WO2024000739A1

WO2024000739A1 - Microfluid device for nanopore sensor, and assembly method therefor

Info

Publication number: WO2024000739A1
Application number: PCT/CN2022/111305
Authority: WO
Inventors: 傅宝柱; 周智
Original assignee: 深圳市梅丽纳米孔科技有限公司
Priority date: 2022-07-01
Filing date: 2022-08-10
Publication date: 2024-01-04
Also published as: CN115121303B; CN115121303A

Abstract

A microfluid device for a nanopore sensor, and an assembly method. The microfluid device comprises a bearing plate (1), a cover plate (2), and a nanopore sensing assembly located in a fluid path, wherein a microfluid cavity (13) having a fluid inlet (11) and a fluid outlet (12) and configured for a sequencing fluid to flow is provided on an upper side of the bearing plate (1); the cover plate (2) is connected to the bearing plate (1); the nanopore sensing assembly comprises a printed circuit board (31), a nanopore chip (32), and an electrically conductive member; the nanopore chip (32) is arranged on an upper side of the printed circuit board (31), and the nanopore chip (32) is provided with a sensing chamber (322) which has a plurality of nanopores (321) and communicates with the microfluid cavity (13); and the electrically conductive member is connected to the printed circuit board (31) and extends into the sensing chamber (322). The microfluid device is easy to realize in terms of structure, is easy to assemble and disassemble, and has good practicability. In addition, by means of a fluid conveying device, film application/pore embedding operations can be completed with the minimum amount of the sequencing fluid, thereby avoiding waste of the sequencing fluid.

Description

Microfluidic devices for nanopore sensors and methods of assembly thereof

This application is based on the Chinese patent application with application number 202210774477.

Technical field

The present application relates to all fields of biological nanopore sensor sequencing, disease analysis and medical disease analysis, and particularly relates to a microfluidic device for a nanopore sensor and its assembly method.

Background technique

At present, biological nanopore technology is usually based on natural pore-forming proteins. The channels formed by pore-forming proteins can be passed through by long DNA chains or polypeptide chain molecules. Due to different bases or amino acids, corresponding current changes will occur, and the instrument will identify them through From changes in the electrical signal, the sequence of bases or amino acids passing through the nanopore can be inferred.

Various microfluidic devices and sensors are known. For example, a microfluidic device disclosed by patent WO2018/007819 is used to prepare a test liquid for sensing analytes present therein. After the microfluidic device is assembled, the nanopore sensor inside the microfluidic device must be soaked in In liquids, there are strict regulations on the time of manufacture and use, that is, they have a certain shelf life. If they cannot be used within the shelf life, the liquid will easily dry out or the nanopores will fall off, etc., resulting in the failure of the nanopore chip sensor.

At the same time, the microfluidic system in the existing technology has complicated operations and will cause a waste of sequencing fluid during the process of coating/embedding sequencing fluid into holes. In other words, based on the value and cost of DNA sequencing fluid in the field of DNA sequencing, It is very high. If the sequencing solution cannot complete the membrane coating/hole inserting operation in a smaller amount, the cost of the microfluidic system in the existing technology will be higher.

Application content

The purpose of this application is to provide a microfluidic device for a nanopore sensor and an assembly method thereof, aiming to solve the problem that existing microfluidic systems cause a waste of sequencing fluid when injecting sequencing fluid.

In order to solve the above technical problems, the purpose of this application is achieved through the following technical solution: providing a microfluidic device for a nanopore sensor, including:

A carrier plate, a microfluidic cavity having a fluid inlet and a fluid outlet and used for flowing sequencing fluid is provided on the upper side of the carrier plate;

a cover plate, which is connected to the carrier plate and used to seal the microfluidic cavity between the fluid inlet and the fluid outlet;

A nanopore sensing component located on the fluid path, the nanopore sensing component includes:

Printed circuit board, the printed circuit board is connected to the lower side of the bearing plate through a fixing plate;

Nanopore chip, the nanopore chip is arranged on the upper side of the printed circuit board and passes through the fixed plate and is in sealing contact with the lower side of the carrier plate. The nanopore chip is arranged with a plurality of nanopores and is connected with the The sensing chamber connected to the microfluidic cavity is used to receive at least a part of the sequencing solution;

A conductive component, the conductive component is connected to the printed circuit board and extends into the sensing chamber.

Furthermore, it also includes:

A first sealing member, the first sealing member is connected to the bearing plate and used to seal the fluid inlet;

A second sealing member is connected to the bearing plate and used to seal the fluid outlet.

Further, the conductive member is a conductive electrode including a contact portion, a connecting portion and a connecting portion connected in sequence;

Wherein, a part of the connecting portion is embedded in the upper side of the bearing plate and exposed from the upper side of the bearing plate;

The contact portion passes through the carrier plate and extends into the sensing chamber;

The connecting portion passes through the carrier board and is connected to the printed circuit board.

Further, all the nanopores are enclosed in a ring shape, the geometric center line of the contact part coincides with the geometric center line formed by all the nanopores, and the end of the contact part has a predetermined distance from the bottom of the sensing chamber. Set a gap.

Further, the conductive member is manufactured by vacuum evaporation, printing, electroplating, or inkjet.

Further, the conductive member is made of one or more precious metals such as ruthenium, rhodium, palladium, platinum, gold or silver, or their compounds.

Further, a third sealing member for sealing the sensing chamber is provided between the sensing chamber and the carrier plate.

Further, a waterproof and breathable membrane is provided at the fluid outlet.

Further, the cover plate is provided with a drip hole connected to the fluid inlet, and the first sealing member closely matches the drip hole for sealing the drip hole;

The drip hole includes a guide portion and a communication portion that are connected. The bottom surface of the guide portion is inclined and extends downward. Two side surfaces of the guide portion are contracted toward the communication portion. The upper end of the communication portion Located at the lowest end of the guide part, the lower end of the communication part is connected with the fluid inlet.

Further, it also includes a fourth sealing member, a liquid inlet column is provided on the lower side of the load-bearing plate, and a liquid inlet hole connected to the fluid inlet is provided on the liquid inlet column, wherein the fourth sealing member is To seal the liquid inlet hole.

Further, the conductive member includes several sets of negative electrodes and positive electrodes arranged on the nanopore chip, wherein the negative electrode is located in the sensing chamber and is used to provide a negative voltage, and the positive electrode is used to provide a positive voltage. Voltage.

Further, the nanopore chip is provided with identification elements for identifying the positions of pins on the nanopore chip.

Embodiments of the present application also provide a method for assembling a microfluidic device for a nanopore sensor as described above, including the following steps:

S101. Fix the pre-assembled nanopore sensing components to the lower side of the fixing plate by bolting;

S102. Fix the fixing plate to the lower side of the load-bearing plate by bolting;

S103. Embed the conductive electrode from the upper side of the carrier plate so that one end of the conductive electrode extends into the sensing chamber and the other end is connected to the printed circuit board;

S104. Fix the cover plate to the upper side of the carrier plate by bonding to form a microfluidic cavity having a fluid inlet and a fluid outlet.

Embodiments of the present application provide a microfluidic device for a nanopore sensor and an assembly method thereof, wherein: the microfluidic device for a nanopore sensor includes a carrier plate, a cover plate, and a nanopore sensor located on a fluid path. Assembly, wherein a microfluidic chamber with a fluid inlet and a fluid outlet for flowing sequencing fluid is provided on the upper side of the carrier plate; the cover plate is connected to the carrier plate for sealing the fluid inlet and fluid outlet. The microfluidic cavity between the outlets; the nanopore sensing assembly includes a printed circuit board, a nanopore chip and a conductive member, wherein the printed circuit board is connected to the lower side of the carrier plate through a fixed plate; the nanopore The hole chip is arranged on the upper side of the printed circuit board and passes through the fixed plate and is in sealing contact with the lower side of the carrier plate. The nanopore chip is arranged with a plurality of nanopores and is connected to the microfluidic cavity. A sensing chamber configured to receive at least a portion of the sequencing fluid; a conductive component connected to the printed circuit board and extending into the sensing chamber.

The microfluidic device of the embodiment of the present application has a simple structure, is easy to assemble and disassemble, and has good practicality.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, which are of great significance to this field. Ordinary technicians can also obtain other drawings based on these drawings without exerting creative efforts.

Figure 1 is a schematic structural diagram of a microfluidic device for a nanopore sensor provided in Embodiment 1 of the present application;

Figure 2 is an exploded schematic diagram of a microfluidic device for a nanopore sensor provided in Embodiment 1 of the present application;

Figure 3 is a top view of a microfluidic device for a nanopore sensor provided in Embodiment 1 of the present application;

Figure 4 is a schematic structural diagram of a nanopore chip in a microfluidic device for a nanopore sensor provided in Embodiment 1 of the present application;

Figure 5 is a partial cross-sectional view of a microfluidic device for a nanopore sensor provided in Embodiment 1 of the present application;

Figure 6 is a bottom view of the microfluidic device used for nanopore sensors provided in Embodiment 1 of the present application;

Figure 7 is a schematic structural diagram of a nanopore chip in a microfluidic device for a nanopore sensor provided in Embodiment 2 of the present application;

FIG. 8 is a flow chart of an assembly method of a microfluidic device for a nanopore sensor provided in Embodiment 3 of the present application.

Description of the marks in the picture:

1. Bearing plate; 11. Fluid inlet; 12. Fluid outlet; 13. Microfluidic cavity; 14. Liquid inlet flow channel; 15. Waste liquid collection flow channel; 2. Cover plate; 3. Fixing plate; 31. Printing Circuit board; 311, contact; 32, nanopore chip; 321, nanopore; 322, sensing chamber; 323, pin; 33, conductive electrode; 331, contact part; 332, connection part; 333, connection part; 34. Fixing bolt; 35. Locking bolt; 36. Negative electrode; 37. Positive electrode; 38. Identification piece; 4. First seal; 41. Second seal; 42. Fitting part; 43. Sealing part ; 5. Third seal; 6. Drip hole; 7. Fourth seal; 71. Liquid inlet column; 72. Liquid inlet hole; 73. Waterproof and breathable membrane; 8. Lower liquid column; 81. Lower liquid hole ; 9. Positioning sink; 10. Allowance hole; 101. Anti-slip protrusion.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of this application.

It should be understood that, when used in this specification and the appended claims, the terms "comprises" and "comprises" indicate the presence of described features, integers, steps, operations, elements and/or components but do not exclude the presence of one or The presence or addition of multiple other features, integers, steps, operations, elements, components and/or collections thereof.

It should also be understood that the terminology used in the specification of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this specification and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly dictates otherwise.

It will be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items. .

Example 1:

With reference to Figures 1 to 6, embodiments of the present application provide a microfluidic device for a nanopore sensor, including:

The carrier plate 1 is provided with a microfluidic cavity 13 having a fluid inlet 11 and a fluid outlet 12 on the upper side for flowing sequencing fluid;

Cover plate 2, which is connected to the carrier plate 1 and used to seal the microfluidic cavity 13 between the fluid inlet 11 and the fluid outlet 12;

Printed circuit board 31, the printed circuit board 31 is connected to the lower side of the carrier plate 1 through the fixing plate 3;

Nanopore chip 32. The nanopore chip 32 is arranged on the upper side of the printed circuit board 31 and passes through the fixed plate 3 and is in sealing contact with the lower side of the carrier plate 1. The nanopore chip 32 is arranged with multiple A nanopore 321 and a sensing chamber 322 connected with the microfluidic chamber 13 are used to receive at least a part of the sequencing solution;

A conductive member is connected to the printed circuit board 31 and extends into the sensing chamber 322 .

In this embodiment, based on the highly transparent nature of PMMA material, the carrier plate 1, cover plate 2, and fixing plate 3 of this application are preferably made of PMMA material to facilitate the user to observe the microfluidic cavity 13 and the flow. Sequencing fluid on the microfluidic chamber 13, but in actual application scenarios, the materials of the carrier plate 1, cover plate 2, and fixed plate 3 can also be of other types, as long as they do not chemically react with the sequencing fluid. , so this application will not elaborate further.

2 and 3 , in this embodiment, the microfluidic cavity 13 has a groove structure, that is, the microfluidic cavity 13 is formed by a groove that is recessed downward from the bearing plate 1 . In this way, the support plate 1 can be ensured. While having structural strength, the thickness and materials of the entire load-bearing plate 1 can be reduced, and the contact area between the cover plate 2 and the load-bearing plate 1 can be increased. More preferably, the cover plate 2 is embedded in the upper side of the load-bearing plate 1, even if The upper surface of the cover plate 2 is flush with the upper surface of the load-bearing plate 1; but in another embodiment, two convex strips can also be formed upward from the upper side of the load-bearing plate 1, and the space between the two convex strips forms The microfluidic cavity 13 of this application will not be described again in this application.

It should be noted that the cover plate 2 can be hollow, and the microfluidic chamber 13 is closed except for the fluid opening and the fluid outlet 12 to ensure that the sequencing liquid entering from the fluid inlet 11 can enter smoothly along the fluid path. The sensing chamber 322 until the sensing chamber 322 is filled with sequencing fluid, during this process, the gas (usually air) in the microfluidic chamber 13 is replaced by the sequencing fluid and discharged through the fluid outlet 12 .

During manufacturing, the fluid inlet 11 and the fluid outlet 12 may be configured at opposite ends of the carrier plate 1 , but in this application: the fluid inlet 11 and the fluid outlet 12 are configured at the same end of the carrier plate 1 to improve the fluid path. The length of the fluid path of the present application may be constructed in a linear manner or in a non-linear manner. In other words, the fluid path may have at least one part of various irregular shapes such as a curved shape. It should be understood that the fluid path of the present application may be constructed in a linear manner or in a non-linear manner. The microfluidic chamber 13 has reasons to change its area on each vertical section based on the required flow rate of the sequencing fluid.

4, it is worth noting that this application does not specifically limit the type of the nanopore chip 32. According to actual detection needs, the nanopore chip 32 can be used for nucleic acid (for example, DNA) sequencing. The nanopore chip 32 includes A large number of sensors (not shown) arranged in an array, so that polynucleotides or polymers of nucleic acids, polypeptides such as proteins, polysaccharides or other polymers fused through the nanopore 321 can come into contact with the sensors, so that the sensors can sense the sequencing fluid , and transmit the detected information to an external equipment analysis instrument. In other words, this application will prepare the number and pore size of the nanopores 321 according to the type of the nanopore chip 32 .

Looking back at Figures 2 and 3, the sensing chamber 322 of this embodiment is located in the middle of the fluid path close to the fluid inlet 11. For convenience, this embodiment uses the sensing chamber 322 as a dividing point, dividing the sensing chamber 322 into The microfluidic cavity 13 between the fluid inlet 11 and the fluid inlet 11 is named the inlet flow channel 14, and the microfluidic cavity 13 between the sensing chamber 322 and the fluid outlet 12 is named the waste liquid collection channel 15. Due to the nanopore The chip 32 is located on the lower side of the carrier plate 1, so the inlet flow channel 14 extends from the fluid inlet 11 to directly above the sensing chamber 322, then extends downward and communicates with the sensor, while the waste liquid collection flow channel 15 extends from the sensing chamber 322 The connected port extends upward and horizontally to the fluid outlet 12. Generally speaking, in the actual sequencing usage scenario, the user introduces the sequencing fluid from the fluid inlet 11 (a pump or other equipment can be used), and the sequencing fluid flows in along the fluid inlet 11. The flow channel 14 enters the sensing chamber 322, fills the sensing chamber 322, and then enters the waste liquid collection flow channel 15 through the port of the waste liquid collection flow channel 15. In this process, through the cooperation of the conductive parts and the nanopore chip 32 Use it to sense the sequencing fluid, which is simple and easy to operate.

More specifically, the cover plate 2 is fixed with the carrier plate 1 by bonding (using chemical adhesive such as shadowless glue) to seal the microfluidic chamber 13, and the fixed plate 3 is bolted ( For example, fixing bolts 34) are fixedly connected to the load-bearing plate 1. The printed circuit board 31 is fixedly connected to the load-bearing plate 1 by bolting (for example, locking bolts 35). The fixing bolts 34 and the locking bolts 35 are evenly arranged. Multiple, for example, 6 of them are provided respectively to ensure the structural strength of the fixed plate 3 and the carrier plate 1, as well as the structural strength of the printed circuit board 31 and the carrier plate 1. The specific assembly steps of the microfluidic device of the present application can be as follows :

First, the nanopore chip 32 is fixed on the corresponding position of the printed circuit board 31. It should be noted that a plurality of contacts 311 are pre-arranged on the lower side of the printed circuit board 31 away from the nanopore chip 32 for communicating with the nanopore chip 32. The pins 323 are connected, and then the printed circuit board 31 is fixedly installed on the lower side of the carrier plate 1 using the locking bolts 35, where the fixing plate 3 is provided with a relief hole 10 for the chip to pass through; and then the fixing bolts 34 are used to secure the The fixed plate 3 is fixedly connected to the carrier plate 1, and finally the cover plate 2 and the carrier plate 1 are fixedly connected using shadowless glue to complete the assembly of the microfluidic device. In general, the structure of this application is simple to implement and easy to assemble and disassemble. Moreover, the nanopore chip 32 itself can be quickly mass-produced, the manufacturing process is simple, and the price is cheap.

Looking back at Figures 2 and 3, in a specific embodiment, the microfluidic device for nanopore sensors of the present application also includes:

The first sealing member 4 is connected to the carrier plate 1 and used to seal the fluid inlet 11;

The second sealing member 41 is connected to the carrier plate 1 and is used to seal the fluid outlet 12 .

In this embodiment, after the microfluidic device is assembled, the first seal 4 is used to seal the fluid inlet 11 and the second seal 41 is used to seal the fluid outlet 12 to prevent foreign matter from entering the microfluidic chamber during transportation. 13. At the same time, in an actual detection scenario, after the sequencing liquid fills the entire microfluidic cavity 13, the first seal 4 and the second seal 41 need to be used to seal the entire microfluidic cavity 13. In addition, it should be noted that , the first seal 4 and the second seal 41 are detachably attached.

In a specific embodiment, a waterproof and breathable membrane 73 is provided at the fluid outlet 12 .

In this embodiment, a lower liquid column 8 is integrally formed on the lower side of the fixed plate 3. The lower liquid column 8 is integrally formed with a lower liquid hole 81 connected to the outlet. The lower liquid hole 81 can be connected to the filling device through a pipeline ( For example, a pump) is connected, and the sequencing fluid is pumped out of the microfluidic chamber 13 through the pump to drain the sequencing fluid located in the microfluidic chamber 13. The second seal 41 is provided on the lower side of the lower liquid hole 81 to seal the lower fluid. Hole 81 is the sealed fluid outlet 12. The waterproof and breathable membrane 73 is embedded on the fixed plate 3, located directly below the fluid outlet 12 and directly above the lower liquid hole 81. The waterproof and breathable membrane 73 can prevent waste liquid from flowing to the microfluidic device. outside to prevent the sequencing fluid from causing safety hazards due to its own corrosiveness, and the waterproof and breathable membrane 73 is breathable and can ensure smooth vacuum and negative pressure drainage.

In a specific embodiment, the conductive element is a conductive electrode 33 including a contact portion 331, a connecting portion 332 and a connecting portion 333 connected in sequence;

Wherein, a part of the connecting portion 332 is embedded in the upper side of the bearing plate 1 and exposed from the upper side of the bearing plate 1;

The contact portion 331 passes through the carrier plate 1 and extends into the sensing chamber 322;

The connecting portion 333 passes through the carrier board 1 and is connected to the printed circuit board 31 .

In this embodiment, the contact portion 331, the connecting portion 332 and the connecting portion 333 connected in sequence form a U-shaped structure, so that the conductive electrode 33 can be inserted into the carrier plate 1 in a U-shaped manner, making the operation simple and convenient.

4 and 5 , in a specific embodiment, all the nanopores 321 are enclosed in a ring shape, the geometric center line of the contact portion 331 coincides with the geometric center line formed by all the nanopores 321 , and the There is a preset gap between the end of the contact portion 331 and the bottom of the sensing chamber 322 .

In this embodiment, the nanopores 321 are arranged on the bottom wall of the sensing chamber 322, and all the nanopores 321 are enclosed in a square shape, but alternatively, all the nanopores 321 can be enclosed in a circular shape, or other special shapes, There are no specific limitations in this application.

In a specific embodiment, a third seal 5 for sealing the sensing chamber 322 is provided between the sensing chamber 322 and the carrier plate 1 .

In this embodiment, the space of the sensing chamber 322 is in the shape of a truncated cone, so the third sealing member 5 is in the shape of a ring. The outside of the third sealing member 5 can be connected to the sensing chamber 322 by bonding to facilitate fixation. When the plate 3 and the carrier plate 1 are fixedly installed, the surface of the third seal 5 will be squeezed by the lower side of the carrier plate 1 and deformed, thereby sealing the sensing chamber 322 and preventing the sequencing liquid from overflowing, so that the sequencing liquid is only in the nanopore. 321 position flow.

In a specific embodiment, a plurality of sets of positioning sinks 9 are provided on the upper side of the fixed plate 3 , and a positioning groove (not shown) for each of the positioning sinks 9 to be embedded is provided on the lower side of the load-bearing plate 1 .

Looking back at Figure 2, in this embodiment, there are 3 groups of positioning sinks 9 evenly arranged. Each group of positioning sinks 9 includes 2 symmetrically arranged positioning sinks 9. Correspondingly, there are 6 positioning sinks 9 on the lower side of the load-bearing plate 1. During the assembly process, directly align the positioning sink 9 with the positioning groove and embed it, so that the relative positions of the fixed plate 3 and the load-bearing plate 1 can be quickly positioned, and the sensing chamber 322 and the waste liquid collection flow channel 15 can be ensured to be in contact with the inlet. The liquid flow channels 14 are connected.

More preferably, the positioning sink 9 of the present application is provided with threaded holes along its length direction for cooperating with the fixing bolts 34 passing through the bearing plate 1 to improve the structural compactness of the microfluidic device of the present application.

1 and 2, in a specific embodiment, the cover plate 2 is provided with a drip hole 6 connected with the fluid inlet 11, and the first seal 4 closely matches the drip hole 6, To seal the dripping hole 6 .

In this embodiment, the position height of the drip hole 6 is higher than the fluid inlet 11, which is convenient for the user to drip the sequencing liquid from the drip hole 6 through a drip tool (such as a dropper tube), so that the sequencing liquid flows from the drip hole 6 under the action of gravity. The drip hole 6 falls into the fluid inlet 11 and the microfluidic chamber 13 .

When it is necessary to seal the microfluidic cavity 13, directly insert the first sealing member 4 into the dripping hole 6. The first sealing member 4 will be deformed after being squeezed by the hole wall of the dripping hole 6, thereby sealing the dripping hole 6. Easy to assemble and disassemble.

In a specific embodiment, the drip hole 6 includes a guide part and a communication part that are connected, the bottom surface of the guide part is inclined and extends downward, and the two side surfaces of the guide part shrink toward the communication part. The upper end of the connecting portion is located at the lowest end of the guide portion, and the lower end of the connecting portion is connected to the fluid inlet 11 .

In this embodiment, the first seal 4 includes a fitting portion 42 that matches the shape of the guide portion and a sealing portion 43 integrally formed on the lower side of the fitting portion 42 . The sealing portion 43 is tightly connected to the connecting portion 333 . The portion 42 is attached to the guide portion. In other words, the first sealing member 4 is embedded in the drip hole 6 and forms a snap-fit relationship with the cover plate 2 , thereby sealing the drip hole 6 . It should be noted that the guide part of this application is in a triangular shape, and the guide part is used to receive the sequencing fluid falling on the surface of the guide part, and guide and collect the sequencing fluid to the connection part 333 until it flows from the connection part 333 to the fluid inlet 11. Therefore, the connecting portion 333 is integrally formed on the lower side of the tip of the guide portion.

More preferably, the microfluidic device for nanopore sensors of the present application also includes a fourth seal 7. A liquid inlet column 71 is provided on the lower side of the carrier plate 1, and a liquid inlet column 71 is provided on the liquid inlet column 71. The fluid inlet 11 communicates with the liquid inlet 72 , wherein the fourth seal 7 is used to seal the liquid inlet 72 .

In this embodiment, the fixed plate 3 is provided with a through hole for the liquid inlet column 71 to pass through. In actual application scenarios, the liquid inlet hole 72 can be connected to a filling device (such as a pump) through a pipeline, and the sequencer can be sequenced through the pump. Injecting the liquid into the microfluidic cavity 13 has the advantage of reducing manual operations and improving detection efficiency compared to the method of using a dropper to drip the sequencing liquid from the dropper hole 6 .

It is worth noting that during the actual design process of the microfluidic device, it can be designed only as a microfluidic device with a drip hole 6, or only as a microfluidic device with a liquid inlet column 71, or even as a microfluidic device. A microfluidic device with both a drip hole 6 and a liquid inlet column 71 is provided to improve the selection diversity and practicality of the microfluidic device.

It should be noted that the first sealing member 4, the second sealing member 41, the third sealing member 5 and the fourth sealing member 7 of the present application can all be made of silicone material, but it should be understood that all seals The parts can also be made of other materials with better sealing performance, and the liquid inlet column 71 and the lower liquid column 8 of this application need to be connected to the corresponding pumps, so the second sealing member 41 and the fourth sealing member 7 are both arranged in an annular shape. .

More preferably, anti-slip protrusions 101 are integrally formed on opposite sides of the load-bearing plate 1 and the fixing plate 3, thereby facilitating the user to assemble the microfluidic device.

In a specific embodiment, the conductive component is manufactured by vacuum evaporation, printing, electroplating, or inkjet.

In this embodiment, the conductive electrode 33 can be manufactured through a vacuum evaporation or printing or electroplating or inkjet process.

In a specific embodiment, the conductive element is made of one or more precious metals such as ruthenium, rhodium, palladium, platinum, gold or silver, or their compounds.

In this embodiment, the conductive electrode 33 is preferably made of platinum material, but it should be understood that during manufacture, the conductive electrode 33 can also be made of other precious metal materials, which is not specifically limited in this application.

Example 2:

7 , the difference between this embodiment and Embodiment 1 lies in the conductive member, specifically: the conductive member includes several groups of negative electrodes 36 and positive electrodes 37 disposed on the nanopore chip 32, wherein the negative electrodes 36 is located in the sensing chamber 322 and is used to provide a negative voltage, and the positive electrode 37 is used to provide a forward voltage.

Since the conductive electrode 33 is mostly made of precious metals (such as platinum, rhodium, palladium, gold), which results in high manufacturing cost of the microfluidic device, this embodiment uses the negative electrode 36 to provide the negative voltage, and the positive electrode 37 to provide Forward voltage to change the situation that the existing nanopore electrode only has microporous negative electrode (the disadvantages of only microporous negative electrode are as follows: after the external reagent completes the micropore coating and embedding the hole, the external electrode still needs to apply the forward pulling voltage , the external circuit can successfully read the DNA step current), that is to say, the microfluidic device obtained in this embodiment can achieve the effects of low manufacturing cost and strong applicability.

Furthermore, the nanopore chip 32 is provided with an identification piece 38 for identifying the position of the pin 323 on the nanopore chip 32 .

In this embodiment, the identification piece 38 is an identification groove provided on a top corner of the nanohole chip 32. It should be noted that during manufacturing, the identification piece 38 can also be a colored identification piece according to actual needs during manufacturing. , as long as it can play a role in positioning the pins, so this application will not go into details.

Embodiment three:

In conjunction with Figure 8, an embodiment of the present application also provides a method for assembling a microfluidic device for a nanopore sensor as described above, including the following steps:

S101. Fix the pre-assembled nanopore sensing components to the lower side of the fixing plate 3 by bolting;

S102. Fix the fixing plate 3 to the lower side of the bearing plate 1 by bolting;

S103. Embed the conductive electrode 33 from the upper side of the carrier plate 1 so that one end of the conductive electrode 33 extends into the sensing chamber 322 and the other end is connected to the printed circuit board 31;

S104. Fix the cover plate 2 to the upper side of the carrier plate 1 by bonding to form a microfluidic cavity 13 having a fluid inlet 11 and a fluid outlet 12.

In this embodiment, the nanopore chip 32 is first fixed on the corresponding position of the printed circuit board 31. It should be noted that a plurality of contacts 311 are pre-arranged on the lower side of the printed circuit board 31 away from the nanopore chip 32. To connect with the pins 323 of the nanopore chip 32, the printed circuit board 31 is then fixedly installed on the underside of the carrier plate 1 using the locking bolts 35, where the fixing plate 3 is provided with a relief hole 10 for the chip to pass through; then Then use the fixing bolts 34 to firmly connect the fixed plate 3 to the carrier plate 1, then insert the conductive electrode 33 into the corresponding position, and finally use shadowless glue to firmly connect the cover plate 2 and the carrier plate 1 to complete the assembly of the microfluidic device. Generally speaking, the structure of the present application is simple to implement, easy to assemble and disassemble, and the nanopore chip 32 itself can be quickly mass-produced, the manufacturing process is simple, and the price is cheap.

The above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person familiar with the technical field can easily think of various equivalent methods within the technical scope disclosed in the present application. Modification or replacement, these modifications or replacements shall be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claims.

Claims

A microfluidic device for a nanopore sensor, characterized by comprising:

A carrier plate (1), a microfluidic cavity (13) having a fluid inlet (11) and a fluid outlet (12) and used for the flow of sequencing fluid is provided on the upper side of the carrier plate (1);

Cover plate (2), which is connected to the carrier plate (1) and used to seal the microfluidic cavity (13) between the fluid inlet (11) and the fluid outlet (12);

A nanopore sensing component located on the fluid path, the nanopore sensing component includes:

Printed circuit board (31), the printed circuit board (31) is connected to the lower side of the carrying plate (1) through the fixing plate (3);

Nanopore chip (32), the nanopore chip (32) is arranged on the upper side of the printed circuit board (31) and passes through the fixed plate (3) and is in sealing contact with the lower side of the carrier plate (1) , the nanopore chip (32) is provided with a sensing chamber (322) having a plurality of nanopores (321) and connected with the microfluidic chamber (13), for receiving at least a part of the sequencing solution;

A conductive member, which is connected to the printed circuit board (31) and extends into the sensing chamber (322).
The microfluidic device for nanopore sensor according to claim 1, further comprising:

A first seal (4), the first seal (4) is connected to the carrier plate (1) and used to seal the fluid inlet (11);

Second sealing member (41), the second sealing member (41) is connected to the carrying plate (1) and is used to seal the fluid outlet (12).
The microfluidic device for nanopore sensor according to claim 1, characterized in that the conductive member is a conductive electrode including a contact portion (331), a connecting portion (332) and a connecting portion (333) connected in sequence. (33);

Wherein, a part of the connecting portion (332) is embedded in the upper side of the bearing plate (1) and exposed from the upper side of the bearing plate (1);

The contact portion (331) passes through the bearing plate (1) and extends into the sensing chamber (322);

The connecting portion (333) passes through the carrier plate (1) and is connected to the printed circuit board (31).
The microfluidic device for nanopore sensor according to claim 3, characterized in that:

All the nanopores (321) are enclosed in a ring shape, the geometric centerline of the contact portion (331) coincides with the geometric centerline formed by all the nanopores (321), and the end of the contact portion (331) There is a preset gap with the bottom of the sensing chamber (322).
The microfluidic device for a nanopore sensor according to claim 1, wherein the conductive member is manufactured by vacuum evaporation, printing, electroplating, or inkjet.
The microfluidic system for nanopore sensors according to claim 1, wherein the conductive member is made of one or more precious metals such as ruthenium, rhodium, palladium, platinum, gold or silver, or their compounds. Become.
The microfluidic device for nanopore sensor according to claim 1, characterized in that: a device for sealing the sensing chamber (322) is provided between the sensing chamber (322) and the carrier plate (1). ) of the third seal (5).
The microfluidic device for nanopore sensor according to claim 1, characterized in that: a waterproof and breathable membrane (73) is provided at the fluid outlet (12).
The microfluidic device for nanopore sensor according to claim 2, characterized in that: the cover plate (2) is provided with a drip hole (6) connected with the fluid inlet (11), and the third A sealing member (4) closely matches the dripping hole (6) for sealing the dripping hole (6);

Wherein, the drip hole (6) includes a guide part and a communication part that are connected, the bottom surface of the guide part is inclined and extends downward, and two side surfaces of the guide part are contracted toward the communication part, The upper end of the communication part is located at the lowest end of the guide part, and the lower end of the communication part is connected with the fluid inlet (11).
The microfluidic device for nanopore sensors according to any one of claims 2 to 9, characterized in that it also includes a fourth seal (7), and a liquid inlet column is provided on the lower side of the carrier plate (1) (71), the liquid inlet column (71) is provided with a liquid inlet (72) connected to the fluid inlet (11), wherein the fourth seal (7) is used to seal the liquid inlet. hole (72).
The microfluidic device for nanopore sensor according to claim 1, characterized in that: the conductive member includes several sets of negative electrodes (36) and positive electrodes (37) arranged on the nanopore chip (32), wherein , the negative electrode (36) is located in the sensing chamber (322) and is used to provide a negative voltage, and the positive electrode (37) is used to provide a forward voltage.
The microfluidic device for a nanopore sensor according to claim 11, characterized in that: the nanopore chip (32) is provided with a pin (323) for identifying the nanopore chip (32). Location identification piece (38).
A method of assembling a microfluidic device for a nanopore sensor according to any one of claims 1 to 12, characterized by comprising the following steps:

S101. Fix the pre-assembled nanopore sensing components to the underside of the fixing plate (3) by bolting;

S102. Fix the fixing plate (3) to the lower side of the load-bearing plate (1) by bolting;

S103. Embed the conductive electrode (33) from the upper side of the carrier plate (1) so that one end of the conductive electrode (33) extends into the sensing chamber (322) and the other end is connected to the printed circuit. Plate (31) connection;

S104. Fix the cover plate (2) to the upper side of the carrier plate (1) by bonding to form a microfluidic cavity (13) having a fluid inlet (11) and a fluid outlet (12).