Composite flexible microfluidic solid-state nanopore detection liquid cell device

Abstract

The application discloses a composite flexible micro-flow channel solid-state nanopore detection liquid pool device, which comprises upper liquid pool boxes, upper sealing films, a nanopore chip, lower sealing films, lower liquid pool boxes, a liquid delivery device and electrodes which are sequentially and sealingly connected; first and second intermediate through holes are arranged on the upper and lower sealing films respectively, and the nanopore chip is arranged between the first and second intermediate through holes; the inner surfaces of the upper and lower liquid pool boxes are respectively provided with upper and lower flow channels; the upper and lower flow channels are outwardly provided with upper and lower injection ports and upper and lower backflow ports; the liquid delivery device is connected with the upper and lower injection ports and the upper and lower backflow ports; and the electrodes are embedded at the upper and lower injection ports; the upper and lower liquid pool boxes and the upper and lower sealing films are bonded to form complete flow channels, and the through holes of the sealing films correspond to the flow channels and are connected with the flow channels, the application meets the requirements of portability and traceability and has better sealing performance, meanwhile, the liquid pool device can complete detection work with a small amount of detection liquid and does not cause waste of the detection liquid.

Description

Technical Field

[0001] This invention relates to nanopore sensor technology, belonging to the technical fields of flexible material fabrication, gene sequencing, protein analysis, disease monitoring, and environmental monitoring, specifically involving a composite flexible microfluidic solid nanopore detection liquid pool device. Background Technology

[0002] With the development of micro- and nanotechnology, single-molecule detection and analysis techniques have become a hot area of ​​scientific research. They are crucial techniques for revealing the properties, interactions, and dynamics of matter, providing unprecedented resolution and sensitivity for life sciences, materials science, and medical diagnostics. Inspired by biological ion channels, nanochannel single-molecule detection technology was proposed in 1996. This technology can detect single DNA or protein molecules in a high-throughput, label-free manner and identify their sequence structure, offering advantages such as high sensitivity, strong selectivity, and rapid response. Its principle involves measuring the passage of biomolecules through nanopores under an electric field, carrying them in an ion solution and generating a corresponding blocking current signal. The changes in this electrical signal characterize the structure and properties of the biomolecule being measured. A common nanopore sensor detection device consists of a solid nanopore chip clamped between two liquid pools, with the chip fixed between the pools by a sealing rubber ring. An Ag / AgCl electrode is inserted into the electrolyte solution in the pool, thus forming a basic nanopore detection system. A probe connected to the electrode is connected to a microcurrent amplifier and a digital-to-analog converter to collect and record the real-time current change signal of the biomolecule to be tested passing through the nanopore. The signal is then analyzed to achieve qualitative and quantitative detection of the biomolecule.

[0003] Traditional nanopore detection liquid cell devices typically have large capacities, require a large number of samples, and are not sealed, making them inconvenient to carry and prone to sample contamination. To meet the growing demand for portability and miniaturization, existing detection liquid cells need to be redesigned and modified to accommodate miniaturized samples and facilitate easy portability, thereby satisfying the development needs of nanopore detection technology in wearable and biomedical detection fields.

[0004] The technical differences compared to existing technologies are as follows:

[0005] Technical comparison with patent CN102866257A "A microfluidic sample boat with a liquid storage chamber and a pump chamber"

[0006] Patent CN102866257A proposes a microfluidic sample boat with a reservoir that also serves as a pump chamber. To address the problems of complex structures and inconvenient manufacturing and use of traditional microfluidic sample boats, it utilizes a flexible sealing layer to achieve dynamic control of the stored liquid, a separate dry powder reagent storage chamber to meet drying requirements, and a filtration structure to filter out substances in the test reagent that interfere with the detection results. The microfluidic sample boat with a reservoir that also serves as a pump chamber, as proposed in patent CN102866257A, aims to optimize fluid flow drive and sealing performance through a combination of rigid and flexible materials.

[0007] This patent proposes a solid-state nanopore detection liquid pool device based on flexible materials, focusing on solving the problems of large volume, high liquid injection volume requirements, and poor sealing of traditional nanopore detection liquid pools. It proposes a five-layer stacked architecture based on flexible material (PDMS), achieving portable, miniaturized, and high-precision detection through a Π-shaped flow channel design, plasma bonding, and injection / return port configuration. The liquid pool device proposed in this patent aims to improve sealing performance, reduce detection liquid waste, and adapt to wearable and real-time detection scenarios.

[0008] The two have fundamental differences in their research objectives.

[0009] Patent CN102866257A employs a four-layer structure (first sealing layer, solution storage chamber layer, second sealing layer, and reaction chamber / microchannel layer). Liquid is released by breaking the second sealing layer with external force. The combination of a rigid chamber layer and a flexible sealing layer achieves the function of the liquid storage chamber. Its technical highlights include the design of the filter membrane, dry powder reagent storage chamber, and pump chamber.

[0010] This patent provides a solid nanopore detection liquid pool device based on flexible materials. It adopts a five-layer stacked architecture (liquid pool box, sealing film, nanopore chip, etc.), and forms a closed flow channel through PDMS plasma bonding. The Π-shaped flow channel design optimizes the liquid flow distribution. An Ag / AgCl electrode is embedded in the injection port to connect to an external signal acquisition system. At the same time, the geometric symmetry design of the flow channel and the through hole, and the use of transparent PDMS material, enable visual detection.

[0011] The two differ fundamentally in their technical solutions and system design.

[0012] The microfluidic sample boat proposed in patent CN102866257A is suitable for scenarios requiring stepwise mixing of multiple reagents. It achieves on-demand release of reagents through dynamic sealing control and is suitable for multi-step biochemical reaction detection (such as blood analysis). However, its structural complexity is still relatively high and it relies on external mechanical intervention.

[0013] The solid nanopore detection liquid pool device based on flexible materials proposed in this patent reduces bubble interference through the design of micro-flow channels and reflux ports. Combined with the high sensitivity detection capability of nanopore chips, it is suitable for single-molecule detection scenarios such as gene sequencing and protein analysis, and supports portability and repeated disassembly.

[0014] The two are fundamentally different in terms of technical effects and application scenarios.

[0015] Technical Comparison with Patent CN101126765A "Microfluidic Sample Boat"

[0016] Patent CN101126765A proposes a microfluidic sample boat to address the problem of slow mixing of microfluidics in practical applications, hindering rapid reaction and efficient analysis. It promotes thorough mixing of microfluidics within microchannels and improves mixing efficiency through a simple microstructure and external pressure drive. The microfluidic sample boat proposed in patent CN101126765A combines a flexible material layer with hydraulic / pneumatic actuation to enhance reaction sensitivity, aiming to meet the rapid mixing requirements in chemical synthesis and biological detection.

[0017] This patent proposes a solid-state nanopore detection liquid pool device based on flexible materials. Addressing the issues of large volume and insufficient sealing in traditional nanopore liquid pools, it achieves miniaturization and high sealing performance through flexible materials and a stacked structure. This patent proposes a detection liquid pool aimed at improving sealing performance, reducing detection liquid waste, and adapting to wearable and real-time detection scenarios, providing a portable and stable solid-state nanopore detection liquid pool device.

[0018] The two differ fundamentally in their application scenarios and research objectives.

[0019] Patent CN101126765A focuses on a design that combines a substrate with flexible material layers, integrating modules such as a reaction chamber, pump chamber, and waste liquid chamber, and driving liquid circulation through hydraulic / pneumatic pressure. Its innovations include a ring-shaped microchannel design to reduce contamination and integrated detection functions within a biochip.

[0020] This patent provides a solid-state nanopore detection liquid pool device based on flexible materials. The key feature is the use of a five-layer PDMS stacked structure. A Π-shaped flow channel and symmetrical through-hole design ensure consistent liquid flow paths, while plasma bonding achieves seamless encapsulation of the nanopore chip. Simultaneously, the use of flexible PDMS material guarantees the flexibility of the liquid pool and its real-time monitoring capabilities.

[0021] The two are fundamentally different in terms of system design and technical effects.

[0022] Patent CN101126765A achieves rapid mixing through cyclic drive, making it suitable for biochemical assays requiring high-throughput reactions (such as PCR amplification), but its structure is complex and it relies on external pressure equipment.

[0023] This patent adopts a five-layer stacked architecture (liquid pool, sealing film, nanoporous chip, etc.), forming a closed flow channel through PDMS plasma bonding. The Π-shaped flow channel design optimizes the liquid flow distribution. The injection port is embedded with an Ag / AgCl electrode to connect to an external signal acquisition system. Through the micro-flow channel and high sealing design, it supports the use of detection liquids at levels as low as microliters, making it suitable for point-of-care testing in laboratory environments (such as pathogen screening), and it also has the advantage of being reusable.

[0024] The two solutions differ fundamentally in their technical approaches.

[0025] Technical Comparison with Patent KR1020220067927A "A Viscous Microfluidic Chip and Its Manufacturing Method"

[0026] Patent KR1020220067927A proposes a viscous microfluidic chip. Conventional microfluidic chip manufacturing methods produce microfluidic chips with microchannels and microcavities formed in a two-dimensional planar structure. However, these chips struggle to adhere to irregular shapes, such as rough surfaces, cylinders, and human skin. This paper aims to develop a microfluidic chip that can not only perform smooth component analysis of trace biochemical samples but also freely adhere to and detach from various types of object surfaces. The microfluidic chip proposed in patent KR1020220067927A aims to improve chip adhesion, adapt to diverse surfaces, and simplify the manufacturing process.

[0027] This patent proposes a solid-state nanopore detection liquid pool device based on flexible materials, focusing on solving the problems of large volume, high liquid volume requirements, and poor sealing of traditional nanopore detection liquid pools. It proposes a five-layer stacked architecture based on flexible material (PDMS), achieving portable, miniaturized, and high-precision detection through a Π-shaped flow channel design, plasma bonding, and injection / return port configuration. The proposed liquid pool device clamps nanopore chips to improve sealing, reduce detection liquid waste, and is designed for wearable and real-time detection scenarios.

[0028] The two differ fundamentally in their application scenarios and research objectives.

[0029] Simultaneously, ensuring that the liquid pool possesses good flexibility and deformation to adapt to different application scenarios is a fundamental problem that urgently needs to be solved for the rapid development of nanopore sensor technology. Summary of the Invention

[0030] The purpose of this invention is to provide a composite flexible microfluidic solid nanopore detection liquid pool device, which aims to solve the problems of traditional liquid pools being too large in size, difficult to assemble, requiring large liquid volume, and being inconvenient to carry.

[0031] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0032] A composite flexible microfluidic solid nanopore detection liquid pool device, characterized in that it comprises, from top to bottom, a top liquid pool box, a top sealing film, a nanopore chip, a bottom sealing film, a bottom liquid pool box, a liquid delivery device, and electrodes, all sealed and connected in sequence. The top and bottom sealing films are respectively provided with a first intermediate through-hole and a second intermediate through-hole. The nanopore chip is disposed between the first and second intermediate through-holes. The inner surfaces of the top and bottom liquid pool boxes are respectively provided with an upper flow channel and a lower flow channel. The upper flow channel has an upper injection port and an upper return port extending outwards. The upper and lower flow channels are provided with a lower inlet and a lower return port. The upper inlet and the lower inlet and the lower return port on the upper and lower flow channels are respectively connected to an infusion device. The electrodes are respectively embedded in the upper and lower inlets of the upper and lower flow channels. The upper liquid pool box, the lower liquid pool box, the upper sealing film, and the lower sealing film are correspondingly bonded to form a complete flow channel. The first intermediate through hole and the second intermediate through hole on the upper and lower sealing films are correspondingly connected to the upper and lower flow channels on the upper and lower liquid pool boxes.

[0033] Furthermore: the upper sealing film, the lower sealing film, the upper liquid pool box, and the lower liquid pool box are all made of a blended flexible material, polydimethylsiloxane. The thickness of the upper sealing film and the lower sealing film is 0.5 mm, and the thickness of the upper liquid pool box and the lower liquid pool box is 2 mm.

[0034] Furthermore, the sealing connection method between the upper liquid pool box and the upper sealing film, the lower sealing film, and the lower liquid pool box is plasma bonding.

[0035] Furthermore: the upper flow channel includes two parallel and symmetrically arranged vertical direct flow channels and a horizontally arranged horizontal flow channel. The horizontal flow channel is connected to the ends of the two vertical direct flow channels to realize the connection between the two vertical direct flow channels. The width of the vertical direct flow channel gradually increases from its center to the bottom. At the bottom opening with the largest opening, an upper injection port and an upper return port are punched on the outer surface of the upper liquid pool box. The infusion device is connected to the upper injection port and the upper return port. The lower flow channel has the same shape and size as the upper flow channel, and the horizontal flow channels of the two are on the same horizontal line. They are staggered in the horizontal position and symmetrical about the center line of the entire microfluidic device.

[0036] Furthermore: the electrode is an Ag / AgCl electrode, which is embedded in the upper and lower injection ports at the bottom of the vertical DC channel in the upper and lower flow channels. It is connected to an external signal acquisition instrument and a small current amplifier, and then connected to a digital-to-analog converter to complete signal analysis.

[0037] Furthermore: the upper flow channel on the upper liquid pool box is machined on its lower surface and a groove is formed on the lower surface, with a groove depth of 0.2mm; the lower flow channel on the lower liquid pool box is machined on its upper surface and a groove is formed on the upper surface, with a groove depth of 0.2mm.

[0038] Furthermore, the first and second intermediate through holes on the upper and lower sealing films are both square holes and circular holes that connect with each other. The thickness of the square hole is 0.08 mm, and the thickness of the circular hole is 0.42 mm. The square hole has the same size as the nanopore chip. The nanopore chip is installed between the square holes on the upper and lower sealing films and is sealed by plasma bonding between the upper and lower sealing films.

[0039] Furthermore: a point on the center line of the horizontal flow channel on the upper layer, the center of the first intermediate through hole, the center of the nanopore on the nanopore chip, the center of the second intermediate through hole, and a point on the center line of the horizontal flow channel of the lower liquid pool box are on the same vertical line.

[0040] In the above structure: This invention proposes a composite flexible microfluidic solid nanopore detection liquid pool device for nanopore sensors. This device is made of a five-layer stacked architecture, including an upper liquid pool box, an upper sealing film, a nanopore chip, a lower sealing film, a lower liquid pool box, a liquid delivery device, and electrodes, which are sequentially and sealed together from top to bottom. The upper and lower sealing films are respectively provided with a first intermediate through-hole and a second intermediate through-hole. The nanopore chip is disposed between the first and second intermediate through-holes. Upper and lower flow channels are respectively provided on the inner surfaces of the upper and lower liquid pool boxes. The upper flow channel has an upper injection port and an upper return port facing outwards, and the lower flow channel has a lower injection port and a lower return port facing outwards. The return port, including the upper injection port and upper return port, and the lower injection port and lower return port, are respectively connected to the infusion device. The electrodes are embedded in the upper injection port and lower injection port respectively. During installation, the upper liquid pool box, the lower liquid pool box and the upper sealing film and the lower sealing film are bonded to form a complete flow channel. The first intermediate through hole and the second intermediate through hole on the upper sealing film and the lower sealing film correspond to the upper flow channel and the lower flow channel on the upper liquid pool box and the lower liquid pool box respectively. The composite flexible microchannel solid nanopore detection liquid pool device meets the requirements of portability and miniaturization and has better sealing performance. At the same time, the detection work can be completed with a small amount of detection liquid through the infusion device, which will not cause waste of the detection sample.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] This invention provides a composite flexible microfluidic solid nanopore detection liquid pool device using flexible PDMS material. Based on its excellent flexibility and elasticity, the volume of the liquid pool is greatly reduced, enabling portable and miniaturized detection. Simultaneously, it reduces the required amount of detection liquid, allowing detection to be completed with a small amount of liquid, avoiding waste. Furthermore, the nanopore chip is bonded and sealed by PDMS, ensuring proper installation of the nanopore chip within the microfluidic liquid pool, resulting in better sealing and assembly stability, preventing leakage and impurity contamination, and ensuring detection accuracy and stability. The shape design of the through-holes in the sealing film layer greatly facilitates the installation of the nanopore chip, reducing assembly difficulty. The five-layer stacked architecture design of this invention allows for direct assembly of individual components without strict control of the tolerances of the liquid pool and the outer shell, achieving excellent sealing effects and significantly reducing operating costs. The use of transparent PDMS material allows observation of the nanopore chip's usage status and the liquid pool's state during detection. If problems with the nanopore chip or leakage are detected, disassembly and reassembly are possible. On the other hand, the flow channel of this microfluidic liquid pool is equipped with both an injection port and a return port. The return of the detection liquid can greatly reduce the phenomenon of bubble generation and further improve the accuracy of detection. Attached Figure Description

[0043] Figure 1 This is a perspective view of the upper and lower isometric angles of the present invention;

[0044] Figure 2 These are top and side sectional views of the present invention;

[0045] Figure 3 This is an exploded view of the present invention;

[0046] Figure 4 This is a front view schematic diagram of the present invention;

[0047] Figure 5 This is a side view schematic diagram of the present invention;

[0048] Figure 6 These are top and front views of the upper liquid tank box of the present invention;

[0049] Figure 7 These are top and front views of the lower liquid tank box of the present invention;

[0050] Figure 8 These are top and front views of the sealing film of the present invention;

[0051] Explanation of reference numerals in the attached figures:

[0052] 1. Upper liquid reservoir box; 101. Upper injection port; 102. Upper reflux port; 103. Upper flow channel; 2. Upper sealing film; 201. First intermediate through hole; 3. Nanoporous chip; 4. Lower sealing film; 401. Second intermediate through hole; 5. Lower liquid reservoir box; 501. Lower injection port; 502. Lower reflux port; 503. Lower flow channel; 6. Infusion device. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0054] like Figure 1-8As shown, the composite flexible microfluidic solid nanopore detection liquid pool device proposed in this invention includes an upper liquid pool box 1, an upper sealing film 2, a nanopore chip 3, a lower sealing film 4, a lower liquid pool box 5, an infusion device 6, and electrodes, which are sequentially and sealed from top to bottom. The upper sealing film 2 and the lower sealing film 4 are respectively provided with a first intermediate through hole 201 and a second intermediate through hole 401. The nanopore chip 3 is disposed between the first intermediate through hole 201 and the second intermediate through hole 401. The inner surfaces of the upper liquid pool box 1 and the lower liquid pool box 5 are respectively provided with an upper flow channel 103 and a lower flow channel 503. The upper flow channel 103 has an upper injection port 101 and an upper return port 102 arranged outwards, and the lower flow channel 503 has an outward... The upper channel 103 is provided with a lower injection port 501 and a lower return port 502. The upper injection port 101 and the upper return port 102 on the upper channel 103, and the lower injection port 501 and the lower return port 502 on the lower channel 503 are respectively connected to the infusion device 6. The electrodes are respectively embedded in the upper injection port 101 and the lower injection port 501 of the upper channel 103 and the lower channel 503. The upper liquid pool box 1 and the lower liquid pool box 5 are correspondingly bonded to the upper sealing film 2 and the lower sealing film 4 to form a complete flow channel. The first intermediate through hole 201 and the second intermediate through hole 401 on the upper sealing film 2 and the lower sealing film 4 are correspondingly connected to the upper channel 103 and the lower channel 503 on the upper liquid pool box 1 and the lower liquid pool box 5. The upper sealing film 2, lower sealing film 4, upper liquid tank 1, and lower liquid tank 5 are all made of a formulated flexible material, polydimethylsiloxane. The thickness of the upper sealing film 2 and lower sealing film 4 is 0.5 mm, and the thickness of the upper liquid tank 1 and lower liquid tank 5 is 2 mm. The sealing connection method between the upper liquid tank 1 and the upper sealing film 2, lower sealing film 4, and lower liquid tank 5 is plasma bonding. The upper flow channel 103 includes two parallel and symmetrically arranged vertical direct current channels and a horizontally arranged horizontal flow channel. The horizontal flow channel is connected to the ends of the two vertical direct current channels to achieve communication between them. The width of the vertical direct current channels gradually increases from the center to the bottom. At the point where the bottom opening is the largest, an upper injection port 101 and an upper return port 102 are formed on the outer surface of the upper liquid pool box 1. The infusion device 6 is connected to the upper injection port 101 and the upper return port 102. The lower flow channel 503 has the same shape and size as the upper flow channel 103, and the horizontal flow channels of both are on the same horizontal line, intersecting each other in the horizontal position and symmetrical about the center line of the entire microfluidic device. The electrode is an Ag / AgCl electrode, which is embedded in the upper injection port 101 and lower injection port 501 at the bottom of the vertical direct current channels in the upper flow channel 103 and lower flow channel 503. It is connected to an external signal acquisition instrument and a small current amplifier, and then connected to a digital-to-analog converter to complete signal analysis.The upper flow channel 103 on the upper liquid tank 1 is machined on its lower surface, forming a groove with a depth of 0.2 mm. The lower flow channel 503 on the lower liquid tank 5 is machined on its upper surface, forming a groove with a depth of 0.2 mm. The first intermediate through hole 201 and the second intermediate through hole 401 on the upper sealing film 2 and the lower sealing film 4 are both square holes with a circular hole connected to each other. The square hole has a thickness of 0.08 mm, and the circular hole has a thickness of 0.42 mm. The square hole has the same size as the nanopore chip 3. The nanopore chip 3 is installed between the square holes on the upper sealing film 2 and the lower sealing film 4 and is sealed by plasma bonding between the upper sealing film 2 and the lower sealing film 4. A point on the center line of the horizontal flow channel on the upper flow channel 103, the center of the first intermediate through hole 201, the center of the nanopore on the nanopore chip 3, the center of the second intermediate through hole 401, and a point on the center line of the horizontal flow channel on the lower liquid tank 5 are on the same vertical line.

[0055] The composite flexible microchannel solid nanopore detection liquid pool device provided by the present invention can effectively solve the problems of large volume, high liquid injection volume and poor sealing effect of the detection liquid pool in the prior art, and uses flexible material PDMS to meet the requirements of convenient and lightweight liquid pool.

[0056] The composite flexible microfluidic solid nanopore detection liquid pool device proposed in this application includes an upper liquid pool box 1, an upper sealing film 2, a nanopore chip 3, a lower sealing film 4, a lower liquid pool box 5, an infusion device 6, and electrodes. The five-layer stacked architecture design allows for direct assembly of each component, making assembly easy. The upper sealing film 2 and the lower sealing film 4 are obtained by casting polydimethylsiloxane (PDMS) with an oligomer and curing agent ratio of 10:1 in a pre-designed mold, resulting in lower costs. A first intermediate through-hole 201 and a second intermediate through-hole 401 are set at the central axis of the upper and lower sealing films. Both the first intermediate through-hole 201 and the second intermediate through-hole 401 are square holes with interconnected circular holes. The square hole thickness is 0.08 mm, and the circular hole thickness is 0.42 mm. The overall thickness of the upper and lower sealing films 4 is 0.5 mm. The square holes on the upper and lower sealing films 4 have the same dimensions as the nanoporous chip 3, which is installed between the square holes of the upper and lower sealing films 4. The upper liquid tank 1 and the lower liquid tank 5 use the same ratio of polydimethylsiloxane (PDMS), exhibiting good flexibility and elasticity and facilitating processing. The thickness of both the upper liquid tank 1 and the lower liquid tank 5 is 2 mm. The inner surfaces of the upper liquid tank 1 and the lower liquid tank 5 are respectively provided with an upper flow channel 103 and a lower flow channel 503. The upper flow channel 103 and the lower flow channel 503 are shaped like a "Π" and include two vertical straight channels. The two vertical straight channels are arranged parallel and symmetrically from left to right, and their tops are connected by a horizontal flow channel. The width of the vertical straight channel gradually increases from the center to the bottom. The width of the horizontal flow channel is 0.3 mm, and the width of the vertical straight channel at its widest point is 1.3 mm. The height of both the vertical straight channel and the horizontal flow channel is 0.2 mm. The design of the small flow channel can reduce the requirement for the amount of detection liquid injected, and the detection work can be completed with a small amount of detection liquid, without causing waste of detection liquid. By designing a microfluidic mold using existing channel dimensions and shapes, the prepared PDMS is poured onto the mold, followed by vacuuming, heating and curing, and demolding to obtain the upper liquid pool box 1 and the lower liquid pool box 5. It is worth noting that the upper channel 103 and the lower channel 503 in the upper liquid pool box 1 and the lower liquid pool box 5 have the same dimensions and can use the same tool. The liquid pool box made of PDMS material is transparent, allowing the usage status of the nanoporous chip 3 and the liquid pool to be observed during the testing process. If any problems are found with the nanoporous chip 3 or leakage occurs, it can be disassembled and reassembled.

[0057] The upper liquid pool box 1 and the upper sealing film 2 are connected by plasma bonding. The lower liquid pool box 5 and the lower sealing film 4 are connected by plasma bonding. The liquid pool box and the sealing film are bonded together to form a complete upper and lower transport channel. The flow channel is formed by plasma bonding, which can ensure that the liquid pool has excellent sealing performance.

[0058] The nanoporous chip 3 is installed between the square holes of the upper sealing film 2 and the lower sealing film 4 via plasma bonding, ensuring the sealing performance of the liquid pool. It is constructed using a five-layer stacked architecture, from top to bottom: upper liquid pool box 1, upper sealing film 2, nanoporous chip 3, lower sealing film 4, and lower liquid pool box 5. Simultaneously, it is ensured that a point on the center line of the horizontal flow channel in the upper liquid pool box 1, the center of the first intermediate through-hole 201, the center of the nanopore, the center of the second intermediate through-hole 401, and a point on the center line of the horizontal flow channel in the lower liquid pool box 5 are all on the same vertical line. The nanopores on the nanoporous chip 3 are the only connection between the upper flow channel 103 and the lower flow channel 503 through the first intermediate through-hole 201 and the second intermediate through-hole 401, guaranteeing the sealing performance of the entire liquid pool and the stability of the detection data.

[0059] At the widest point at the bottom of the vertical flow channels 103 and 503, upper inlet 101, upper reflux inlet 102, lower inlet 501, and lower reflux inlet 502 are punched outwards, and Ag / AgCl electrodes are embedded therein. The infusion device 6 is connected to the upper inlet 101 and upper reflux inlet 102, and the lower inlet 501 and lower reflux inlet 502. Simultaneously, the Ag / AgCl electrodes are embedded at the upper inlet 101 and lower inlet 501 at the bottom of the vertical flow channels, and are connected to external signal acquisition instruments and a small current amplifier, then to a digital-to-analog converter to complete signal analysis, ensuring the accuracy of the acquired signals.

[0060] The composite flexible microfluidic solid nanopore detection liquid pool device provided by this invention has excellent flexibility and elasticity, greatly reducing the volume of the liquid pool and enabling portable and miniaturized detection. It also reduces the required amount of detection liquid, allowing detection to be completed with a small amount of liquid, thus avoiding waste. Furthermore, the nanopore chip 3 is bonded and sealed by PDMS, ensuring proper installation of the nanopore chip 3 in the microfluidic liquid pool, providing better sealing and assembly stability, preventing leakage and impurity contamination, and ensuring the accuracy and stability of the detection. The shape design of the through-holes in the sealing film layer greatly facilitates the installation of the nanopore chip 3 and reduces assembly difficulty. The five-layer stacked architecture design of this invention allows for direct assembly of individual components without strict control of the tolerances of the liquid pool and the outer shell, achieving a good sealing effect and greatly reducing operating costs. Simultaneously, the use of transparent PDMS material allows observation of the nanopore chip 3's usage status and the liquid pool's status during detection. If a problem with the nanopore chip 3 or leakage is found, it can be disassembled and reassembled. On the other hand, the flow channel of this microfluidic liquid pool is equipped with both an injection port and a return port. The return of the detection liquid can greatly reduce the phenomenon of bubble generation and further improve the accuracy of detection.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any modifications or equivalent changes made based on the technical essence of the present invention shall still fall within the scope of protection claimed by the present invention.

Claims

1. A composite flexible microfluidic solid nanopore detection liquid pool device, characterized in that: The system comprises, from top to bottom, a sealed upper liquid tank (1), an upper sealing film (2), a nanoporous chip (3), a lower sealing film (4), a lower liquid tank (5), an infusion device (6), and electrodes, all connected in a sealed manner. The upper sealing film (2) and the lower sealing film (4) are respectively provided with a first intermediate through-hole (201) and a second intermediate through-hole (401). The nanoporous chip (3) is disposed between the first intermediate through-hole (201) and the second intermediate through-hole (401). The inner surfaces of the upper liquid tank (1) and the lower liquid tank (5) are respectively provided with an upper flow channel (103) and a lower flow channel (503). The upper flow channel (103) has an upper injection port (101) and an upper return port (102) extending outwards, and the lower flow channel (503) has a lower injection port (501) and a lower return port extending outwards. The upper injection port (101) and upper return port (102) on the upper flow channel (103), and the lower injection port (501) and lower return port (502) on the lower flow channel (503) are respectively connected to the infusion device (6). The electrodes are respectively embedded in the upper injection port (101) and lower injection port (501) of the upper flow channel (103) and the lower flow channel (503). The upper liquid pool box (1) and the lower liquid pool box (5) are bonded to the upper sealing film (2) and the lower sealing film (4) to form a complete flow channel. The first intermediate through hole (201) and the second intermediate through hole (401) on the upper sealing film (2) and the lower sealing film (4) are correspondingly connected to the upper flow channel (103) and the lower flow channel (503) on the upper liquid pool box (1) and the lower liquid pool box (5). The upper flow channel (103) includes two parallel and symmetrically arranged vertical straight channels and a horizontally arranged horizontal flow channel. The horizontal flow channel is connected to the ends of the two vertical straight channels to realize the connection between the two vertical straight channels. The width of the vertical straight channel gradually increases from its center to the bottom. At the bottom opening with the largest opening, an upper injection port (101) and an upper return port (102) are punched on the outer surface of the upper liquid pool box (1). The infusion device (6) is connected to the upper injection port (101) and the upper return port (102). The lower flow channel (503) has the same shape and size as the upper flow channel (103), and the horizontal flow channels of the two are on the same horizontal line. They are staggered in the horizontal position and symmetrical about the center line of the entire microfluidic device. The upper sealing film (2), the lower sealing film (4), the upper liquid pool box (1) and the lower liquid pool box (5) are all polydimethylsiloxane, a flexible material with an oligomer and curing agent ratio of 10:

1.

2. The composite flexible microfluidic solid nanopore detection liquid pool device according to claim 1, characterized in that: The thickness of the upper sealing film (2) and the lower sealing film (4) is 0.5 mm, and the thickness of the upper liquid pool box (1) and the lower liquid pool box (5) is 2 mm.

3. The composite flexible microfluidic solid nanopore detection liquid pool device according to claim 1, characterized in that: The sealing connection method between the upper liquid pool box (1), the upper sealing film (2), the lower sealing film (4), and the lower liquid pool box (5) is plasma bonding.

4. The composite flexible microfluidic solid nanopore detection liquid pool device according to claim 1, characterized in that: The electrode is an Ag / AgCl electrode, which is embedded in the upper injection port (101) and lower injection port (501) at the bottom of the vertical DC channel in the upper channel (103) and lower channel (503). It is connected to an external signal acquisition instrument and a small current amplifier, and then connected to a digital-to-analog converter to complete signal analysis.

5. The composite flexible microfluidic solid nanopore detection liquid pool device according to claim 1, characterized in that: The upper flow channel on the upper liquid pool box (1) is machined on its lower surface and a groove is formed on the lower surface, with a groove depth of 0.2 mm. The lower flow channel (503) on the lower liquid pool box (5) is machined on its upper surface and a groove is formed on the upper surface, with a groove depth of 0.2 mm.

6. The composite flexible microfluidic solid nanopore detection liquid pool device according to claim 1, characterized in that: The first intermediate through hole (201) and the second intermediate through hole (401) on the upper sealing film (2) and the lower sealing film (4) are both square holes and circular holes connected to each other. The thickness of the square hole is 0.08 mm and the thickness of the circular hole is 0.42 mm. The square hole is the same size as the nanopore chip (3). The nanopore chip (3) is installed between the square holes on the upper sealing film (2) and the lower sealing film (4) and is sealed by plasma bonding of the upper sealing film (2) and the lower sealing film (4).

7. The composite flexible microfluidic solid nanopore detection liquid pool device according to claim 6, characterized in that: A point on the center line of the horizontal flow channel of the upper flow channel (103), the center of the first intermediate through hole (201), the center of the nanopore on the nanopore chip (3), the center of the second intermediate through hole (401), and a point on the center line of the horizontal flow channel of the lower liquid pool box (5) are on the same vertical line.

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