Composite flexible micro-channel solid nanopore detection liquid pool device

Abstract

The invention discloses a composite flexible micro-channel solid nanopore detection liquid pool device which comprises an upper-layer liquid pool box, an upper-layer sealing film, a nanopore chip, a lower-layer sealing film, a lower-layer liquid pool box, a liquid conveying device and an electrode which are sequentially connected in a sealing manner, and the upper-layer sealing film and the lower-layer sealing film are respectively provided with a first middle through hole and a second middle through hole; the nanopore chip is arranged between the first middle through hole and the second middle through hole; an upper-layer runner and a lower-layer runner are respectively arranged on the inner surfaces of the upper-layer liquid pool box and the lower-layer liquid pool box, upper-layer and lower-layer injection ports and upper-layer and lower-layer backflow ports are outwards formed in the upper-layer and lower-layer runners, the liquid conveying device is connected with the upper-layer and lower-layer injection ports and the upper-layer and lower-layer backflow ports, and the electrodes are embedded in the upper-layer and lower-layer injection ports; the upper liquid pool box, the lower liquid pool box, the upper sealing film and the lower sealing film are bonded to form a complete flow channel, the through holes of the sealing films are correspondingly connected with the flow channel, the portable and micro-scale requirements are met, better sealing performance is achieved, meanwhile, detection work can be completed with a small amount of detection liquid through the liquid pool device, and waste of the detection liquid is avoided.

Description

Technical Field

[0001] The present invention relates to nanopore sensor technology and belongs to the technical fields of flexible material fabrication, gene sequencing, protein analysis, disease monitoring, environmental monitoring, etc. Specifically, it relates to a composite flexible microchannel solid-state nanopore detection liquid cell device. Background Art

[0002] With the development of micro-nano technology, single-molecule detection and analysis technology has become a hot field of scientific research. It is an important technical means to reveal the characteristics, interactions, and kinetic behaviors of substances, providing unprecedented resolution and sensitivity for fields such as life science, materials science, and medical diagnosis. Inspired by biological ion channels, the nanochannel single-molecule detection technology was proposed in 1996. This technology can detect single DNA or protein molecules with high throughput and without labeling, and can identify their sequence structures, with advantages such as high sensitivity, strong selectivity, and rapid response. Its principle is that under the drive of an electric field, biological molecules are transported through a nanopore by an ionic solution and generate corresponding blocking current signals, and the structure and properties of the measured biological molecules are characterized by changes in the electrical signals. A common nanopore sensor detection device is to clamp a solid-state nanopore chip between two liquid cells. The nanopore chip is fixed between the two liquid cells through a sealing rubber ring, and Ag / AgCl electrodes are inserted into the electrolyte solution in the liquid cells, thus forming a basic nanopore detection system. The probe connected to the electrode is connected to a microcurrent amplifier and an analog-to-digital converter to collect and record the real-time current change signals of the biological molecules to be measured passing through the nanopore, and qualitative and quantitative detection of biological molecules is achieved through signal analysis.

[0003] The capacity of traditional nanopore detection liquid cell devices is usually large, requiring a relatively large amount of samples. Moreover, the liquid cells are not sealed, which is not convenient to carry and is prone to sample contamination. In order to meet the growing demands for portability and miniaturization, it is necessary to redesign and transform the existing detection liquid cells to meet the requirements of sample miniaturization and convenient carrying, so as to meet the development needs of nanopore detection technology in the fields of wearable and biomedical detection.

[0004] The technical differences compared with the prior art are as follows: Technical comparison with the patent CN102866257A "A microfluidic sample boat with a liquid storage chamber and a pump chamber" Patent CN102866257A proposes a microfluidic sample boat with a liquid storage chamber and a pump chamber, aiming to solve the problems of complex structure, inconvenient manufacturing and use of traditional microfluidic sample boats. The dynamic control of liquid storage is realized through a flexible sealing layer, and an independent dry reagent storage chamber is designed to meet the drying requirements. A filtering structure is designed to filter out substances that interfere with the test results in the reagent to be tested. The microfluidic sample boat with a liquid storage chamber and a pump chamber proposed in Patent CN102866257A aims to optimize the liquid flow driving and sealing performance through the combination of rigid materials and flexible materials.

[0005] This patent proposes a solid-state nanopore detection liquid cell device based on flexible materials, focusing on solving the problems of large volume, high liquid injection requirement and poor sealing performance of traditional nanopore detection liquid cells. A five-layer stacked structure based on flexible materials (PDMS) is proposed. Through the design of Π-shaped flow channels, plasma bonding and the setting of injection ports / reflux ports, portable and micro-scale high-precision detection is achieved. The liquid cell device proposed in this patent aims to improve the sealing performance, reduce the waste of detection liquid, and adapt to wearable and point-of-care testing scenarios.

[0006] There are essential differences in their research purposes.

[0007] Patent CN102866257A adopts a four-layer structure (the first sealing layer, the solution storage chamber layer, the second sealing layer, the reaction chamber and microchannel layer), releases the liquid by externally damaging the second sealing layer, and realizes the function of the liquid storage chamber by the combination of the rigid chamber layer and the flexible sealing layer. Its technical highlights include the design of the filter membrane, the dry reagent storage chamber and the pump chamber structure.

[0008] This patent provides a solid-state nanopore detection liquid cell device based on flexible materials, adopting a five-layer stacked structure (liquid cell box, sealing film, nanopore chip, etc.). The closed flow channel is formed by 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 the external signal acquisition system. At the same time, the geometric symmetry of the flow channel and the through hole is designed, and a transparent PDMS material is used to realize visual detection.

[0009] There are essential differences in their technical solutions and system designs.

[0010] The microfluidic sample boat proposed in Patent CN102866257A is applicable to scenarios where multiple reagents need to be mixed step by step. The on-demand release of reagents is realized through dynamic sealing control, which is applicable to multi-step biochemical reaction detection (such as blood analysis). However, its structural complexity is still relatively high, and it relies on external mechanical intervention.

[0011] The solid-state nanopore detection liquid cell device based on flexible materials proposed in this patent reduces bubble interference through the design of microfluidic channels and reflux ports, and combines the high-sensitivity detection ability of nanopore chips. It is applicable to single-molecule detection scenarios such as gene sequencing and protein analysis, and supports portability and repeated disassembly.

[0012] There are essential differences between the two in terms of technical effects and application scenarios.

[0013] Technical comparison with the patent CN101126765A "Microfluidic sample boat" The patent CN101126765A proposed a microfluidic sample boat to solve the problems of slow mixing of trace fluids, inability to react quickly and analyze efficiently in practical applications. And through a micro-structure with a simple structure and an external pressure-driven method to promote the full mixing reaction of microfluids in the microchannel and improve the mixing efficiency. The microfluidic sample boat proposed in the patent CN101126765A aims to combine a flexible material layer with hydraulic / pneumatic drive to improve the reaction sensitivity and meet the rapid mixing requirements in chemical synthesis and biological detection.

[0014] This patent proposes a solid-state nanopore detection liquid cell device based on flexible materials. Aiming at the problems of large volume and insufficient sealing of traditional nanopore liquid cells, miniaturization and high sealing are achieved through flexible materials and a stacked architecture. The detection liquid cell proposed in this patent aims to improve the sealing performance, reduce the waste of detection liquid, and adapt to wearable and point-of-care testing scenarios, providing a portable and stable solid-state nanopore detection liquid cell device.

[0015] There are essential differences between the two in terms of application scenarios and research purposes.

[0016] The patent CN101126765A focuses on the combined design of a substrate and a flexible material layer, integrating modules such as a reaction chamber, a pump chamber, and a waste liquid chamber, and driving the liquid to circulate through hydraulic / pneumatic pressure. Its innovations include a ring-shaped microchannel design to reduce contamination and the integrated detection function of a biochip.

[0017] A solid-state nanopore detection liquid cell device based on flexible materials provided by this patent focuses on *adopting a five-layer PDMS stacked structure, ensuring the consistency of the liquid flow path through the design of Π-shaped channels and symmetric through-holes, and realizing the seamless encapsulation of the nanopore chip through plasma bonding. At the same time, the flexible material PDMS is used to ensure the flexibility and real-time monitoring ability of the liquid cell.

[0018] There are essential differences between the two in terms of system design and technical effects.

[0019] The patent CN101126765A achieves rapid mixing through cyclic drive and is applicable to biochemical detection (such as PCR amplification) that requires high-throughput reactions, but its structure is complex and relies on external pressure equipment.

[0020] This patent adopts a five-layer stacked architecture (liquid cell cassette, sealing film, nanopore chip, etc.), forms a closed flow channel through PDMS plasma bonding, optimizes the liquid flow distribution with a Π-shaped flow channel design, embeds an Ag / AgCl electrode at the injection port to connect to an external signal acquisition system, and supports the use of detection liquid as low as the microliter level through a micro-channel and high-sealing design, is suitable for point-of-care testing (such as pathogen screening) in an out-of-lab environment, and has the advantage of being reusable.

[0021] There are essential differences between the two in terms of technical solutions.

[0022] Technical comparison with the patent KR1020220067927A "A sticky microfluidic chip and its manufacturing method" The patent KR1020220067927A proposes a sticky microfluidic chip. For a microfluidic chip manufactured by a conventional microfluidic chip manufacturing method, it has micro-channels and micro-cavities formed in a two-dimensional planar structure. For various irregular shapes, such as a rough surface with irregularities, a cylinder, and the human skin, there is a problem of difficulty in adhering to the object surface. It attempts to develop a microfluidic chip that can not only perform smooth component analysis on trace biochemical samples but also freely adhere to and separate from various types of object surfaces. The microfluidic chip proposed by the patent KR1020220067927A aims to improve the chip adhesion, adapt to diverse surfaces, and simplify the manufacturing process.

[0023] This patent proposes a solid-state nanopore detection liquid cell device based on flexible materials, focusing on solving the problems of large volume, high liquid injection volume requirement, and poor sealing of traditional nanopore detection liquid cells. It proposes a five-layer stacked architecture based on flexible materials (PDMS), and through a Π-shaped flow channel design, plasma bonding, and the setting of injection ports / return ports, realizes portable and micro-scale high-precision detection. The liquid cell device proposed in this patent aims to clamp a nanopore chip to improve the sealing performance, reduce the waste of detection liquid, and adapt to wearable and point-of-care testing scenarios.

[0024] There are essential differences between the two in terms of application scenarios and research purposes.

[0025] At the same time, enabling the liquid cell to have 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

[0026] The purpose of the present invention is to provide a composite flexible micro-channel solid-state nanopore detection liquid cell device, aiming to solve the problems of too large volume, difficult assembly, large liquid injection volume, and inconvenient carrying of traditional liquid cells.

[0027] To achieve the above purpose, the technical solution adopted by the present invention is: A composite flexible microfluidic solid-state nanopore detection liquid pool device is characterized in that it includes an upper liquid pool box, an upper sealing film, a nanopore chip, a lower sealing film, a lower liquid pool box, an infusion device and an electrode that are sealed and connected in sequence from top to bottom, the upper sealing film and the lower sealing film are respectively provided with a first middle through hole and a second middle through hole, the nanopore chip is arranged between the first middle through hole and the second middle through hole, the inner surfaces of the upper liquid pool box and the lower liquid pool box are respectively provided with an upper flow channel and a lower flow channel, the upper flow channel is provided with an upper injection port and an upper reflux port outwardly, and the lower A lower layer injection port and a lower layer return port are provided outwardly on the laminar flow channel, the upper layer injection port and the upper layer return port on the upper layer flow channel, the lower layer injection port and the lower layer return port on the lower layer flow channel are respectively connected to an external infusion device, the electrodes are respectively embedded in the upper layer injection port and the lower layer injection port of the upper layer flow channel and the lower layer flow channel, the upper layer liquid pool box and the lower layer liquid pool box are bonded to the upper layer sealing film and the lower layer sealing film to form a complete flow channel, the first middle through hole and the second middle through hole on the upper layer sealing film and the lower layer sealing film are correspondingly connected to the upper layer flow channel and the lower layer flow channel on the upper layer liquid pool box and the lower layer liquid pool box.

[0028] Furthermore: the upper sealing film, the lower sealing film, the upper liquid pool box and the lower liquid pool box are all made of the formulated flexible material polydimethylsiloxane, the thickness of the upper sealing film and the lower sealing film are both 0.5 mm, and the thickness of the upper liquid pool box and the lower liquid pool box are both 2 mm.

[0029] 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.

[0030] Furthermore: the upper flow channel includes two parallel and symmetrically arranged vertical flow channels and a horizontal flow channel arranged laterally, the horizontal flow channels are respectively connected to the ends of the two vertical flow channels to realize the connection between the two vertical flow channels, the width of the vertical flow channel gradually widens from the center to the bottom, and the upper injection port and the upper return port are punched out on the outer surface of the upper liquid pool box at the largest opening of the bottom, the infusion device is connected to the upper injection port and the upper return port, the lower flow channel and the upper flow channel have the same shape and size and the horizontal flow channels of the two are on the same horizontal line, staggered with each other in the horizontal position and symmetrical about the center line of the entire microfluidic device.

[0031] Furthermore: the electrode is an Ag / AgCl electrode, which is embedded in the upper injection port and the lower injection port at the bottom of the vertical flow channel in the upper flow channel and the lower flow channel, 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.

[0032] Further: The upper flow channel on the upper liquid pool box is processed on its lower surface and forms a groove on the lower surface, with the groove depth being 0.2 mm. The lower flow channel on the lower liquid pool box is processed on its upper surface and forms a groove on the upper surface, with the groove depth being 0.2 mm.

[0033] Further: The shapes of the first intermediate through holes and the second intermediate through holes on the upper sealing film and the lower sealing film are both a square hole and a circular hole 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 size of the square hole is the same as that of the nanopore chip. The nanopore chip is installed between the square holes on the upper sealing film and the lower sealing film and is sealed through plasma bonding of the upper sealing film and the lower sealing film.

[0034] Further: A certain point on the center line of the horizontal flow channel on the upper flow channel, the center of the first intermediate through hole, the center of the nanopores on the nanopore chip, the center of the second intermediate through hole, and a certain point on the center line of the horizontal flow channel of the lower liquid pool box are on the same vertical line.

[0035] In the above structure: The present invention provides a composite flexible microchannel solid-state nanopore detection liquid pool device for nanopore sensors. The device is made of a five-layer stacked structure, including an upper liquid pool box, an upper sealing film, a nanopore chip, a lower sealing film, a lower liquid pool box, an infusion device, and electrodes, which are hermetically connected in sequence from top to bottom. The upper sealing film and the lower sealing film are respectively provided with a first intermediate through hole and a second intermediate through hole. The nanopore chip is arranged between the first intermediate through hole and the second intermediate through hole. An upper flow channel and a lower flow channel are respectively arranged on the inner surfaces of the upper liquid pool box and the lower liquid pool box. Among them, an upper injection port and an upper return port are arranged outward on the upper flow channel, and a lower injection port and a lower return port are arranged outward on the lower flow channel. Among them, the upper injection port and the upper return port, the lower injection port and the lower return port are respectively externally connected to the infusion device, and the electrodes are respectively embedded at the upper injection port and the lower injection port. During installation, the upper liquid pool box, the lower liquid pool box are key-bonded with the upper sealing film and the lower sealing film correspondingly 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 and connect with 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-state nanopore detection liquid pool device meets the requirements of portability and miniaturization and has better sealing performance. At the same time, through the infusion device, the detection work can be completed with a small amount of detection liquid, without causing waste of the detection sample.

[0036] Compared with the prior art, the beneficial effects of the present invention are: The present invention provides a composite flexible microchannel solid-state nanopore detection liquid cell device. Using the flexible material PDMS, based on its good flexibility and elasticity, the volume of the liquid cell is greatly reduced, enabling portable and miniaturized detection. At the same time, the requirement for the injection volume of the detection liquid is reduced, and the detection work can be completed with a small amount of detection liquid, without causing waste of the detection liquid. Meanwhile, the nanopore chip is bonded and sealed with PDMS. On the basis of ensuring that the nanopore chip is correctly installed in the microfluidic liquid cell, it has better sealing performance and assembly stability, avoiding liquid leakage and mixing of impurities, and ensuring the accuracy and stability of detection. The shape design of the through holes in the sealing film layer can greatly facilitate the installation of the nanopore chip and reduce the assembly difficulty. The five-layer stacked architecture design of the present invention allows direct assembly of each component, and without strictly controlling the tolerances of the liquid cell and the housing, a good sealing effect can be achieved, greatly reducing the use cost. At the same time, the use of transparent PDMS material can observe the usage status of the nanopore chip and the status of the liquid cell during the detection process. Once problems are found with the nanopore chip or liquid leakage occurs, it can be disassembled and reassembled. On the other hand, the flow channel of the microfluidic liquid cell is provided with an injection port and a reflux port at the same time. By the reflux of the detection liquid, the phenomenon of bubble generation can be greatly improved, further enhancing the detection accuracy. Description of the Drawings

[0037] Figure 1 is the upper and lower isometric axonometric perspective schematic diagram of the present invention; Figure 2 is the top view and side sectional view schematic diagram of the present invention; Figure 3 is the exploded schematic diagram of the present invention; Figure 4 is the front view schematic diagram of the present invention; Figure 5 is the side view schematic diagram of the present invention; Figure 6 is the top view and front view schematic diagram of the upper liquid cell box of the present invention; Figure 7 is the top view and front view schematic diagram of the lower liquid cell box of the present invention; Figure 8 is the top view and front view schematic diagram of the sealing film of the present invention; Description of the Reference Numerals: 1. Upper liquid cell box; 101. Upper injection port; 102. Upper reflux port; 103. Upper flow channel; 2. Upper sealing film; 201. First intermediate through hole; 3. Nanopore chip; 4. Lower sealing film; 401. Second intermediate through hole; 5. Lower liquid cell box; 501. Lower injection port; 502. Lower reflux port; 503. Lower flow channel; 6. Liquid infusion device. Detailed Embodiments

[0038] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments: Such as Figure 1-8As shown, the composite flexible microfluidic solid-state nanopore detection liquid pool device proposed in the present 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 an electrode that are sealed and connected in sequence from top to bottom, the upper sealing film 2 and the lower sealing film 4 are respectively provided with a first middle through hole 201 and a second middle through hole 401, the nanopore chip 3 is arranged between the first middle through hole 201 and the second middle 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 is provided with an upper injection port 101 and an upper reflux port 102 outwardly, and the lower flow channel 503 is provided with an upper injection port 101 and an upper reflux port 102 outwardly. A lower layer injection port 501 and a lower layer return port 502 are provided, and the upper layer injection port 101 and the upper layer return port 102 on the upper layer flow channel 103, and the lower layer injection port 501 and the lower layer return port 502 on the lower layer flow channel 503 are respectively connected to the infusion device 6, and the electrodes are respectively embedded in the upper layer injection port 101 and the lower layer injection port 501 of the upper layer flow channel 103 and the lower layer flow channel 503, and the upper layer liquid pool box 1 and the lower layer liquid pool box 5 are bonded to the upper layer sealing film 2 and the lower layer sealing film 4 to form a complete flow channel, and the first middle through hole 201 and the second middle through hole 401 on the upper layer sealing film 2 and the lower layer sealing film 4 are connected to the upper layer flow channel 103 and the lower layer flow channel 503 on the upper layer liquid pool box 1 and the lower layer liquid pool box 5. The upper sealing film 2, lower sealing film 4, upper liquid pool box 1, and lower liquid pool box 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 pool box 1 and lower liquid pool box 5 is 2 mm. The upper liquid pool box 1, upper sealing film 2, lower sealing film 4, and lower liquid pool box 5 are sealed together using plasma bonding. The upper flow channel 103 includes two parallel and symmetrical vertical flow channels and a horizontal flow channel arranged horizontally. The horizontal flow channels are respectively connected to the ends of the two vertical flow channels to achieve connectivity between the two vertical flow channels. The width of the vertical flow channel gradually widens from its center to the bottom. At the largest opening at the bottom, an upper injection port 101 and an upper return port 102 are punched out 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, interlaced with each other in the horizontal position and symmetrical about the center line of the entire microfluidic device. The electrodes are Ag / AgCl electrodes, which are embedded in the upper injection port 101 and the lower injection port 501 at the bottom of the vertical flow channels in the upper flow channel 103 and the lower flow channel 503, and are connected to an external signal acquisition instrument and a low current amplifier, and then connected to a digital-to-analog converter to complete signal analysis.The upper flow channel 103 on the upper liquid pool box 1 is processed on its lower surface and forms a groove on the lower surface, with the groove depth being 0.2 mm. The lower flow channel 503 on the lower liquid pool box 5 is processed on its upper surface and forms a groove on the upper surface, with the groove depth being 0.2 mm. The shapes of 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 a square hole plus a circular hole 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 size of the square hole is the same as that of 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 through the plasma bonding of the upper sealing film 2 and the lower sealing film 4. A certain 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 nanopores on the nanopore chip 3, the center of the second intermediate through-hole 401, and a certain point on the center line of the horizontal flow channel on the lower liquid pool box 5 are on the same vertical line.

[0039] The composite flexible microchannel solid-state nanopore detection liquid pool device provided by the present invention can effectively solve the problems of large volume of the detection liquid pool, high requirement for liquid injection volume, and poor sealing effect in the prior art, and uses the flexible material PDMS to meet the requirements of convenience and light weight of the liquid pool.

[0040] The composite flexible microfluidic solid-state nanopore detection liquid cell device proposed in this application includes an upper liquid cell box 1, an upper sealing film 2, a nanopore chip 3, a lower sealing film 4, a lower liquid cell box 5, an infusion device 6 and electrodes. The five-layer stacked architecture design allows for direct assembly of each component, with low assembly difficulty. The upper sealing film 2 and the lower sealing film 4 are obtained by casting a mixture of oligomer and curing agent in a ratio of 10:1 of polydimethylsiloxane (PDMS) in a designed mold, which is of low cost. At the same time, a first intermediate through-hole 201 and a second intermediate through-hole 401 are provided at the central axis positions of the upper sealing film and the lower sealing film. The shapes of the first intermediate through-hole 201 and the second intermediate through-hole 401 are both a square hole connected to a circular hole. The thickness of the square hole is 0.08 mm, the thickness of the circular hole is 0.42 mm, and the overall thickness of the upper sealing film and the lower sealing film 4 is 0.5 mm. The size of the square holes on the upper sealing film and the lower sealing film 4 is the same as that of the nanopore chip 3, and the nanopore chip 3 is installed between the square holes of the upper sealing film and the lower sealing film 4. The upper liquid cell box 1 and the lower liquid cell box 5 use polydimethylsiloxane (PDMS) with the same formulation ratio, which has good flexibility and elasticity and is easy to process. The thicknesses of both the upper liquid cell box 1 and the lower liquid cell box 5 are 2 mm. Upper flow channels 103 and lower flow channels 503 are respectively provided on the inner surfaces of the upper liquid cell box 1 and the lower liquid cell box 5. The shapes of the upper flow channels 103 and the lower flow channels 503 are similar to the shape of "Π", including two vertical flow channels. The two vertical flow channels are arranged parallel and symmetrically on the left and right, and their tops are connected by a horizontal flow channel. The width of the vertical flow channels gradually widens from the center to the bottom. The width of the horizontal flow channel is 0.3 mm, the width of the widest part of the vertical flow channels is 1.3 mm, and the heights of both the vertical flow channels and the horizontal flow channel are 0.2 mm. The design of the small flow channels can reduce the requirement for the injection volume of the detection liquid, enabling the detection work to be completed with a small amount of detection liquid without causing waste of the detection liquid. By designing a microfluidic mold based on the existing flow channel dimensions and shapes, the prepared PDMS is cast on the mold and then undergoes vacuum pumping, heat curing, and demolding processes to obtain the upper liquid cell box 1 and the lower liquid cell box 5. It should be noted that the sizes and dimensions of the upper flow channels 103 and the lower flow channels 503 in the upper liquid cell box 1 and the lower liquid cell box 5 are the same, and the same mold can be used. The liquid cell boxes prepared using PDMS materials are transparent, and the usage status of the nanopore chip 3 and the status of the liquid cell can be observed during the detection process. Once problems are found with the nanopore chip 3 or there is a liquid leakage phenomenon, it can be disassembled and reassembled again.

[0041] The inverted mold surface of the upper liquid pool box 1 and the upper sealing film 2 are connected by plasma bonding. The inverted mold surface of 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 to form a complete upper conveying channel and a lower conveying channel. Forming the channel by plasma bonding can ensure excellent sealing performance of the liquid pool.

[0042] The nanopore chip 3 is installed between the square holes of the upper sealing film 2 and the lower sealing film 4 by plasma bonding, ensuring the sealing of the liquid pool. It is made of a five-layer stacked structure, which is, from top to bottom, the upper liquid pool box 1, the upper sealing film 2, the nanopore chip 3, the lower sealing film 4, and the lower liquid pool box 5. At the same time, ensure that a certain point on the center line of the horizontal 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 certain point on the center line of the horizontal channel in the lower liquid pool box 5 are on the same vertical line. The nanopore on the nanopore chip 3 is the only connection between the upper channel 103 and the lower channel 503 through the first intermediate through hole 201 and the second intermediate through hole 401, ensuring the sealing performance of the entire liquid pool and the stability of the detection data.

[0043] At the widest part of the bottom of the vertical channels of the upper channel 103 and the lower channel 503, punch out the upper injection port 101, the upper return port 102, the lower injection port 501, and the lower return port 502 with holes on the outside, and embed Ag / AgCl electrodes. The infusion device 6 is connected to the upper injection port 101 and the upper return port 102, the lower injection port 501 and the lower return port 502. At the same time, the Ag / AgCl electrodes are embedded at the upper injection port 101 and the lower injection port 501 at the bottom of the vertical channel, and are connected to an external signal acquisition instrument and a small current amplifier, and then connected to an analog-to-digital converter to complete signal analysis, ensuring the accuracy of the acquired signal.

[0044] The composite flexible microfluidic solid-state nanopore detection liquid cell device provided by the present invention has good flexibility and elasticity, greatly reduces the volume of the liquid cell, and can achieve portable and microscale detection. At the same time, the requirement for the injection volume of the detection liquid is reduced, and the detection work can be completed with a small amount of detection liquid, without causing waste of the detection liquid. At the same time, the nanopore chip 3 is bonded and sealed by PDMS. On the basis of ensuring that the nanopore chip 3 is correctly installed in the microfluidic liquid cell, it has better sealing performance and assembly stability, avoiding liquid leakage and mixing of impurities, and ensuring the accuracy and stability of detection. The shape design of the through hole of the sealing film layer can greatly facilitate the installation of the nanopore chip 3 and reduce the assembly difficulty. The five-layer stacked architecture design of the present invention allows direct assembly of each component, and without strictly controlling the tolerances of the liquid cell and the housing, a good sealing effect can be achieved, greatly reducing the use cost. At the same time, the use of transparent PDMS material can observe the usage status of the nanopore chip 3 and the status of the liquid cell during the detection process. Once problems are found with the nanopore chip 3 or liquid leakage occurs, it can be disassembled and reassembled. On the other hand, the flow channel of the microfluidic liquid cell is provided with an injection port and a reflux port at the same time. By refluxing the detection liquid, the phenomenon of bubble generation can be greatly improved, and the detection accuracy can be further improved.

[0045] The above are only preferred embodiments of the present invention, and do not constitute any other form of limitation to the present invention. Any modification or equivalent change made according to the technical essence of the present invention still falls within the scope of protection required by the present invention.

Claims

1. A composite flexible microchannel solid-state nanopore detection liquid cell device, characterized in that: The invention comprises 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 an electrode, which are sealed and connected in sequence 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 arranged 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) is provided with an upper injection port (101) and an upper reflux port (102) facing outwards. The lower flow channel (503) is provided with a lower injection port (501) and a lower reflux port (503) facing outwards. The upper injection port (101) and the upper return port (102) on the upper flow channel (103), and the lower injection port (501) and the lower return port (502) on the lower flow channel (503) are respectively connected to an infusion device (6); the electrodes are respectively embedded in the upper injection port (101) and the 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 middle through hole (201) and the second middle through hole (401) on the upper sealing film (2) and the lower sealing film (4) are 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) to each other.

2. The composite flexible microchannel solid-state nanopore detection liquid cell device according to claim 1, wherein: 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 made of a formulated flexible material, polydimethylsiloxane. The thickness of the upper sealing film (2) and the lower sealing film (4) are both 0.5 mm, and the thickness of the upper liquid pool box (1) and the lower liquid pool box (5) are both 2 mm.

3. The composite flexible microchannel solid-state nanopore detection liquid cell device according to claim 1, wherein: 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 microchannel solid-state nanopore detection liquid cell device according to claim 1, wherein: The upper flow channel (103) includes two parallel and symmetrically arranged vertical flow channels and a horizontal flow channel arranged transversely. The horizontal flow channels are respectively connected to the ends of the two vertical flow channels to realize the connection between the two vertical flow channels. The width of the vertical flow channel gradually widens from the center to the bottom. At the largest opening of the bottom, an upper injection port (101) and an upper return port (102) are punched out toward 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) and the upper flow channel (103) have the same shape and size and the horizontal flow channels of the two are on the same horizontal line. They are staggered with each other in the horizontal position and are symmetrical about the center line of the entire microfluidic device.

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

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

7. The composite flexible microchannel solid-state nanopore detection liquid cell device according to claim 1, wherein: The shapes of 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 a square hole plus a circular hole 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 size of the square hole is the same as that of 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).

8. The composite flexible microchannel solid-state nanopore detection liquid cell device according to claim 4 or 7, characterized in that: A certain point on the horizontal flow channel center line of the upper flow channel (103), the center of the first intermediate through hole (201), the center of the nanopores on the nanopore chip (3), the center of the second intermediate through hole (401), and a certain point on the horizontal flow channel center line of the lower liquid cell box (5) are on the same vertical line.

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