phase, the situation of incomplete replacement of the oil phase may occur at one end of the chip region 40 close to the chip outlet region 60 (i.e., the end of the chip region 40), resulting in the retention of the oil phase at both ends of the chip region 40 in the third direction D3. For the retention situation, see Figure 11 as shown, thus causing the oil phase to not be effectively displaced, the oil film in the sequencing holes 41 to thicken, and resulting in the inability to obtain an amphiphilic molecular film 4 with consistent film formation position and morphology.

[0141] Combined with Figures 11 to 15Analysis of the deformation and evolution of the contact part 6 of the gas-oil interface shown until abnormal fracture occurs within the chip channel structure. As described above, since the introduced substance is a fluid, the contact part 6 is actually formed into a curved surface approaching a plane. The curved phase interface will generate a Laplace pressure difference ΔP (also known as the additional pressure) on both sides of different phases. Its magnitude is related to the interfacial tension γ and the radius of curvature R. The formula for the pressure difference ΔP is:

[0142]

[0143] In the formula: R1 and R2 are any two orthogonal radii of curvature of ΔP acting on a certain point on the curved surface. The additional pressure points to the center of curvature of the phase interface, that is, the pressure on the convex surface side (the side where the gas 3 is located) is greater than the pressure on the concave surface side (the side where the non-polar solvent 2 is located), and the difference is ΔP. It can be seen from the formula that the greater the pressure difference between the two sides of the curved surface with a smaller radius of curvature R.

[0144] Furthermore, when the contact part 6 (i.e., the gas-oil interface) is about to flow out of the chip area 40, as Figure 12 in the process e and Figure 13 shown, the radii of curvature of the middle area of the gas-oil interface in the third direction D3 and the two end areas in the third direction D3 are the same, making the additional pressure evenly distributed on the gas-oil interface. In one embodiment, the non-polar solvent 2 is silicone oil AR20, and the amphiphilic molecule dissolved in it is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (abbreviated as diphytoyl phosphatidylcholine, abbreviated as DPhPC). At this time, the gas-oil interfacial tension is about 20 mN / m. When the size of the chip area 40 in the second direction D2 is 0.4 mm and the size in the third direction D3 is 7 mm, R1 in the above formula corresponds to 3.5 mm, R2 corresponds to 0.2 mm, and the additional pressure ΔP obtained based on the formula is 106 Pa; and when the gas-oil interface flows to the intersection of the chip area 40 and the chip outlet area 60, as Figure 12 in the process f and Figure 14 shown, the middle area of the gas-oil interface in the third direction D3 first enters the intersection of the chip area 40 and the chip outlet area 60, and when it first enters, the radius of curvature of the gas-oil interface in the third direction D3 can be considered unchanged, but in the cross-section shown in Figure 14 the radius of curvature will suddenly become larger, resulting in a sudden decrease in the additional pressure. At this time, the maximum height dimension of the intersection of the chip area 40 and the chip outlet area 60 (see the dimension of h Figure 19 indicated by the arrow in max2 ) is 1.7 mm. In the above formula, R1 corresponds to 3.5 mm, R2 corresponds to 0.85 mm, and the calculated additional pressure is reduced to about 29 Pa. At this time, the morphological profile diagrams of both sides of the gas-oil interface in the third direction D3 are still as Figure 13As shown, the additional pressure is still around 106 Pa. That is, for gas 3, a pressure relief area is formed at the intersection of the chip area 40 and the chip outlet area 60 at this time. The gas 3 located upstream of the chip flow channel structure will quickly enter and gradually fill this area. At this time, the moving speed of the gas-oil interfaces on both sides in the third direction D3 will slow down, and even stop moving forward until the situation of the gas-oil interface breakage shown in Figure 12 occurs in process g of the figure, resulting in the incomplete replacement of the oil phase at the end of the chip area 40 close to the chip outlet area 60, and finally leading to a decrease in the film formation rate in this area.

[0145] To solve the possible situation that the non-polar solvent 2 is likely to remain, in this embodiment, as shown in Figure 16 and Figure 17 , along the flow direction of the introduced substance, an outlet intersection part 50 communicating with the chip area 40 is formed at the beginning of the chip outlet area 60; through the research of the inventor, when the ratio of the equivalent diameter of the outlet intersection part 50 to the equivalent diameter of the chip area 40 is not greater than 3, the reliability of the effective replacement of the non-polar solvent 2 during the replacement of the oil phase by gas 3 can be improved. Among them, the introduction of the equivalent diameter (i.e., the hydraulic diameter) in fluid mechanics aims to equivalent the non-circular pipe flow to the circular pipe flow, which is an equivalent transformation for the flow channel cross-section. The equivalent diameter can be understood as: for the irregular-shaped flow channel cross-section, the diameter of the circle with the same area as it.

[0146] The formula for the equivalent diameter D is:

[0147]

[0148] In the formula, A is the cross-sectional area of the chip flow channel structure;

[0149] P is the cross-sectional perimeter of the chip flow channel structure;

[0150] h is the height dimension of A;

[0151] w is the width dimension of A;

[0152] The cross-section is a plane perpendicular to the flow direction of the introduced substance.

[0153] The specific analysis is as follows: According to the requirements of sequencing, the size of the chip area 40 in the third direction D3 is usually much larger than the size in the second direction D2, so that in the ΔP formula, the curvature in the third direction D3 can be ignored compared with the curvature in the second direction D2. It should be noted that the curvature is the reciprocal of the curvature radius, and the curvature radius in the second direction D2 is half of the size in this direction (i.e., h / 2). Therefore:

[0154]

[0155] In the formula for the equivalent diameter D, the reciprocal of the width dimension w can also be neglected compared to the reciprocal of the height dimension h. Therefore:

[0156]

[0157] Combining Equation (1) and Equation (2) gives: ΔP≈4γ / D, that is, the additional pressure ΔP is approximately inversely proportional to the equivalent diameter D.

[0158] In addition, it has been found through research that when the contact part 6 is the gas-oil interface, if the additional pressure suddenly changes to less than 1 / 3 at the intersection of the inlet and outlet, it will cause the interface to break and the oil phase to remain.

[0159] Furthermore, in this embodiment, as Figure 18 and Figure 19 shown, the connection line between the end of the upper surface of the chip area 40 located downstream and the end of the same side and located at the bottom wall of the chip flow channel structure forms the maximum height dimension h of the outlet intersection part 50 max2 . When the ratio of the equivalent diameter of the cross-section where h max2 is located to the equivalent diameter of the chip area 40 is not greater than 3, the ratio of the equivalent diameter of the cross-section at each location of the outlet intersection part 50 to the equivalent diameter of the chip area 40 is not greater than 3. Therefore, when calculating, only h max2 needs to be substituted into the formula for the equivalent diameter D. When the ratio of the equivalent diameters is limited to not greater than 3, the movement of the contact part 6 of the gas-oil interface is as Figure 21 shown, and it can be seen that the effect of replacing it with a film is effectively improved.

[0160] It should be noted that as described above, the chip flow channel structure is preferably an axisymmetric structure, so that the flow channel structure through which the introduced substance flows before entering the chip area 40 is mirror-symmetrical to the flow channel structure after flowing out of the chip area 40 (see Figure 19 and Figure 20 ). Therefore, by limiting the ratio of the equivalent diameter of the cross-section where the maximum height dimension h max1 is located and the ratio of the equivalent diameter of the cross-section where the maximum height dimension h max2 is located to the equivalent diameter of the chip area 40 to be not greater than 3, it is ensured that no matter whether the introduced substance is introduced into the chip flow channel structure from the inlet part 80 or from the outlet part 90 (both the inlet part 80 and the outlet part 90 can be used as inlets or outlets, and can also be called in-out ports and can be swapped and replaced with each other), the effect of replacing it with a film can be effectively improved.

[0161] As described above, since both the inlet part 80 and the outlet part 90 can be used as inlets or outlets, in this embodiment, as Figure 16 and Figure 17As shown, an inlet intersection 30 is formed at the end of the chip inlet region 20 and is in communication with the chip region 40. The ratio of the equivalent diameter of the inlet intersection 30 to the equivalent diameter of the chip region 40 is not greater than 3. In this way, even if the introduced substance enters the chip flow channel structure through the outlet portion 90, there will be no incomplete replacement of the oil phase at the end of the chip flow channel structure, thereby further ensuring the reliability of film formation by replacement.

[0162] Furthermore, in this embodiment, as Figure 20 shown, along the flow direction of the introduced substance, the connection line between the end of the upper surface of the chip region 40 located upstream and the end of the bottom wall of the chip flow channel structure on the same side forms the maximum height dimension h of the inlet intersection 30 max1 . When the ratio of the equivalent diameter of the cross-section where h max1 is located to the equivalent diameter of the chip region 40 is not greater than 3, the ratio of the equivalent diameter of each cross-section of the inlet intersection 30 to the equivalent diameter of the chip region 40 is not greater than 3; therefore, in the calculation, only h max1 needs to be substituted into the formula of the equivalent diameter D.

[0163] If only the ratio of the equivalent diameter of the outlet intersection 50 to the equivalent diameter of the chip region 40 is limited to not greater than 3, in the extreme case where the outlet intersection 50 is very short, an overly large equivalent diameter of the chip outlet region 60 may also cause residual non-polar solvent 2. In order to further improve the reliability of the effective replacement of the non-polar solvent 2 during the gas 3 replacement of the oil phase, in this embodiment, the ratio of the equivalent diameter of the chip outlet region 60 to the equivalent diameter of the chip region 40 is not greater than 3.

[0164] Similarly, if only the ratio of the equivalent diameter of the inlet intersection 30 to the equivalent diameter of the chip region 40 is limited to not greater than 3, in the extreme case where the inlet intersection 30 is very short, an overly large equivalent diameter of the chip inlet region 20 may also cause residual non-polar solvent 2. In order to further improve the reliability of the effective replacement of the non-polar solvent 2 during the gas 3 replacement of the oil phase, in this embodiment, the ratio of the equivalent diameter of the chip inlet region 20 to the equivalent diameter of the chip region 40 is not greater than 3.

[0165] It should be noted that when the introduced substance introduced for the (N + 1)-th time is the oil phase and the introduced substance introduced for the N-th time is the water phase, the tension of the contact portion 6 is small, resulting in a small additional pressure difference. Therefore, the limitation that the ratio of the equivalent diameters is not greater than 3 has little impact on the process of oil phase replacement of water phase.

[0166] However, the dimensions of the above chip flow channel structure are only an optional implementation manner, and the dimensions of each region can be adjusted according to the requirements for film formation or sequencing.

[0167] Embodiment III

[0168] Embodiment 3 is a further improvement based on the chip flow channel structure in Embodiment 2. The structure disclosed in Embodiment 2 is applied to Embodiment 3 and will not be repeated here.

[0169] In this embodiment, as Figures 22 to 26 shown, along the flow direction of the inflow object, the end of the inlet intersection 30 is formed as a first transition section 31 extending vertically downward, so that the dimension of h max1 is reduced (the position of h max2 is shown by the arrow indication in Figure 26 ), thereby reducing the ratio of the equivalent diameter of the inlet intersection 30 to the equivalent diameter of the chip area 40, and effectively controlling the ratio within a range not greater than 3; similarly, the beginning of the outlet intersection 50 is formed as a second transition section 51 extending vertically upward, so that the dimension of h max2 is reduced (the position of h max2 is shown by the arrow indication in Figure 25 ), thereby reducing the ratio of the equivalent diameter of the outlet intersection 50 to the equivalent diameter of the chip area 40. Furthermore, on the premise of not changing the equivalent diameter of the chip area 40, the ratio is effectively controlled within a range not greater than 3, and since the control of this ratio is not achieved by increasing the height dimension of the chip area 40 in the second direction D2 as Figure 27 shown to increase the equivalent diameter of the chip area 40, the addition amount of the library sample in the sequencing stage will not be increased.

[0170] In an alternative embodiment, the dimensions of the chip flow channel structure are as follows: The length dimension of the first straight-through region 11 in the first direction D1 is 6 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 is 1.2 mm; the second straight-through region 71 may have the same dimensions as the first straight-through region 11; the length dimension of the gradually expanding region 12 in the first direction D1 is 5.3 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 gradually expands from 1.2 mm to 7 mm; the length dimension of the gradually contracting region 72 in the first direction D1 is 5.3 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 gradually contracts from 7 mm to 1.2 mm; the length dimension of the chip inlet region 20 in the inclined direction forming an angle of 5 with the chip region 40 is 4 mm, the thickness dimension in the direction perpendicular to both the inclined direction and the third direction D3 is 0.6 mm, and the width dimension in the third direction D3 is 7 mm. It should be noted that the length direction of the chip inlet region 20 is the same as the flow direction of the inflow substance in the chip inlet region 20, and the thickness is the distance between the top wall and the bottom wall of the chip inlet region 20 in the direction perpendicular to the top wall and the bottom wall of the chip inlet region 20; the chip outlet region 60 has the same dimensions as the chip inlet region 20; the length dimension of the chip region 40 in the first direction D1 is 20 mm, the height dimension in the second direction D2 is 0.4 mm, and the width dimension in the third direction D3 is 7 mm; the length dimension of the inlet intersection 30 in the first direction D1 is 0.5 mm, the height dimension in the second direction D2 is 0.6 mm, and the width dimension in the third direction D3 is 7 mm; the outlet intersection 50 has the same dimensions as the inlet intersection 30, h max1 = h max2 = 0.78 mm.

[0171] After calculation, the equivalent diameter of the chip region 40 is 0.76 mm, the equivalent diameter of the chip outlet region 60 or the chip inlet region 20 is 1.11 mm, and the maximum equivalent diameter of the inlet intersection 30 or the outlet intersection 50 is 1.4 mm. Therefore, the ratio of the equivalent diameter of the inlet intersection 30 or the outlet intersection 50 to the equivalent diameter of the chip region 40 is 1.8, and the ratio of the equivalent diameter of the chip outlet region 60 or the chip inlet region 20 to the equivalent diameter of the chip region 40 is 1.5. The additional pressure of the gas-oil interface contact part 6 before entering the outlet intersection 50 is about 106 Pa, and when entering the intersection, the additional pressure drops to about 57 Pa. Figure 28 Shows a top-down microscopic image of the amphiphilic molecular film 4 formed in the sequencing pore 41 at the end of the chip region 40 in this chip flow channel structure. The circular black boundary in the middle of the sequencing pore 41 is the edge region of the amphiphilic molecular film 4. It can be seen that the film-forming positions and morphologies in each sequencing pore 41 are in good agreement.

[0172] According to the chip flow channel structure of the present invention, through the inclined chip inlet area and chip outlet area, during the process of replacing the polar solvent with the non-polar solvent, the contact part is formed into a quasi-planar structure and can translate uniformly within the chip area. In addition, by limiting the equivalent diameter ratio to not more than 3, the situation of non-polar solvent remaining in the chip area during the process of replacing the non-polar solvent with gas is avoided. In this way, it is ensured that all the introduced substances can sweep across the sequencing pores at a uniform speed during the entire replacement process, thereby achieving effective replacement, forming an amphiphilic molecular film with good morphology and position consistency in each sequencing pore, which can significantly improve the film formation rate and further increase the sequencing throughput.

[0173] According to a second aspect of the present invention, a nanopore sequencing device is provided, which includes a housing assembly, a sequencing chip 300, and a sealing gasket 400.

[0174] Hereinafter, the specific structures of the above components of the nanopore sequencing device according to the present invention will be described in detail.

[0175] In this embodiment, as Figure 29 and Figure 30 shown, the housing assembly includes a base 100 and an upper cover 200 that are assembled with each other. The chip flow channel structure is formed between the buckled base 100 and upper cover 200. Specifically, the first transition area 10, the chip inlet area 20, the chip outlet area 60, and the second transition area 70 in the chip flow channel structure are formed on the upper cover 200; the sequencing chip 300 is disposed above the base 100. The sequencing chip 300 and the upper cover 200 enclose a chip area 40, and sequencing pores 41 are disposed on the surface of the sequencing chip 300; the sealing gasket 400 is clamped between the sequencing chip 300 and the upper cover 200. In this way, the chip flow channel structure is enclosed by the assembly method of the housing assembly, the sequencing chip 300, and the sealing gasket 400, and the forming method of the chip flow channel structure is simple and convenient. In addition, the housing assembly, the sequencing chip 300, and the sealing gasket 400 are detachably connected. After use, a new sequencing chip 300 can be replaced, thereby realizing repeated use and saving costs.

[0176] In this embodiment, as Figure 30 shown, a through hole 401 penetrating through its main body is formed on the sealing gasket 400. The side wall of the through hole 401 encloses a part of the chip flow channel structure. The bottom of the sealing gasket 400 is attached to the sequencing chip 300, and the top of the sealing gasket 400 is attached to the upper cover 200. In an alternative embodiment, as Figures 31 to 34 shown, the side walls of the through hole 401 are all vertically arranged (i.e., extending along the second direction D2), which is convenient for the assembly of the nanopore sequencing device; in another alternative embodiment, as Figure 35As shown, the two side walls of the through hole 401 are inclined on both sides in the first direction D1, and the inclination angle of the side wall is the same as that of the hole wall on the same side in the flow channel hole 2021 described below. Compared with the vertically arranged side walls, the inclined side walls reduce the equivalent diameter of the end part of the chip inlet area 20 and the start part of the chip outlet area 60 in the flow direction of the introduced substance, but the assembly accuracy requirements are relatively high.

[0177] Furthermore, in this embodiment, as Figures 29 to 35 shown, the upper cover 200 includes a first upper cover 201 and a second upper cover 202 that are fastened to each other up and down; the first upper cover 201 is formed with a protruding part 2011 extending towards the second upper cover 202, and the second upper cover 202 is formed with a flow channel hole 2021 corresponding to the protruding part 2011 for the protruding part 2011 to extend into; when the first upper cover 201 and the second upper cover 202 are fastened together, a gap is formed between them, and this gap corresponds to a part of the chip flow channel structure. Specifically, a first transition area 10 and a second transition area 70 are formed by the bottom part of the first upper cover 201 and the top part of the second upper cover 202; the two side walls in the length direction of the protruding part 2011 (i.e., in the first direction D1) are inclined, so that the two side walls in the length direction of the protruding part 2011 and the two side walls in the length direction of the flow channel hole 2021 respectively enclose the chip inlet area 20 and the chip outlet area 60, and the inclined side walls make the included angles 5 formed between the chip inlet area 20 and the chip outlet area 60 and the chip area 40 respectively greater than 90°. The sequencing chip 300 and the bottom wall of the protruding part 2011 enclose the chip area 40.

[0178] Even further, in this embodiment, as Figure 29 、 Figure 31 、 Figure 33 and Figure 35 shown, the inlet part 80 and the outlet part 90 are opened on the top of the first upper cover 201 to facilitate the connection of the nanopore sequencing device with the container containing the introduced substance and the suction pump, so that the introduced substance flows along a stepped route extending downward to the sequencing chip 300 through the inlet part 80, and then flows out of the outlet part 90 along a stepped route extending upward, thereby completing the replacement and film formation. It should be noted that the size and shape of the inlet part 80 and the outlet part 90 can be set according to the size of the hose used to connect the container or the suction pump.

[0179] In a preferred embodiment, as Figures 29 to 35 shown, the upper cover 200 is formed of a transparent material, such as plastic material, so that the preparer can directly observe the internal situation of the nanopore sequencing device, thereby improving the success rate of replacement and film formation in the chip flow channel structure.

[0180] In addition, in this embodiment, as Figure 30As shown, during the assembly process of each component in the nanopore sequencing device, positioning is achieved through hole pins. For example, a positioning pin protruding upward is formed on the top of the base 100, and a positioning hole for the positioning pin to extend into is provided at the bottom of the upper cover 200. In this way, the installation accuracy between components is improved, so that after the nanopore sequencing device is assembled, the chip flow channel structure enclosed inside it is consistent with the actual requirements, thus meeting the sequencing requirements.

[0181] It should be noted that Figure 31 and Figure 32 The chip flow channel structure enclosed by the nanopore sequencing device shown corresponds to the second embodiment above. Specifically, the two side walls on both sides in the length direction of the protruding portion 2011 (i.e., in the first direction D1) are symmetrically inclined, so that the size of the bottom of the protruding portion 2011 is smaller than the size of the top of the protruding portion 2011; Figure 33 and Figure 34 The chip flow channel structure enclosed by the nanopore sequencing device shown corresponds to the third embodiment above. Specifically, the two side walls on both sides in the length direction of the protruding portion 2011 (i.e., in the first direction D1) are symmetrically inclined, so that the size of the bottom of the protruding portion 2011 is smaller than the size of the top of the protruding portion 2011, and the bottom of the protruding portion 2011 extends downward. This downward extension structure can reduce the distance between the side wall at the bottom of the protruding portion 2011 and the second upper cover 202 and the sealing gasket 400, so that the dimensions of h max1 and h max2 can be relatively reduced.

[0182] In the nanopore sequencing device according to the present invention, the chip flow channel structure is enclosed inside the housing assembly by assembling each component, making the flow channel forming method simple and convenient to prepare. In addition, through the detachable connection between each component, after sequencing is completed, by replacing a new sequencing chip, the housing assembly and the sealing gasket can be reused to save costs.

[0183] According to the third aspect of the present invention, a film-forming method is provided. The film-forming method is applied to the nanopore sequencing device, and the film-forming method mainly includes the following steps:

[0184] Pretreatment: Coating a non-polar solvent 2 on the side wall of the sequencing pore 41 to achieve the purpose of surface modification, and non-polar solvents 2 such as n-hexadecane and silicone oil can be selected.

[0185] Replacement film formation: Different introduced substances are sequentially introduced into the chip flow channel structure. The introduced substance introduced for the (N + 1)th time can replace part of the introduced substance introduced for the Nth time in the sequencing pore 41, or can replace the introduced substance introduced for the Nth time in the sequencing pore 41 and part of the introduced substance introduced for the (N - 1)th time, so as to form an amphiphilic molecular film 4 in the sequencing pore 41.

[0186] In one embodiment, as Figure 37 shown, the circumferential side wall of the sequencing hole 41 is formed into a toothed structure. In the pretreatment step, the non-polar solvent 2 is coated inside the toothed structure. Optionally, the method of completing the pretreatment step may be to contact a porous medium (such as a filter membrane, a porous plate) containing the non-polar solvent 2 with the upper surface of the sequencing chip 300, so that the non-polar solvent 2 is transferred into the sequencing hole 41. For example, it can be printed on the side wall of the sequencing hole 41 by the method of printing plate 500 transfer, as Figure 36 shown. The non-polar solvent 2 is first coated on the printing plate 500, and the printing plate 500 is pressed against the sequencing chip 300. During the contact process between the printing plate 500 and the sequencing chip 300, the non-polar solvent 2 on the printing plate 500 is transferred to the side wall of the sequencing hole 41.

[0187] Furthermore, in this embodiment, as Figure 36 and Figure 37 shown, the side wall of the sequencing hole 41 is formed into a stepped structure to divide the sequencing hole 41 into upper and lower layers. The radial dimension of the upper part 42 of the sequencing hole 41 is larger than the radial dimension of the lower part 43 of the sequencing hole 41; the amphiphilic molecular film 4 is formed between the upper and lower layers of the sequencing hole 41.

[0188] In this embodiment, as Figure 38 shown, the specific process of the film replacement step includes:

[0189] S10. The introduced substance is the first polar solvent 1. The first polar solvent 1 enters from the inlet part 80 of the chip flow channel structure and flows out from the outlet part 90 of the chip flow channel structure, so that the first polar solvent 1 fills the sequencing hole 41. Among them, the first polar solvent 1 is an aqueous solution, that is, the above-mentioned aqueous phase.

[0190] S20. The introduced substance is the non-polar solvent 2. The non-polar solvent 2 contains amphiphilic molecules such as phospholipids and / or block polymers, that is, the above-mentioned oil phase. Specifically, the non-polar solvent 2 enters the inlet part 80 at a first flow rate. When the non-polar solvent 2 flows through the sequencing hole 41, the non-polar solvent 2 can push the first polar solvent 1 located in the upper part 42 of the sequencing hole 41 out of the sequencing hole 41 and remain in the upper part 42 of the sequencing hole 41, so as to realize the replacement of part of the first polar solvent 1 in the sequencing hole 41 by the non-polar solvent 2, so that the non-polar solvent 2 covers the top of the first polar solvent 1 located in the lower part 43, and the replaced first polar solvent 1 flows out from the outlet part 90.

[0191] In step S20, the optional setting range of the first flow rate is 50 μL / min to 8000 μL / min. For example, it can be set to 2000 μL / min; the junction between the first polar solvent 1 and the non-polar solvent 2 forms the lower surface of the amphiphilic molecular film 4.

[0192] S30. The introduced substance is gas 3, and gas 3 is preferably an inert gas 3 such as air or nitrogen. Specifically, gas 3 enters the inlet part 80 at a second flow rate. When gas 3 flows through the sequencing holes 41, gas 3 can push out a part of the non-polar solvent 2 in the upper part 42 of the sequencing holes 41 and remain in the upper part 42 of the sequencing holes 41, so as to realize the replacement of a part of the non-polar solvent 2 in the sequencing holes 41 by gas 3, making the non-polar solvent 2 sandwiched between the gas 3 in the upper part 42 and the first polar solvent 1 in the lower part 43. Only an oil phase film covers the upper part of the first polar solvent 1, and the replaced non-polar solvent 2 is discharged from the outlet part 90.

[0193] In step S30, the optional setting range of the second flow rate is 10 μL / min to 500 μL / min. For example, it can be set to 40 μL / min.

[0194] S40. The introduced substance is the second polar solvent 1, and the second polar solvent 1 can be the same as the first polar solvent 1. Specifically, the second polar solvent 1 enters from the inlet part 80. When the second polar solvent 1 flows through the sequencing holes 41, it can replace all the gas 3 and part of the non-polar solvent 2 in the sequencing holes 41, so that the amphiphilic molecular film 4 reaches the required thickness. The replaced gas 3 and non-polar solvent 2 are discharged from the outlet part 90, and thus a qualified amphiphilic molecular film 4 is prepared.

[0195] In step S40, the junction between the non-polar solvent 2 and the second polar solvent 1 forms the upper surface of the amphiphilic molecular film 4.

[0196] In addition, in order to facilitate the preparation personnel to determine the time point to stop introducing substances into the chip flow channel structure, so as to avoid waste of introduced substances on the basis of ensuring effective replacement, in step S20, it further includes: S21. When the non-polar solvent 2 flows out from the outlet part 90, stop introducing the non-polar solvent 2 into the chip flow channel structure;

[0197] In step S30, it further includes: S31. When the gas 3 flows out from the outlet part 90, stop introducing the gas 3 into the chip flow channel structure;

[0198] In step S40, it further includes: S41. When the second polar solvent 1 flows out from the outlet part 90, stop introducing the second polar solvent 1 into the chip flow channel structure.

[0199] According to the film-forming method of the present invention, by introducing different introduced substances into the chip flow channel structure, the introduced substances are sequentially replaced layer by layer during the process of flowing through the sequencing holes in turn, so as to obtain the amphiphilic molecular film required for sequencing.

[0200] Finally, it should be noted that the above-described embodiments are only specific implementation manners of the present application, used to illustrate the technical solutions of the present application, rather than limiting it. The protection scope of the present application is not limited thereto. Although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any person skilled in the art within the technical scope disclosed by the present application can still modify the technical solutions recorded in the foregoing embodiments or can easily think of changes, or perform equivalent replacements on some of the technical features; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A chip flow channel structure, characterized in that, Along the flow direction of the introduced substance within the chip flow channel structure, the chip flow channel structure includes: A first transition zone, a chip inlet zone, a chip zone, a chip outlet zone, and a second transition zone arranged in sequence; The first transition zone is arranged at an angle with the chip inlet zone, and the second transition zone is arranged at an angle with the chip outlet zone; Sunken sequencing holes are formed in the chip zone; The chip inlet zone and the chip outlet zone respectively form an angle greater than 90° with the chip zone; The first transition zone, the second transition zone, and the chip zone are parallel to each other; Along the flow direction of the introduced substance, the first transition zone includes a first straight-through zone and a gradually expanding zone that are connected in sequence, and the gradually expanding zone is formed into a flared structure that communicates with the chip inlet zone; along the flow direction of the introduced substance, the second transition zone includes a gradually shrinking zone and a second straight-through zone that are connected in sequence, and the gradually shrinking zone is formed into a constricted structure that communicates with the chip outlet zone; The gradually expanding zone and the first straight-through zone are coplanar, and the gradually shrinking zone and the second straight-through zone are coplanar.

2. The chip flow channel structure according to claim 1, characterized in that, Along the first direction, the second direction, and the third direction, the chip flow channel structure is an axisymmetric structure; The first direction, the second direction, and the third direction are perpendicular to each other in pairs, and the first direction is the same as the flow direction of the introduced substance.

3. The chip flow channel structure according to claim 1, characterized in that, The setting range of the angle is 110° to 160°.

4. The chip flow channel structure according to claim 1, characterized in that, The chip zone is located at the bottommost part of the chip flow channel structure and is horizontally arranged; A plurality of the sequencing holes are arranged at the bottom of the chip zone.

5. The chip flow channel structure according to claim 1, characterized in that, The plurality of the sequencing holes are distributed in an array; The introduced substance flows through each of the sequencing holes in each row in the direction perpendicular to the flow direction in the array at the same speed.

6. The chip flow channel structure according to claim 1, characterized in that, The successively introduced substances do not blend, such that a contact part is formed between the (N + 1)-th introduced substance and the N-th introduced substance, and the contact part translates within the chip flow channel structure, such that the contact part is formed into a plane-like shape perpendicular to the flow direction of the introduced substance.

7. The chip flow channel structure according to claim 1, characterized in that, The introduced substance is a gas or the introduced substance is a liquid of a polar solvent or a non-polar solvent.

8. The chip flow channel structure according to claim 1, characterized in that, The chip flow channel structure further includes: An inlet part, arranged upstream of the first transition zone, the inlet part is arranged at an angle with the first transition zone, and the channel for the introduced substance to flow from the inlet part to the chip zone is formed into a stepped structure extending downward; An outlet part, arranged downstream of the second transition zone, the outlet part is arranged at an angle with the second transition zone, and the channel for the introduced substance to flow from the chip zone to the outlet part is formed into a stepped structure extending upward.

9. The chip flow channel structure according to any one of claims 1 to 8, characterized in that, Along the flow direction of the introduced substance, the starting end of the chip outlet zone is formed into an outlet intersection part that communicates with the chip zone; The ratio of the equivalent diameter of the outlet intersection part to the equivalent diameter of the chip zone is not greater than 3; The formula for the equivalent diameter D is: In the formula, A is the cross-sectional area of the chip flow channel structure; P is the cross-sectional perimeter of the chip flow channel structure; h is A height dimension; w is A width dimension; The cross-section is a plane perpendicular to the flow direction of the introduced substance.

10. The chip flow channel structure according to claim 9, wherein, Along the flow direction of the introduced substance, the end of the chip inlet zone is formed into an inlet intersection part that communicates with the chip zone; The ratio of the equivalent diameter of the inlet intersection part to the equivalent diameter of the chip zone is not greater than 3.

11. The chip flow channel structure according to claim 10, wherein, The ratio of the equivalent diameter of the chip inlet region to the equivalent diameter of the chip region is not greater than 3.

12. The chip flow channel structure according to claim 9, wherein, The ratio of the equivalent diameter of the chip outlet region to the equivalent diameter of the chip region is not greater than 3.

13. The chip flow channel structure according to claim 10, wherein, Along the flow direction of the introduced substance, the connection line between the end of the upper surface of the chip area and the end of the same side and located at the bottom wall of the chip flow channel structure forms the maximum height dimension of the inlet intersection part h max1 ; or forms the maximum height dimension of the outlet intersection part h max2 .

14. The chip flow channel structure according to claim 13, wherein, Along the flow direction of the introduced substance, the end of the inlet intersection portion is formed as a first transition section extending vertically downward; the beginning of the outlet intersection portion is formed as a second transition section extending vertically upward.

15. The chip flow channel structure according to claim 9, wherein, The fact that the ratio is not greater than 3 is not achieved by increasing the equivalent diameter of the chip region.

16. A nanopore sequencing device, wherein, Including the chip flow channel structure according to any one of claims 1 to 15, and: A housing assembly, including a base and an upper cover assembled with each other, the first transition region, the chip inlet region, the chip outlet region, and the second transition region are formed in the upper cover; A sequencing chip, disposed above the base, the sequencing chip and the upper cover enclose the chip region, and the sequencing holes are disposed on the surface of the sequencing chip; A sealing gasket, clamped between the sequencing chip and the upper cover.

17. The nanopore sequencing device according to claim 16, wherein, The upper cover includes a first upper cover and a second upper cover which are fastened up and down; The first upper cover is formed with a protruding portion extending towards the second upper cover, and the second upper cover is formed with a flow channel hole corresponding to the protruding portion for the protruding portion to extend into; A part of the bottom of the first upper cover and the top of the second upper cover enclose the first transition region and the second transition region; The two side walls in the length direction of the protruding portion and the two side walls in the length direction of the flow channel hole respectively enclose the chip inlet region and the chip outlet region; The sequencing chip and the bottom wall of the protruding portion enclose the chip region.

18. The nanopore sequencing device according to claim 16, wherein, The upper cover is formed of a transparent material.

19. A film forming method, wherein, The film-forming method is applied to the nanopore sequencing device according to any one of claims 16 to 18, and the film-forming method includes the following steps: Pretreatment: Coating a non-polar solvent on the side wall of the sequencing hole; Replacement film formation: Different introduced substances are sequentially introduced into the chip flow channel structure, and the introduced substance introduced at the (N + 1)-th time can replace part of the introduced substance introduced at the N-th time in the sequencing hole, or can replace the introduced substance introduced at the N-th time in the sequencing hole and part of the introduced substance introduced at the (N - 1)-th time, so as to form an amphiphilic molecular film in the sequencing hole.

20. The film forming method according to claim 19, wherein, The circumferential side wall of the sequencing hole is formed as a toothed structure, and the non-polar solvent in the pretreatment step is coated in the toothed structure.

21. The film forming method according to claim 19, wherein, The side wall of the sequencing hole is formed as a stepped structure to divide the sequencing hole into upper and lower layers; the amphiphilic molecular film is formed between the upper and lower layers of the sequencing hole.

22. The film forming method according to claim 19, wherein, The replacement film formation step includes: S10. The introduced substance is a first polar solvent, and the first polar solvent enters from the inlet portion of the chip flow channel structure and flows out from the outlet portion of the chip flow channel structure, so that the first polar solvent fills the sequencing hole; S20. The introduced substance is a non-polar solvent, and the non-polar solvent enters the inlet portion at a first flow rate. When the non-polar solvent flows through the sequencing hole, it can replace part of the first polar solvent in the sequencing hole, and the replaced first polar solvent is discharged from the outlet portion; S30. The introduced substance is a gas. The gas enters the inlet part at a second flow rate. When the gas flows through the sequencing pores, it can displace part of the non-polar solvent in the sequencing pores, and the displaced non-polar solvent is discharged from the outlet part. S40. The introduced substance is a second polar solvent. The second polar solvent enters from the inlet part. When the second polar solvent flows through the sequencing pores, it can displace the gas and part of the non-polar solvent in the sequencing pores, and the displaced gas and non-polar solvent are discharged from the outlet part.

23. The film forming method according to claim 22, characterized in that, In step S20, the junction between the first polar solvent and the non-polar solvent forms the lower surface of the amphiphilic molecular film. In step S40, the junction between the non-polar solvent and the second polar solvent forms the upper surface of the amphiphilic molecular film.

Images