A fully integrated nucleic acid detection chip based on silicon membrane method

Abstract

The present invention provides a flexible thin-film microfluidic chip and a method for nucleic acid extraction and detection, which can achieve nucleic acid extraction based on the silicon membrane method. The flexible thin-film microfluidic chip includes a laminated membrane, at least one fixed binding area, multiple functional chambers, a fluid channel, at least one silicon membrane chamber, at least one valve area, and at least one liquid inlet. The fixed binding area, functional chamber, fluid channel, silicon membrane chamber, and valve area are all distributed within the laminated membrane, and the size and shape of the functional chamber, fluid channel, and silicon membrane chamber are defined by the boundaries of the fixed binding area. The microfluidic chip of the present invention achieves nucleic acid extraction and amplification detection based on the silicon membrane method in a closed flexible thin-film microfluidic chip.

Description

Technical Field

[0001] The present invention relates to a fully integrated nucleic acid detection chip based on a silicon membrane method, belonging to the technical field of microfluidic chips. Background Art

[0002] Since the advent of polymerase chain reaction (PCR) technology, genetic analysis through DNA amplification has become a common technique. However, due to structural limitations, traditional PCR tube-based amplification devices suffer from high energy consumption, long detection times, cross-contamination, and the inability to perform real-time on-site detection. These devices are increasingly unable to meet the demands of diverse applications.

[0003] With the advent of microfluidics technology, its advantages—integration, miniaturization, full enclosure, and diverse application scenarios—have garnered widespread attention. Over the past decade or so, a large number of microfluidic chips with diverse structures and thermal cycling implementations have been developed and widely used in pathogen detection. The advantages of microfluidic testing products in point-of-care testing (POCT) scenarios have been fully demonstrated worldwide.

[0004] Traditional microfluidic chips are primarily made from cured polydimethylsiloxane (PDMS). Its soft-etching process makes it well-suited for laboratory-based conceptual research and small-batch production. However, this production process is not suitable for large-scale production, especially automated mass production. Furthermore, as a rubber material, its use in in vitro diagnostic (IVD) testing products has been difficult for the industry to accept.

[0005] CN2022109249601 discloses a microfluidic chip made of a flexible film, which can be used for cell lysis and nucleic acid extraction. It is made by overlapping a first flexible film and a second flexible film, and setting multiple functional chambers that are interconnected between the films. Microfluidic channels can be set between the chambers. High-speed liquid flow is obtained through short microfluidic channels to enhance the lysis effect, and nucleic acid extraction is achieved using magnetic beads.

[0006] CN2008801038390 discloses a multi-chamber container made of a flexible sheet for nucleic acid amplification, which uses a magnetic bead method to extract and elute nucleic acids. In order to facilitate the efficient flow of magnetic beads or fluids between chambers, the chambers are directly connected to each other at the shortest possible distance or connected through a valve structure.

[0007] CN2022110178734 discloses a flexible film-based microfluidic chip for nucleic acid amplification. The microfluidic chip includes a composite membrane, a fixed binding area, multiple functional chambers, a disposable valve, and a liquid- and gas-blocking fluid channel. Under pressure, a first flexible film and a second flexible film within the functional chamber and the liquid- and gas-blocking fluid channel can switch between separate and attached states. When the first and second flexible films within the liquid- and gas-blocking fluid channel separate and form a fluid channel, the channel allows liquid to pass through but blocks isolated bubbles in the liquid.

[0008] The magnetic bead method is currently the most widely used method for nucleic acid extraction. The core of the magnetic beads typically used is composed of tiny (20 to 30 nanometers) magnetic iron oxide particles, such as ferroferric oxide (Fe₃O₄). Unlike conventional magnetic materials, the tiny size of these particles imparts superparamagnetism. They gravitate toward one pole of the magnetic field only in the presence of a strong magnetic field. Outside of the magnetic field, they are not attracted to each other, effectively preventing clumping caused by bead aggregation. This property makes magnetic beads ideal for surface chemistry-based macromolecular separation or surface-specific adsorption in complex samples. The chemical compositions resulting from various surface modifications have led to the widespread use of magnetic bead methods for the separation and purification of nucleic acids, proteins, and other biomolecules in in vitro testing, gene sequencing, environmental protection, and food safety. When used for nucleic acid adsorption, the surface of the magnetic beads is typically coated with silica (SiO₂) molecules. Under appropriate salt concentrations, pH values, and nucleic acid binding buffer, the magnetic beads can efficiently adsorb nucleic acid macromolecules in the reaction system and, under the influence of the strong magnetic field, aggregate at specific locations in the reactor, facilitating the removal of waste liquids. In this way, during the nucleic acid extraction process, magnetic bead enrichment and release can be achieved repeatedly, impurities non-specifically adsorbed on the surface of the magnetic beads can be washed away, and the purified nucleic acid template can be obtained after elution.

[0009] When the above-mentioned magnetic bead method for nucleic acid extraction is expanded and applied to microfluidic devices in POCT scenarios, the structural design, manufacturing process and coordination with the controller of the consumable nucleic acid extraction part all face huge challenges due to the high integration of the detection device, the tiny size of the fluid structure and the almost stringent cost control requirements.

[0010] Another widely used, but limited, method for nucleic acid extraction is the silica membrane spin column method. Similar to the magnetic bead method, the substance that adsorbs nucleic acids from the liquid during nucleic acid extraction using the silica membrane spin column method is also SiO2. The process also utilizes varying salt ion concentrations and pH values ​​to achieve binding and release of DNA, thereby extracting and purifying nucleic acids. A fundamental requirement for nucleic acid extraction using the silica membrane spin column method is that the reaction solution passes through the silica membrane under the action of centrifugal force and then enters a waste container, ensuring sufficient contact between the sample nucleic acids and the SiO2 substance, completing the nucleic acid adsorption process. Obviously, when using this method, the extract, as well as the subsequent rinse and eluents, are uniformly passed through the silica membrane under the action of centrifugal force, perpendicular to the plane of the membrane, achieving efficient and rapid nucleic acid extraction.

[0011] Another implementation of the silica membrane spin column method uses vacuum suction instead of centrifugal force. In this method, multiple silica membrane spin columns are typically placed in a sealed chamber with multiple outlets. After adding the sample or reagents, negative pressure is introduced into the vacuum chamber. Under this negative pressure, the liquid in the silica membrane spin column flows through the silica membrane into the vacuum chamber, achieving nucleic acid adsorption and rinsing.

[0012] In this way, the adsorption gradient of the silicon membrane is inversely proportional to its thickness. Since the thickness of the membrane can usually be very thin, the adsorption working surface is large, the gradient decrease is very small, and the adsorption efficiency is very high. How to achieve nucleic acid adsorption, rinsing and elution based on the silicon membrane method in a closed two-dimensional flexible thin film microfluidic chip and complete nucleic acid detection is difficult.

[0013] In principle, it is possible to extract and purify nucleic acids from complex samples by placing a silicone membrane flat in the channel of a flexible thin-film microfluidic chip and sequentially passing a sample solution, rinse solution, and eluent with an appropriate salt ion concentration. However, in practice, because the flexible thin-film microfluidic chip is a two-dimensional planar structure, the microfluidic channel embedded with the silicone membrane, while having a certain thickness, has a cross-sectional dimension much smaller than the cross-sectional dimension of a conventional silicone membrane centrifugal column. When a fluid permeates through the silicone membrane along the thickness direction, the adsorption gradient of the silicone membrane is inversely proportional to the length of the silicone membrane. In this case, when the fluid flows through the nucleic acid extraction silicone membrane along the microfluidic channel, in order to fully adsorb the nucleic acids in the sample on the silicone membrane, the length of the flow channel containing the silicone membrane needs to be as long as possible, thus far exceeding the thickness of the silicone membrane through which the liquid flows when using the centrifugal column method. This not only hinders the design of the microfluidic chip size, but also increases the flow resistance generated during the process as the amount of liquid flowing through the silicone membrane increases. Furthermore, thin-film microfluidic chips have a limited flexibility, both due to their materials and fabrication methods. Under sustained and increasing pressure, the microchannels can potentially deform destructively, leading to experimental failure. Therefore, the existing centrifugal column silicon membrane method cannot be directly applied to thin-film microfluidic chips. Summary of the Invention

[0014] The main purpose of the present invention is to provide a flexible thin film microfluidic chip and a nucleic acid extraction method, which can realize nucleic acid extraction based on the silicon membrane method to expand the application of existing microfluidic technology.

[0015] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include:

[0016] One aspect of the present invention provides a flexible thin film microfluidic chip, comprising a laminated membrane, at least one fixed bonding area, multiple functional chambers, a fluid channel, at least one silicon membrane chamber, at least one valve area, and at least one liquid inlet, wherein the fixed bonding area, functional chamber, fluid channel, silicon membrane chamber, and valve area are all distributed within the laminated membrane, and the size and shape of the functional chamber, fluid channel, and silicon membrane chamber are defined by the boundaries of the fixed bonding area. The liquid inlet is connected to at least one functional chamber and is sealed after liquid is introduced.

[0017] The laminated film comprises a first film and a second film that are stacked, and at least one of the first film and the second film is a flexible film;

[0018] The first film and the second film are irreversibly bonded in the fixed bonding area;

[0019] The maximum width of the silicon membrane chamber is greater than the width of the fluid channel, and a silicon membrane having a shape matching that of the fluid channel is accommodated therein;

[0020] The first film and the second film of the functional chamber can switch between two states of separation and mutual adhesion under pressure. When the first film and the second film in the functional chamber are separated from each other, the functional chamber can contain fluid;

[0021] The functional chamber includes at least one sample chamber, at least one rinsing liquid chamber, at least one waste liquid chamber and at least two eluent chambers, wherein the two eluent chambers are respectively a first eluent chamber and a second eluent chamber, preferably one sample chamber, two rinsing liquid chambers, one waste liquid chamber and two eluent chambers, wherein the two rinsing liquid chambers are respectively a first rinsing liquid chamber and a second rinsing liquid chamber;

[0022] The functional chamber is connected to the silicon membrane chamber via a fluid channel, and the fluid channel allows the fluid to pass through in a controllable manner at a low speed;

[0023] Preferably, the fluid channel is divided into a plurality of sections based on the functional chambers connected thereto with the silicone membrane chamber as a dividing point, and the plurality of sections include at least a first fluid channel and a second fluid channel, wherein the first fluid channel is connected to the sample chamber, and the second fluid channel is connected to the waste liquid chamber;

[0024] There is no restriction on the connection method between the functional chamber and the silicon membrane chamber through the fluid channel. The functional chambers can be directly connected to the silicon membrane chamber through the fluid channel independently. However, this connection method is not preferred because the silicon membrane chamber is very small and it is difficult to provide enough space to connect with each fluid channel, which will also limit the size of the fluid channel.

[0025] Preferably, each chamber is connected to the silicon membrane chamber by sharing a portion of the fluid channels. Preferably, the number of fluid channels directly connected to the silicon membrane chamber is no more than 5, 4, 3 or 2;

[0026] Preferably, each functional chamber is connected to the silicon membrane chamber by sharing the first fluid channel and / or the second fluid channel, and the number of fluid channels directly connected to the first fluid channel and / or the second fluid channel is no less than 5, 4, 3, 2, or 1. Most preferably, the sample chamber is connected to the silicon membrane chamber via the first fluid channel, the waste liquid chamber is connected to the silicon membrane chamber via the second fluid channel, and the remaining functional chambers are connected to the first or second fluid channel via their own fluid channels, wherein the rinse liquid chamber is connected to the first fluid channel; the first eluent chamber is connected to the second fluid channel, and the second eluent chamber is connected to the first fluid channel; or the first eluent chamber is connected to the first fluid channel, and the second eluent chamber is connected to the second fluid channel.

[0027] Furthermore, the functional chamber further includes a PCR amplification detection chamber, the PCR amplification detection chamber includes a PCR reaction liquid chamber and an optional PCR reaction chamber, and the PCR reaction liquid chamber and the PCR reaction chamber are respectively connected to the second eluent chamber;

[0028] The first film and the second film of the fluid channel can switch between two states of separation and mutual adhesion under the action of pressure. When the fluid flows out of the functional chamber under the action of pressure, the first film and the second film of the fluid channel separate from each other under the action of the fluid pressure, and the fluid enters the other functional chamber from one functional chamber at a low speed and controllably through the fluid channel;

[0029] The key to achieving adsorption and elution by the silicon membrane method of the present invention is that the fluid channel allows the liquid to flow in a controlled manner at a low speed. The lower speed allows the liquid flowing through the silicon membrane to have sufficient adsorption and elution time with the silicon membrane, thereby minimizing the degree of gradient decline in adsorption and elution. As a result, the silicon membrane can fully adsorb the nucleic acid in the liquid and fully elute the eluted liquid in a shorter adsorption path, achieving an effect similar to that of the liquid passing vertically through the silicon membrane.

[0030] The low-speed controllable flow described in the present invention refers to the liquid flowing in the fluid channel at an average flow rate of less than 45ul / s, preferably less than 40ul / s, 30ul / s, 20ul / s, 15ul / s, 10ul / s, or 5ul / s.

[0031] In the microfluidic chip of the applicant's prior patent CN2022109249601, microfluidic channels can also be set between each chamber, and the width of the channel is limited to 0.1-10mm and the length is 0.1mm-50mm. It is recorded that the liquid passes through the microfluidic channel at a high speed (5-20m / s) to enhance the lysis effect of cells and other substances. The high-speed flow of liquid in the microfluidic channel is obviously not applicable to the present invention. The applicant's further research found that fluid channels with different structures have different effects on the liquid flow rate. Specifically, the liquid flow rate in the channel is related to the following factors:

[0032] a. The maximum angle between the fluid channel film and the microfluidic chip plane after the fluid channel is stretched open by the liquid;

[0033] b. The width of the fluid channel;

[0034] c. Length of the fluid channel.

[0035] When positive pressure is applied to squeeze the liquid storage chamber, the liquid in the chamber gains a certain liquid pressure, which drives the liquid to start flowing. Since the fluid channel is in a close fit before the fluid passes through, after the fluid flows out of the liquid storage chamber and into the fluid channel, it needs to overcome the tension of the film to open the fluid channel:

[0036] First, after the fluid channel is opened, the smaller the maximum angle between the fluid channel film and the plane of the microfluidic chip, the greater the hydraulic pressure required to open the fluid channel when the elastic modulus of the film remains unchanged, which means that the greater the resistance that needs to be overcome. The size of the maximum angle is usually related to the hardness of the film material, and the hardness is related to the elastic modulus and thickness. Generally speaking, the larger the elastic modulus, the smaller the maximum angle, and the smaller the elastic modulus, the larger the maximum angle.

[0037] Second, the smaller the width of the fluid channel, the smaller the cross-sectional area after the channel is expanded, the greater the hydraulic pressure on the cross section, and the greater the resistance that needs to be overcome.

[0038] Third, the longer the length of the fluid channel, the greater the resistance given by the entire fluid channel.

[0039] In the microfluidic chip of the applicant's prior patent CN2022109249601, the microfluidic channel actually used is very short, tending to the lower limit of the length range. Driven by a large hydraulic pressure, the liquid can pass through the channel at high speed. Obviously, this is not what the present invention expects.

[0040] The present invention can select and match the membrane material, fluid channel width and length settings so that the flow rate of the liquid in the fluid channel meets the above definition, and can be used as the fluid channel of the present invention for low-speed controllable flow of liquid.

[0041] In the present invention, when using a common flexible film (elastic modulus 400-6000 MPa, thickness 0.05-2 mm), in order to match the actual product size, the channel length can be set to 5-150 mm, preferably 20-100 mm. Under the above dimensions, the communication width can be 0.5-2 mm, preferably 1.3-1.8 mm. It should be noted that the above dimensions are not limitations on the fluid channel structure of the present invention, but only provide an implementable range, and do not limit the possibility of implementation outside this range, as long as the low-speed controllable effect described in the present invention can be achieved.

[0042] From the above description, it can be seen that when the fluid channel described in the present invention is in use, the resistance encountered by the fluid in the channel is dynamically variable. It is very difficult to use the existing technology to achieve the liquid pre-filled in the liquid storage chamber of the thin film microfluidic chip to flow in the microchannel in a repeatable, controllable, slow and uniform manner. When mechanical extrusion is used, the total volume of the chamber after filling will be different because the amount of air encapsulated in the liquid storage chamber is different each time filling. As a result, the same extrusion speed will produce different extrusion effects. In addition, the special shape of the chamber also causes the flow rate of the liquid in the channel to be significantly different when the thickness of the driving liquid chamber changes, and even the occurrence of tearing of the welding parts at different positions.

[0043] In the solution of the present invention, after applying positive pressure to squeeze the liquid storage chamber, the liquid in the chamber obtains a certain liquid pressure, thereby driving the liquid to start flowing. Because the elongated fluid channel is in a close-fitting state before the fluid passes through, the fluid needs to overcome resistance to open the fluid channel after flowing out of the liquid storage chamber and into the fluid channel. Moreover, when the flexible film of the fluid channel is stretched, the film in the channel part is very narrow, and the deformation caused by the stretching of the film will generate flow resistance to the fluid due to the large pressure. Therefore, the fluid needs to overcome resistance to flow all the time, and the flow rate in the fluid channel is slow. As the liquid flows out of the liquid storage chamber, the pressure is released when the extrusion device does not increase the extrusion stroke. When the pressure is released or the pressure is not enough to drive the liquid out, the liquid stops flowing. Therefore, it is necessary for the extrusion device to continuously maintain a constant pressure on the chamber to make the liquid flow at a uniform speed, or to intermittently increase the extrusion stroke to squeeze the chamber to make the liquid flow in waves. Take intermittent extrusion as an example, such as using a pulsed extrusion method. For the first time, a pressure is applied to the chamber, and the liquid generates internal pressure, and the liquid begins to flow. However, due to the existence of a narrow closed fluid channel, the liquid flows out very slowly, so the pressure inside the liquid is also released slowly. After the internal pressure of the liquid is released to a certain value (for example, when the liquid cannot be driven to flow out of the narrow fluid channel), because the extrusion device does not increase the extrusion stroke, as the volume of the chamber becomes smaller, the extrusion device and the side wall of the chamber have no force contact or the mutual force is weak; at this time, the extrusion stroke is increased, and pressure is applied a second time to continue to maintain the internal pressure of the liquid, so that the liquid can continue to flow out of the narrow fluid channel slowly. In this cycle, the liquid can intermittently maintain a slow speed to flow out of the narrow fluid channel. When the interval of intermittent extrusion continues to shrink, it actually becomes a continuous extrusion method.

[0044] A valve area is provided between the functional chamber and the fluid channel as required, including the requirement that the functional chamber be pre-filled with liquid. An irreversible, disposable valve structure is distributed within the valve area. When the disposable valve structure is switched from a closed state to an open state, for example due to fluid pressure, fluid can flow from the functional chamber into the fluid channel or vice versa under pressure.

[0045] Another aspect of the present invention further provides a fluid sample processing device, comprising:

[0046] The flexible thin film microfluidic chip;

[0047] One or more first squeezing mechanisms, each of which is positioned and geometrically shaped to correspond to a functional chamber of the microfluidic chamber and is configured to selectively apply pressure to the corresponding functional chamber to open a disposable valve or force fluid into the fluid channel and flow to a desired location;

[0048] One or more physical valves that selectively cooperate with disposable valves on a microfluidic chip to repeatedly physically control the opening and closing of a fluid channel that does not have a disposable valve or, after a disposable valve is opened, to repeatedly physically continue to control the opening and closing of the fluid channel corresponding to the disposable valve.

[0049] In conjunction with the pressure control mechanism, a special control sequence is used to adjust the switching mode and time of the gas source solenoid valve to achieve the purpose of slowly applying band pressure to the corresponding functional chamber.

[0050] Another aspect of the present invention further provides a use of the fluid sample processing device, such as use in nucleic acid extraction and nucleic acid PCR amplification detection.

[0051] Another aspect of the present invention further provides a method for extracting nucleic acid, which is implemented based on the fluid sample processing device, and comprises:

[0052] S1. Sample loading and liquid injection step: injecting a nucleic acid sample into the sample chamber of the microfluidic chip, injecting a rinse liquid into the rinse liquid chamber, and injecting an eluent into at least one of the first eluent chamber and the second eluent chamber; preferably, the rinse liquid chamber is divided into a first rinse liquid chamber and a second rinse liquid chamber, and the rinse liquids are the first rinse liquid and the second rinse liquid, respectively;

[0053] The sample here is the target nucleic acid, and it is necessary to dissolve or infiltrate the cell to first release the target nucleic acid and make it available for amplification detection. If the cell is dissolved, the material contained in the lysate also includes, in addition to the nucleic acid, organelles, proteins (including enzymes such as proteases and nucleases), carbohydrates, and lipids, which require further purification and extraction of nucleic acid. The sample needs to be subjected to the cracking of these cell tissues before being loaded into the fluid sample processing device of the present invention, or after being loaded into the fluid sample processing device of the present invention, the cracking of the cell tissue is completed in the sample chamber or other functional chambers, for example, together with lysate or separately loaded into the sample chamber or other functional chambers. The cell can be cracked by various methods well known to those skilled in the art, including chemical methods, mechanical methods (for example, ultrasound) and / or thermal methods.

[0054] S2, nucleic acid capture step: closing the physical valve, leaving only the passage from the sample chamber through the silicon membrane chamber to the waste chamber open, squeezing the sample chamber with a set squeezing force, so that the liquid sample flows through the fluid channel through the silicon membrane chamber and then into the waste chamber, completing nucleic acid capture;

[0055] S3, nucleic acid rinsing step: adjusting the physical valve to open only the passage from the rinsing liquid chamber through the silicon membrane chamber to the waste liquid chamber, and squeezing the rinsing liquid chamber with a set squeezing force, so that the rinsing liquid flows through the fluid channel through the silicon membrane chamber and then into the waste liquid chamber, completing the nucleic acid rinsing;

[0056] Preferably, when the rinsing liquid chamber is set as a first rinsing liquid chamber and a second rinsing liquid chamber, only the passage from the first rinsing liquid chamber to the waste liquid chamber through the silicon membrane chamber is opened first, and the first rinsing liquid chamber is squeezed with a set extrusion force, so that the first rinsing liquid flows through the silicon membrane chamber through the fluid channel and then is discharged into the waste liquid chamber, and then only the passage from the second rinsing liquid chamber to the waste liquid chamber through the silicon membrane chamber is opened, and the second rinsing liquid chamber is squeezed with a set extrusion force, so that the second rinsing liquid flows through the silicon membrane chamber through the fluid channel and then is discharged into the waste liquid chamber, thereby completing the nucleic acid rinsing.

[0057] S4, nucleic acid elution step:

[0058] Adjusting the physical valve to open only the passages of the first and second eluent chambers through the silicon membrane chamber, squeezing the eluent chamber filled with eluent at a set squeezing force, causing the eluent to flow through the silicon membrane chamber through the fluid channel and then into the other eluent chamber, then alternately squeezing the two eluent chambers at the set squeezing force until the nucleic acid adsorbed on the silicon membrane is completely eluted and the eluent carrying the nucleic acid is ultimately deposited in the first eluent chamber or the second eluent chamber, thereby completing the nucleic acid extraction;

[0059] Preferably, when the rinsing liquid chamber is provided as a first rinsing liquid chamber and a second rinsing liquid chamber, the rinsing liquid chamber may be the first rinsing liquid chamber or the second rinsing liquid chamber.

[0060] Another aspect of the present invention further provides a method for nucleic acid PCR amplification detection, which is implemented based on the fluid sample processing device. After the nucleic acid extraction method, the method further includes:

[0061] S5. Quantification of nucleic acid eluate:

[0062] S5a: Forward nucleic acid quantification step: When the nucleic acid is stored in the first eluent chamber, the physical valve is adjusted to open only the passages between the first eluent chamber and the second eluent chamber;

[0063] The first eluent chamber is squeezed continuously or intermittently at a set pressure, so that the liquid flows from the first eluent chamber into the second eluent chamber at a uniform speed or in a wave band. The squeezing time is controlled so that a fixed amount of nucleic acid eluent flows into the second eluent chamber and is stored;

[0064] Alternatively, S5b: a reverse nucleic acid quantification step: when the nucleic acid is stored in the second eluent chamber, adjusting the physical valve to open only the passage to the second eluent chamber and one other chamber, wherein the other chamber is selected from the group consisting of: the sample chamber, the first rinse chamber, the second rinse chamber, the waste chamber, and the first eluent chamber; continuously or intermittently squeezing the second eluent chamber at a set pressure to allow liquid to flow from the second eluent chamber into the other chamber at a uniform speed or in a wave pattern, controlling the squeezing time to quantitatively discharge excess eluent, and obtaining a quantitative nucleic acid eluate in the second eluent chamber and storing it;

[0065] In the prior art, the chambers are directly connected or connected by channels. The channels in the prior art are either rigid pipes that are conducive to fluid flow, or short-stroke channels (such as disclosed in CN2022109249601). At this time, after the liquid passes through the connecting port or channel under the action of pressure, it enters another chamber immediately or in a short time. Due to the huge pressure difference on both sides, the liquid outflow speed is very fast and changes rapidly, and quantitative control within unit time cannot be achieved.

[0066] The channels of the present invention utilize fluid channels that can switch between separate and attached states. By selecting the parameters described above and coordinating the pressure applied to the liquid storage chamber, the liquid can flow at a slow, uniform rate or in waves, further enabling quantitative liquid discharge. In the solution of the present invention, applying positive pressure to squeeze the liquid storage chamber creates a certain liquid pressure within the chamber, thereby driving the liquid to begin flowing. Because the elongated fluid channel is in a closed state before the fluid passes through it, the fluid needs to overcome resistance to expand the channel after flowing out of the liquid storage chamber and into the fluid channel. Furthermore, when the flexible membrane of the fluid channel is expanded, the membrane in the channel portion is very narrow, and the deformation of the membrane also exerts a large pressure on the fluid. Therefore, the fluid constantly needs to overcome resistance to flow, and the flow rate in the fluid channel is slow. As the liquid flows out of the liquid storage chamber, the pressure is released without increasing the extrusion stroke of the extrusion device. When the pressure is released or the pressure is insufficient to drive the liquid out, the liquid stops flowing. Therefore, it is necessary for the extrusion device to maintain a constant pressure in the chamber to ensure a uniform flow of the liquid, or to intermittently increase the extrusion stroke to squeeze the chamber to achieve a wave-like flow of the liquid. Taking intermittent extrusion as an example, such as using a pulsed extrusion method, a pressure is first applied to the chamber, internal pressure is generated in the liquid, and the liquid begins to flow. However, due to the narrow and closed fluid channel, the liquid flows out very slowly, and therefore the internal pressure of the liquid is also released slowly. When the internal pressure of the liquid is released to a certain value (for example, when the liquid cannot be driven to flow out of the narrow fluid channel), because the extrusion device does not increase the extrusion stroke, as the volume of the chamber decreases, the extrusion device and the side wall of the chamber have no force contact or the mutual force is weak; at this time, the extrusion stroke is increased and pressure is applied a second time to continue to keep the internal pressure of the liquid, so that the liquid can continue to flow out of the narrow fluid channel slowly. In this cycle, the liquid can intermittently maintain a slow speed to flow out of the narrow fluid channel. When the interval of intermittent extrusion continues to shrink, it actually becomes a continuous extrusion method.

[0067] The structure and operation mode of the present invention convert the phenomenon of draining all the liquid in the chamber in an instant in the prior art into a phenomenon of draining the liquid slowly, evenly or in waves over a longer period of time.

[0068] When a quantitative amount of liquid is required, the present invention only needs to limit the discharge time. For example, under a certain extrusion method, the time for slowly discharging 100uL of liquid is 10s. Then, when we need 50uL of liquid, we can limit the time of the extrusion method to 5s.

[0069] S6. Amplification solution mixing: Adjust the physical valves to open only the second eluent chamber and the PCR reaction solution chamber, and sequentially squeeze the second eluent chamber and the PCR reaction solution chamber to achieve mixing of the nucleic acid and the PCR reaction solution. The nucleic acid mixture is stored in the second eluent chamber;

[0070] S7: PCR Amplification: Transfer the nucleic acid mixture from the second elution chamber to the PCR reaction chamber. Alternatively, if the mixture is not transferred, apply the preset PCR temperature ramp program to the chamber containing the nucleic acid mixture and collect fluorescence at the set temperature points. Plot the PCR fluorescence curve and calculate the Ct value to complete the nucleic acid detection process.

[0071] Compared with the prior art, the present invention has at least the following beneficial effects:

[0072] (1) The microfluidic chip of the present invention realizes nucleic acid extraction and amplification detection based on the silicon membrane method in a closed two-dimensional planar flexible thin film microfluidic chip.

[0073] (2) The flexible thin film microfluidic chip of the present invention is provided with a fluid channel. In addition to connecting each functional chamber and the silicon membrane chamber to form a liquid passage, the fluid channel can also provide space for the setting of the silicon membrane chamber. The silicon membrane chamber is equivalent to being set on a fluid channel. Each of the other functional chambers is connected to the fluid channel provided with the silicon membrane chamber through a fluid channel or is directly connected to the silicon membrane chamber. The silicon membrane chamber requires as small an expansion deformation as possible, and its projected area is much smaller than that of each functional chamber. In the silicon membrane method, the types of liquids flowing through the silicon membrane chamber in batches are relatively large, so the silicon membrane chamber actually becomes the core chamber. In the prior art, each functional chamber is directly or connected with a very short connecting channel. The connection method of the prior art cannot realize the reliable connection between each functional chamber and the smaller silicon membrane chamber, and cannot arbitrarily switch the liquid passage flowing through the silicon membrane chamber by physical valves or other means. It is even more impossible to ensure the possibility of cross contamination between the liquids due to residue or rapid vibration or movement of the sample gun after the liquid passage is switched.

[0074] (3) A flexible and slender fluid channel that can be opened and closed is set between the two chambers. The opening and closing tension of the slender channel and the continuous resistance of the long stroke are used to achieve quantitative and controllable low-speed flow of liquid between the chambers. This enables the silicon membrane to fully adsorb and elute nucleic acids to be detected in a two-dimensional plane, and solves the technical problem of quantitatively obtaining amplified nucleic acids in PCR amplification experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The following describes exemplary embodiments of the present invention with reference to the accompanying drawings. It should be understood that these specific descriptions are only used to teach those skilled in the art how to implement the present invention, and are not intended to exhaust all possible embodiments of the present invention, nor to limit the scope of protection of the present invention.

[0076] Figure 1 1 is a schematic structural diagram of a flexible thin film microfluidic chip according to an embodiment of the present invention;

[0077] Figure 2The figure is a schematic diagram of the layout of the liquid inlet and valve area in a flexible thin film microfluidic chip according to one embodiment of the present invention. DETAILED DESCRIPTION

[0078] Some embodiments of the present invention provide a flexible thin film microfluidic chip, comprising a laminated membrane, at least one fixed bonding area, multiple functional chambers, a fluid channel, at least one silicon membrane chamber, at least one valve area, and at least one liquid inlet, wherein the fixed bonding area, the functional chamber, the fluid channel, the silicon membrane chamber, and the valve area are all distributed within the laminated membrane, and the size and shape of the functional chamber, the fluid channel, and the silicon membrane chamber are defined by the boundaries of the fixed bonding area. The liquid inlet is connected to the at least one functional chamber and is sealed after liquid is introduced.

[0079] The laminated film comprises a first film and a second film that are stacked, and at least one of the first film and the second film is a flexible film;

[0080] The first film and the second film are irreversibly bonded in the fixed bonding area;

[0081] The maximum width of the silicon membrane chamber is greater than the width of the fluid channel, and a silicon membrane having a shape matching that of the fluid channel is accommodated therein;

[0082] The functional chamber can switch between two states of separation and adhesion under the action of pressure, wherein the first film and the second film in the functional chamber are separated from each other, and the functional chamber can contain fluid;

[0083] The functional chamber comprises at least one sample chamber, at least one rinse liquid chamber, at least one waste liquid chamber and at least two eluent chambers, namely a first eluent chamber and a second eluent chamber.

[0084] In one embodiment, the functional chamber includes a sample chamber, two rinsing liquid chambers, a waste liquid chamber and two eluent chambers, wherein the two rinsing liquid chambers are respectively a first rinsing liquid chamber and a second rinsing liquid chamber;

[0085] The functional chamber is connected to the silicon membrane chamber via a fluid channel, and the fluid channel allows the fluid to pass through at a low speed and in a controllable manner;

[0086] The fluid channel can be divided into a plurality of sections according to the functional chambers connected thereto, the plurality of sections including at least a first fluid channel and a second fluid channel, wherein the first fluid channel is connected to the sample chamber and the second fluid channel is connected to the waste chamber;

[0087] There is no restriction on the connection method between the functional chamber and the silicon membrane chamber through the fluid channel. In one embodiment, the functional chambers can be independently connected to the silicon membrane chamber directly through the fluid channel. However, this connection method is not preferred because the silicon membrane chamber is very small and it is difficult to provide enough space to connect with each fluid channel, which will also limit the size of the fluid channel.

[0088] In one embodiment, each chamber is connected to the silicon membrane chamber by sharing a portion of fluid channels, wherein the number of fluid channels directly connected to the silicon membrane chamber is no more than 5, 4, 3 or 2;

[0089] In one embodiment, the sample chamber is connected to the silicon membrane chamber through a first fluid channel, the waste liquid chamber is connected to the silicon membrane chamber through a second fluid channel, and the remaining functional chambers are connected to the first or second fluid channel through their respective fluid channels, wherein the rinse liquid chamber is connected to the first fluid channel, the first eluent chamber is connected to the second fluid channel, and the second eluent chamber is connected to the first fluid channel;

[0090] Furthermore, the functional chamber further includes a PCR amplification detection chamber, the PCR amplification detection chamber includes a PCR reaction liquid chamber and an optional PCR reaction chamber, and the PCR reaction liquid chamber and the PCR reaction chamber are respectively connected to the second eluent chamber;

[0091] The first film and the second film of the fluid channel can switch between two states of separation and mutual adhesion under the action of pressure. When the fluid flows out of the functional chamber under the action of pressure, the first film and the second film of the fluid channel separate from each other under the action of the fluid pressure, and the fluid enters from one functional chamber to another functional chamber through the fluid channel;

[0092] A valve area is provided between the functional chamber and the fluid channel as required, wherein an irreversible disposable valve structure is distributed in the valve area. When the disposable valve structure is changed from a closed state to an open state due to fluid pressure, for example, fluid can enter the fluid channel from the functional chamber or from the fluid channel into the functional chamber under the action of pressure;

[0093] The disposable valve structure can adopt a variety of weak connection structures known in the art, which are used to isolate the functional chamber and the fluid channel from each other, and can be destroyed under certain heat, electromagnetic radiation or force to change from a closed state to an open state.

[0094] Preferably, the first and second films in the valve area are detachably combined to form the disposable valve structure, which can be destroyed by the impact of a fluid at a certain pressure, thereby changing from a closed state to an open state. For example, when a set pressure is applied to a functional chamber containing a fluid, the fluid can be impacted and destroyed by the irreversible disposable valve structure disposed between the functional chamber and the fluid channel, thereby allowing the fluid to enter the fluid channel. The disposable valve structure can physically constitute a "valve" between the liquid storage chamber and the fluid channel. These "valves" can be closed simply and quickly by selective squeezing, and the "valves" do not have any physical space after closing. Compared with traditional valves, they are easier to operate and do not cause fluid waste.

[0095] In the present invention, the pressure effect may be pressure provided by a force-applying mechanism (e.g., a squeezing mechanism) associated with each functional chamber or fluid channel. In some cases, the pressure effect may also include stress generated by deformation of the flexible film in the first film or the second film.

[0096] In the present invention, at least one positioning hole may be formed on the laminated membrane. Providing the positioning hole allows workers to more quickly and accurately place the microfluidic chamber into a corresponding detection device, and more accurately perform operations such as selectively squeezing different functional chambers in the microfluidic chamber.

[0097] In the present invention, the first film and the second film include, but are not limited to, any of PET film, PE film, PP film, PA film, PS film, and PI film, an aluminized film of any of these films, a composite film of any of these films and aluminum foil, a combination of multiple films, or a composite film of a combination of multiple films and aluminum foil. Preferably, both the first film and the second film are flexible films.

[0098] Another aspect of the present invention further provides a fluid sample processing device, comprising:

[0099] The flexible thin film microfluidic chip;

[0100] One or more first squeezing mechanisms, each first squeezing mechanism being provided corresponding to a functional chamber of the microfluidic chamber and being used to selectively apply pressure to the corresponding functional chamber to open a disposable valve or drive fluid into the fluid channel and flow to a desired location;

[0101] One or more physical valves that selectively cooperate with disposable valves or fluid channels on a microfluidic chip to repeatedly and physically control the opening and closing of fluid channels that do not have disposable valves or, after a disposable valve is opened, to repeatedly and physically continue to control the opening and closing of the fluid channel corresponding to the disposable valve.

[0102] In one embodiment, the physical valve includes one or more second squeezing mechanisms, each second squeezing mechanism is provided corresponding to a valve area of ​​the microfluidic chamber and is used to selectively apply pressure to the corresponding valve area to open or close the corresponding valve.

[0103] In one embodiment, the fluid sample processing device further includes a control unit, the control unit being connected to at least the first extrusion mechanism and configured to regulate the operating state of the first extrusion mechanism. Furthermore, the second extrusion mechanism may also be connected to the control unit, and its operating state may be regulated by the control unit. The control unit may be, but is not limited to, a PLC, an MCU, or a computer.

[0104] Furthermore, the fluid sample processing device may also include other auxiliary mechanisms, such as a drive mechanism, base, and cover plate of the aforementioned extrusion mechanism. These auxiliary mechanisms may be configured in accordance with methods well known in the mechanical field. The drive mechanism may be, but is not limited to, a pneumatic telescopic mechanism, an electromagnetic telescopic mechanism, a hydraulic telescopic mechanism, a linear motor, or other mechanical drive mechanisms.

[0105] Another aspect of the present invention further provides a use of the fluid sample processing device, such as use in nucleic acid extraction and nucleic acid PCR amplification detection.

[0106] Another aspect of the present invention further provides a method for extracting nucleic acid, which is implemented based on the fluid sample processing device, and comprises:

[0107] S1. Sample loading and liquid injection step: injecting a sample into the sample chamber of the microfluidic chip, injecting a rinsing liquid into the rinsing liquid chamber, and injecting an eluent into at least one of the first eluent chamber and the second eluent chamber; preferably, the rinsing liquid chamber is divided into a first rinsing liquid chamber and a second rinsing liquid chamber, and the rinsing liquids are the first rinsing liquid and the second rinsing liquid, respectively;

[0108] The sample here is the target nucleic acid, and it is necessary to dissolve or infiltrate the cell to first release the target nucleic acid and make it available for amplification detection. If the cell is dissolved, the material contained in the lysate also includes, in addition to the nucleic acid, organelles, proteins (including enzymes such as proteases and nucleases), carbohydrates, and lipids, which require further purification and extraction of nucleic acid. The sample needs to be cracked by these cells before being loaded into the fluid sample processing device of the present invention, or after being loaded into the fluid sample processing device of the present invention, the cracking of the cell tissue is completed in the sample chamber, for example, loaded into the sample chamber together with a lysate. The cell can be cracked by various methods well known to those skilled in the art, including chemical methods, mechanical methods (for example, ultrasound) and / or thermal methods.

[0109] S2, nucleic acid capture step: closing the physical valve, leaving only the passage from the sample chamber through the silicon membrane chamber to the waste chamber open, squeezing the sample chamber with a set squeezing force, so that the liquid sample flows through the fluid channel through the silicon membrane chamber and then into the waste chamber, completing nucleic acid capture;

[0110] S3, nucleic acid rinsing step: adjusting the physical valve to open only the passage from the rinsing liquid chamber through the silicon membrane chamber to the waste liquid chamber, and squeezing the rinsing liquid chamber with a set squeezing force, so that the rinsing liquid flows through the fluid channel through the silicon membrane chamber and then into the waste liquid chamber, completing the nucleic acid rinsing;

[0111] In one embodiment, when the rinsing liquid chamber is set as a first rinsing liquid chamber and a second rinsing liquid chamber, only the passage from the first rinsing liquid chamber to the waste liquid chamber through the silicon membrane chamber is opened first, and the first rinsing liquid chamber is squeezed with a set extrusion force, so that the first rinsing liquid flows through the silicon membrane chamber through the fluid channel and then is discharged into the waste liquid chamber. Then, only the passage from the second rinsing liquid chamber to the waste liquid chamber through the silicon membrane chamber is opened, and the second rinsing liquid chamber is squeezed with a set extrusion force, so that the second rinsing liquid flows through the silicon membrane chamber through the fluid channel and then is discharged into the waste liquid chamber, thereby completing the nucleic acid rinsing.

[0112] S4, nucleic acid elution step:

[0113] Adjusting the physical valve to open only the passages of the first and second eluent chambers through the silicon membrane chamber, squeezing the eluent chamber filled with eluent at a set squeezing force, causing the eluent to flow through the silicon membrane chamber through the fluid channel and then into the other eluent chamber, then alternately squeezing the two eluent chambers at the set squeezing force until the nucleic acid adsorbed on the silicon membrane is completely eluted and the eluent carrying the nucleic acid is ultimately deposited in the first eluent chamber or the second eluent chamber, thereby completing the nucleic acid extraction;

[0114] In one embodiment, when the rinsing liquid chamber is provided as a first rinsing liquid chamber and a second rinsing liquid chamber, the rinsing liquid chamber may be the first rinsing liquid chamber or the second rinsing liquid chamber.

[0115] Another aspect of the present invention further provides a method for nucleic acid PCR amplification detection, which is implemented based on the fluid sample processing device. After the nucleic acid extraction method, the method further includes:

[0116] S5. Quantification of nucleic acid eluate:

[0117] S5a: Forward nucleic acid quantification step: When the nucleic acid is stored in the first eluent chamber, the physical valve is adjusted to open only the passages between the first eluent chamber and the second eluent chamber;

[0118] The first eluent chamber is squeezed continuously or intermittently at a set pressure, so that the liquid flows from the first eluent chamber into the second eluent chamber at a uniform speed or in a wave band. The squeezing time is controlled so that a fixed amount of nucleic acid eluent flows into the second eluent chamber and is stored;

[0119] Or S5b: Reverse nucleic acid quantification step: When the nucleic acid is stored in the second eluent chamber, adjust the physical valve to open only the passage to the second eluent chamber and one other chamber, and the other chamber is selected from: the sample chamber, the first rinse liquid chamber, the second rinse liquid chamber, the waste liquid chamber and the first eluent chamber; continuously or intermittently squeeze the second eluent chamber at a set pressure to make the liquid flow from the second eluent chamber into one other chamber at a uniform speed or in a band manner, control the squeezing time, quantitatively discharge the excess eluent, and obtain a quantitative nucleic acid eluent in the second eluent chamber and save it.

[0120] In the present invention, the set pressure refers to a pressure exceeding a threshold value, and the threshold value varies with the specifications and materials of the microfluidic chamber.

[0121] The technical solution of the present invention will be explained in more detail below in conjunction with several embodiments. However, these specific descriptions are only used to teach those skilled in the art how to implement the present invention, rather than to exhaustively enumerate all feasible methods of the present invention, nor to limit the scope of the present invention.

[0122] Example 1: Flexible thin film microfluidic chip

[0123] See also Figure 1-Figure 2 This embodiment provides a flexible thin film microfluidic chip, which is mainly composed of a laminated film 1000 and a liquid inlet interface 2000. The laminated film 1000 and the liquid inlet interface 2000 can be combined into a whole by hot pressing welding, ultrasonic welding, chemical bonding, etc.

[0124] The laminated film 1000 is primarily composed of a first film 1001 and a second film 1002. At least one of the first film 1001 and the second film 1002 is preferably a flexible film having longitudinal stretchability. For example, the first film includes, but is not limited to, a composite film formed by combining one or more of PET film, PE film, PP film, PA film, PS film, and PI film, preferably a composite film. The second film includes, but is not limited to, a composite film formed by coextrusion or composite bonding of one or more of PET film, PE film, PP film, PA film, PS film, and PI film, such as an aluminized film or aluminum foil film, preferably a composite film.

[0125] In this embodiment, one or more fixed bonding areas and multiple functional chambers can be defined in the laminated film 1000 along the film plane. Furthermore, the fixed bonding areas divide the entire laminated film 1000 into several functional chambers, fluid channels 1110, and silicon membrane chambers 1201 along the film plane. That is, the size and shape of the functional chambers, fluid channels 1110, and silicon membrane chambers 1201 are defined by the boundaries of the fixed bonding areas. Within the fixed bonding areas, the first and second films are irreversibly bonded by ultrasonic welding, bonding, hot pressing, or other methods.

[0126] The first and second films within the functional chamber and fluid channel 1110 can switch between separation and adhesion under the action of external force. Specifically, taking the functional chamber as an example, when a fluid at a certain pressure is injected into the functional chamber, the first and second films within the functional chamber deform and separate under the action of the fluid pressure, giving the functional chamber a chamber structure capable of containing liquid and gas. When sufficient pressure is applied to the functional chamber using a squeezing mechanism, for example, the first and second films within the functional chamber adhere to each other, and the physical space within the functional chamber used to contain the fluid and gas disappears.

[0127] In this embodiment, multiple functional chambers primarily serve as liquid storage chambers or reaction chambers, including a sample chamber 1101, a first rinse liquid chamber 1102, a second rinse liquid chamber 1103, a first eluent chamber 1104, a second eluent chamber 1105, a waste liquid chamber 1106, a PCR reaction liquid chamber 1107, and a PCR reaction liquid chamber 1108. Each of these liquid storage chambers has a corresponding liquid inlet, such as a sample inlet 1301, a first rinse liquid inlet 1302, a second rinse liquid inlet 1303, a first eluent inlet 1304, a second eluent inlet 1305, and a PCR reaction liquid inlet 1306. These inlets can be sealed after liquid is filled, and the sample inlet 1301 can be connected to the liquid inlet interface 2000.

[0128] Before liquid and gas are injected, the first and second films are tightly attached to each other within these functional chambers, leaving no physical space between them. Once the corresponding liquid and gas are injected into the corresponding functional chambers through the sample injection holes, the longitudinal stretchability of the flexible films generates internal pressure within the liquid, causing the first and second films to deform and separate, forming liquid storage chambers. Once the liquid within the liquid storage chambers is expelled due to pressure from the squeezing mechanism and the inherent stress of the flexible films, the first and second films reattach to each other within the corresponding functional chambers, eliminating the physical space between them.

[0129] In this embodiment, the functional chamber is connected to the silicon membrane chamber through a fluid channel 1110, and the fluid channel is elongated in shape; the width of the fluid channel is not greater than 5 mm, preferably not greater than 2 mm; the length of the fluid channel is not less than 2 mm, preferably not less than 5 mm, and most preferably not less than 10 mm.

[0130] It should be noted that the above-mentioned size selection in this embodiment is not a limitation of the scheme of the present invention. Those skilled in the art can determine the appropriate film material and channel size based on the relationship between the size, material and flow rate disclosed in the present invention, so that the flow rate in the fluid channel is lower than 45ul / s, preferably lower than 40ul / s, 30ul / s, 20ul / s, 15ul / s, 10ul / s, 5ul / s, that is, the low-speed controllable as described in the present invention.

[0131] In this embodiment, the sample chamber 1301 is connected to the silicon membrane chamber 1201 through the first fluid channel 1111, the waste liquid chamber 1106 is connected to the silicon membrane chamber 1201 through the second fluid channel 1112, and the remaining functional chambers are connected to the first or second fluid channel through their respective fluid channels, wherein the first rinse liquid chamber 1102, the second rinse liquid chamber 1106, and the second eluent chamber 1105 are connected to the first fluid channel 1111 through their respective fluid channels, and the first eluent chamber 1104 is connected to the second fluid channel 1112 through a fluid channel.

[0132] The silicon membrane chamber 1201 can be in various shapes, such as circular, elliptical, diamond-shaped, etc., and its maximum width must be greater than the width of the fluid channel, and contains a silicon membrane with a matching shape;

[0133] Silicon membrane chamber 1201 is different from a functional chamber. It is not desirable for the flexible membrane to undergo significant elastic deformation upon fluid entry, thereby increasing the volume of the silicon membrane chamber. Because the silicon membrane is flat, the larger the chamber, the greater the proportion of fluid that does not flow through the membrane, and the lower the adsorption and elution efficiency. The deformation of the silicon membrane chamber upon fluid entry can be reduced by changing the material of the silicon membrane chamber wall. For example, a material with a greater elastic modulus than the flexible membrane can be used as the silicon membrane chamber wall, or a material with a greater elastic modulus than the flexible membrane can be attached to the outside of the silicon membrane chamber wall.

[0134] In this embodiment, one or more valve regions can be defined within the laminated film 1000 along the film plane. Within each valve region, an irreversible, disposable valve structure is disposed, connecting between a functional chamber and the fluid channel 1110, or between two functional chambers. When the disposable valve structure is switched from a closed state to an open state, for example due to fluid pressure, fluid can enter the fluid channel from the functional chamber or vice versa under the action of pressure. In this embodiment, within the valve region, a first film and a second film are releasably bonded to form the disposable valve structure. "Releasably bonded" herein means that this bond can be broken under certain conditions, such as a certain external force, thereby separating the first and second films within the valve region. Preferably, the first and second films within the valve region can be bonded by ultrasonic welding, adhesive bonding, hot press welding, or other methods, and this bond can be broken by heat, electromagnetic radiation, or force from the fluid. Generally, the width of the disposable valve structure is equal to or slightly greater than the width of the fluid channel and significantly smaller than the diameter of the functional chamber. After the functional chamber and the fluid channel are connected, the communication port is only the torn valve.

[0135] In this embodiment, there can be multiple valve areas, among which the multiple irreversible disposable valve structures can be named as sample valve 1401, first rinse liquid valve 1402, second rinse liquid valve 1403, first eluent valve 1404, second eluent valve 1405, PCR reaction liquid valve 1407, etc.

[0136] In this embodiment, the PCR reaction liquid chamber 1107 and the PCR reaction chamber 1108 are respectively connected to the second eluent chamber 1105 via fluid channels 1110 .

[0137] Also, please refer again to Figure 1-Figure 2 A number of positioning holes, such as a first positioning hole 1501 and a second positioning hole 1502, may be formed on the laminated membrane, especially on its edge, to guide the positioning of the microfluidic chamber and occupy less effective space on the microfluidic chamber.

[0138] Example 2: Fluid Sample Processing Device

[0139] This embodiment provides a fluid sample processing device, comprising the flexible thin film microfluidic chip described in Example 1, and a first extrusion mechanism positioned above or below each liquid storage chamber. When liquid flow is required, the corresponding first extrusion mechanism is pushed, causing the liquid in the corresponding functional chamber to be subjected to pressure. Each liquid storage area has its own corresponding valve area. The valve within each valve area (i.e., the aforementioned irreversible disposable valve structure) will tear open when subjected to fluid pressure exceeding its rupture threshold, thereby allowing the liquid storage chamber and fluid channel connected by the valve to communicate with each other. Liquid can then enter the fluid channel from the liquid storage chamber, and the liquid can continue to flow as needed until the valve structure connected to the other end of the fluid channel is torn open.

[0140] In this embodiment, a corresponding second squeezing mechanism can be provided above or below each valve area to serve as a physical valve. If the valve within each valve area is damaged by liquid pressure, the corresponding physical valve can be used to provide a barrier function. Specifically, the second squeezing mechanism can squeeze the corresponding valve area to form a seal, thereby blocking the flow of liquid.

[0141] The fluid sample processing device also includes a control unit, which is connected to at least the first extrusion mechanism and is used to control the operating state of the first extrusion mechanism. Furthermore, the second extrusion mechanism may also be connected to the control unit, and its operating state may be controlled by the control unit. The control unit may be, but is not limited to, a PLC, an MCU, or a computer.

[0142] Furthermore, the fluid sample processing device may also include other auxiliary mechanisms, such as a drive mechanism, base, and cover plate of the aforementioned extrusion mechanism. These auxiliary mechanisms may be configured in accordance with methods well known in the mechanical field. The drive mechanism may be, but is not limited to, a pneumatic telescopic mechanism, an electromagnetic telescopic mechanism, a hydraulic telescopic mechanism, a linear motor, or other mechanical drive mechanisms.

[0143] The specific mechanical structure of the above-mentioned fluid sample processing device can be implemented using existing technologies, such as those described in CN2022110178734.

[0144] Example 3a: Nucleic acid extraction method

[0145] A method for extracting nucleic acid using the microfluidic chip and fluid sample processing device of Examples 1-2 may include the following steps:

[0146] S1, sample loading and liquid injection step: inject a sample into the sample chamber 1101 of the microfluidic chip, inject a first rinse liquid into the first rinse liquid chamber 1102, inject a second rinse liquid into the second rinse liquid chamber 1103, inject eluent into the first eluent chamber 1104 and the second eluent chamber 1105, and inject a nucleic acid reaction solution into the PCR reaction solution chamber 1107;

[0147] The sample here is a mixture of lysed target nucleic acids containing cell debris and other impurities.

[0148] S2, nucleic acid capture step: Close the physical valve, leaving only the passage from sample chamber 1101 through silicon membrane chamber 1201 to waste liquid chamber 1106 open. Squeeze sample chamber 1101 with a set extrusion force, allowing the liquid sample to flow through first fluid channel 1111, through the silicon membrane chamber, and then through second fluid channel 1112 into waste liquid chamber 1106, completing nucleic acid capture.

[0149] S3. Nucleic acid rinsing step: adjust the physical valve to open only the passage from the first rinsing liquid chamber 1102 to the waste liquid chamber 1106 through the silicon membrane chamber 1201, and squeeze the first rinsing liquid chamber 1102 with a set extrusion force, so that the first rinsing liquid flows through the silicon membrane chamber 1201 through the fluid channel 1110 and then is discharged into the waste liquid chamber 1106, then only open the passage from the second rinsing liquid chamber 1103 to the waste liquid chamber 1106 through the silicon membrane chamber 1201, and squeeze the second rinsing liquid chamber 1103 with a set extrusion force, so that the second rinsing liquid flows through the silicon membrane chamber 1201 through the fluid channel 1110 and then is discharged into the waste liquid chamber 1106, thereby completing the nucleic acid rinsing.

[0150] S4, nucleic acid elution step:

[0151] Adjust the physical valve to open only the passages of the first eluent chamber 1104 and the second eluent chamber 1105 through the silicon membrane chamber 1201, and squeeze the first eluent chamber 1104 with the set squeezing force, so that the eluent flows through the silicon membrane chamber 1201 through the fluid channel 1110 and then flows into the second eluent chamber 1105. Then, squeeze the two chambers alternately with the set squeezing force until the nucleic acid adsorbed on the silicon membrane is completely eluted, and the eluent carrying the nucleic acid is finally stored in the first eluent chamber 1104, completing the nucleic acid extraction.

[0152] Example 3b: Nucleic acid extraction method

[0153] The difference between this embodiment and embodiment 3a is only in the following steps S1 and S4:

[0154] S1, sample loading and liquid injection step: inject the sample into the sample chamber 1101 of the microfluidic chip, inject the first rinse liquid into the first rinse liquid chamber 1102, inject the second rinse liquid into the second rinse liquid chamber 1103, inject the eluent into the first eluent chamber 1104, and inject the nucleic acid reaction solution into the PCR reaction solution chamber 1107;

[0155] The sample here is a mixture of lysed target nucleic acids containing cell debris and other impurities.

[0156] S4, nucleic acid elution step:

[0157] Adjust the physical valve to open only the passages of the first eluent chamber 1104 and the second eluent chamber 1105 through the silicon membrane chamber 1201, and squeeze the first eluent chamber 1104 with the set squeezing pressure to allow the eluent to flow through the silicon membrane chamber 1201 through the fluid channel 1110 and then flow into the second eluent chamber 1105. Then, alternately squeeze the two eluent chambers with the set squeezing pressure until the nucleic acid adsorbed on the silicon membrane is completely eluted, and the eluent carrying the nucleic acid is finally stored in the second eluent chamber 1105, completing the nucleic acid extraction.

[0158] Example 4a: Nucleic Acid Amplification Detection Method

[0159] This embodiment provides a method for nucleic acid PCR amplification detection, which is implemented based on the fluid sample processing device. The method, after the nucleic acid extraction method described in Example 3a, further includes:

[0160] S5. Quantification of nucleic acid eluent: Adjust the physical valve to open only the passages of the first eluent chamber 1104 and the second eluent chamber 1105;

[0161] The first eluent chamber 1104 is squeezed continuously or intermittently at a set pressure, so that the liquid flows from the first eluent chamber 1104 into the second eluent chamber 1105 at a uniform speed or in a wave-like manner. The squeezing time is controlled so that a fixed amount of nucleic acid eluent flows into the second eluent chamber 1105 and is stored;

[0162] S6. Amplification solution mixing: Adjust the physical valves to open only the second eluent chamber 1105 and the PCR reaction solution chamber 1107. The second eluent chamber 1105 and the PCR reaction solution chamber 1107 are squeezed in sequence to achieve mixing of the nucleic acid and the PCR reaction solution. The nucleic acid mixture is stored in the second eluent chamber 1105.

[0163] S7: PCR Amplification: The nucleic acid mixture is transferred from the second eluent chamber 1105 to the PCR reaction chamber 1108. Alternatively, the mixture is not transferred. The chamber containing the nucleic acid mixture is subjected to a preset PCR temperature ramp program, and fluorescence is collected at the set temperature points. A PCR fluorescence curve is plotted, and the Ct value is calculated, completing the nucleic acid detection process.

[0164] Example 4b: Nucleic Acid Amplification Detection Method

[0165] The method described in this embodiment further includes subsequent steps after the nucleic acid extraction method described in Example 3b. The subsequent steps are substantially the same as those in Example 4a, with the only difference being the following step S5:

[0166] S5: Quantification of nucleic acid eluent: Adjust the physical valve to open only the passage between the second eluent chamber 1105 and the sample chamber 1101; continuously or intermittently squeeze the second eluent chamber 1105 at a set pressure to allow the liquid to flow from the second eluent chamber 1105 into the sample chamber 1101 at a uniform speed or in a wave band, control the squeezing time, quantitatively discharge the excess eluent, and obtain a quantitative amount of nucleic acid eluent in the second eluent chamber 1105 and save it.

[0167] Example 5: Fluid channel flow rate and silicon membrane adsorption experiment

[0168] The experimental sample was 50 ng / ul human genomic DNA, with a sample volume of 200 ul. A total of 10,000 ng of DNA was added. Nucleic acid extraction was performed using a commercially available DNA nucleic acid extraction kit.

[0169] Repeat the nucleic acid capture steps: Close the physical valve, leaving only the passage from sample chamber 1101 through silicon membrane chamber 1201 to waste chamber 1106. Apply a set pressure to squeeze sample chamber 1101, allowing 200 μl of liquid sample to flow through first fluid channel 1111, through the silicon membrane chamber, and then through second fluid channel 1112 into waste chamber 1106, completing nucleic acid capture. When the sample chamber is completely drained, record the drain time. Following rinsing and elution, measure the DNA content in the eluate (using a Qubit or Nanodrop device) and convert this into a DNA recovery rate.

[0170] In one set of experimental schemes, the total length of the fluid channel between the sample chamber 1101 and the waste liquid chamber 1106 (including the length of the silicon membrane chamber) is 100 mm. In multiple schemes, the width of the fluid channel is 1.0 mm, 1.3 mm, 1.5 mm, 1.7 mm, 1.8 mm, and 2.0 mm, respectively.

[0171] The experimental results are shown in Table 1

[0172] Table 1

[0173]

[0174] The estimated relationship between flow rate and flow velocity is: flow velocity × 3.14 × (L / 2) 2 = flow rate, where L is the width of the fluid channel.

[0175] From the above experiments, it can be seen that the adsorption effect of the silicon membrane is significantly different under different flow rates and flow rates. When the flow rate is greater than 45ul / s, the adsorption efficiency of the flat-laid silicon membrane drops sharply to below 20%. It is generally believed in this field that a 20% adsorption rate is the minimum requirement for some low-precision nucleic acid analysis. Therefore, a flow rate greater than 45ul / s will basically not meet the requirements of nucleic acid adsorption. The present invention sets the fluid channel so that the flow rate in the fluid channel is lower than 45ul / s, preferably lower than 40ul / s, 30ul / s, 20ul / s, 15ul / s, 10ul / s, and 5ul / s, that is, the low-speed controllable method described in the present invention, so that the silicon membrane can still effectively complete nucleic acid adsorption in the flat state. The above effect also applies to the desorption process.

[0176] Example 6: Relationship between fluid channel length and flow rate

[0177] In order to study the relationship between the length of the fluid channel and the flow rate, the following experiment was designed:

[0178] The silicon membrane chamber was removed, the physical valve was closed, and only the passage from sample chamber 1101 to waste chamber 1106 was left open. Sample chamber 1101 was squeezed with a set pressure, allowing 200 μl of liquid sample to drain through the fluid channel into waste chamber 1106. When the sample chamber was completely drained, the drain time was recorded. The fluid channel between sample chamber 1101 and waste chamber 1106 was 1.5 mm wide, and in various designs, the lengths were 1 mm, 2 mm, 5 mm, 10 mm, 20 mm, and 40 mm, respectively. The experimental results are shown in Table 2.

[0179] Table 2

[0180]

[0181] Through experiments, we also found that in the range of 2mm or more, the sensitivity of the change in channel length to the flow rate is significantly less than the change in width.

[0182] Although the present invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions, and / or additions may be made and that substantial equivalents may be substituted for the elements of the embodiments without departing from the spirit and scope of the present invention. Additionally, many modifications may be made to adapt specific circumstances or materials to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended herein to limit the present invention to the disclosed specific embodiments for carrying out the present invention, but rather to include all embodiments within the scope of the appended claims. Furthermore, unless specifically stated, any use of the terms first, second, etc. does not indicate any order or importance, but rather uses the terms first, second, etc. to distinguish one element from another.

Claims

1. A flexible thin film microfluidic chip comprising a laminated membrane, at least one fixed bonding region, a plurality of functional chambers, a fluid channel, at least one silicon membrane chamber, at least one valve region, and at least one liquid inlet, wherein the fixed bonding region, functional chamber, fluid channel, silicon membrane chamber, and valve region are all distributed within the laminated membrane, and the size and shape of the functional chamber, fluid channel, and silicon membrane chamber are defined by the boundaries of the fixed bonding region. The liquid inlet is connected to the at least one functional chamber and is sealed after liquid is introduced. in, The laminated film includes a first film and a second film that are stacked, and at least one of the first film and the second film is a flexible film; The first film and the second film are irreversibly bonded in the fixed bonding area; The maximum width of the silicon membrane chamber is greater than the width of the fluid channel, and a silicon membrane having a shape matching that of the fluid channel is accommodated therein in a flat manner; The first film and the second film of the functional chamber can switch between two states of separation and mutual adhesion under pressure. When the first film and the second film in the functional chamber are separated from each other, the functional chamber can contain fluid; The functional chamber includes at least one sample chamber, at least one rinse liquid chamber, at least one waste liquid chamber and at least two eluent chambers, wherein the two eluent chambers are respectively a first eluent chamber and a second eluent chamber; The functional chamber is connected to the silicon membrane chamber via a fluid channel, and the fluid channel allows the fluid to pass through in a controllable manner at a low speed; The first film and the second film of the fluid channel can switch between two states of separation and mutual adhesion under the action of pressure. When the fluid flows out of the functional chamber under the action of pressure, the first film and the second film of the fluid channel separate from each other under the action of the fluid pressure, and the fluid enters the other functional chamber from one functional chamber at a low speed and controllably through the fluid channel; The valve area is arranged between the functional chamber and the fluid channel, and an irreversible disposable valve structure is distributed in the valve area.

2. The flexible thin film microfluidic chip according to claim 1, wherein: The fluid channel is divided into multiple sections based on the functional chambers connected to it using the silicone membrane chamber as a dividing point. The multiple sections include at least a first fluid channel and a second fluid channel, wherein the first fluid channel is connected to the sample chamber and the second fluid channel is connected to the waste liquid chamber.

3. The flexible thin film microfluidic chip according to claim 1 or 2, characterized in that: The functional chamber includes a sample chamber, two rinsing liquid chambers, a waste liquid chamber and two elution liquid chambers, wherein the two rinsing liquid chambers are respectively a first rinsing liquid chamber and a second rinsing liquid chamber.

4. The flexible thin film microfluidic chip according to claim 1, wherein: The functional chamber is connected to the silicon membrane chamber by sharing a portion of fluid channels, and the number of fluid channels directly connected to the silicon membrane chamber is no more than 5, 4, 3 or 2.

5. The flexible thin film microfluidic chip according to claim 2, wherein: The functional chamber is connected to the silicon membrane chamber by sharing the first fluid channel and / or the second fluid channel, and the number of fluid channels directly connected to the first fluid channel and / or the second fluid channel is no less than 5, 4, 3, 2 or 1.

6. The flexible thin film microfluidic chip according to claim 2, wherein: The sample chamber is connected to the silicon membrane chamber through a first fluid channel, the waste liquid chamber is connected to the silicon membrane chamber through a second fluid channel, and the remaining functional chambers are connected to the first or second fluid channel through their own fluid channels.

7. The flexible thin film microfluidic chip according to claim 3, wherein: The rinse liquid chamber is connected to the first fluid channel; the first eluent chamber is connected to the second fluid channel, the second eluent chamber is connected to the first fluid channel, or the first eluent chamber is connected to the first fluid channel, the second eluent chamber is connected to the second fluid channel.

8. The flexible thin film microfluidic chip according to claim 1, wherein: The functional chambers are independently connected to the silicon membrane chamber directly through fluid channels.

9. The flexible thin film microfluidic chip according to claim 1, wherein: The functional chamber further includes a PCR amplification detection chamber, which includes a PCR reaction liquid chamber, and the PCR reaction liquid chamber is connected to the second eluent chamber.

10. The flexible thin film microfluidic chip according to claim 9, characterized in that: The PCR amplification detection chamber further includes a PCR reaction chamber, and the PCR reaction chamber is connected to the second eluent chamber.

11. A fluid sample processing device, comprising: The flexible thin film microfluidic chip according to any one of claims 1 to 10.

12. The fluid sample processing device according to claim 11, wherein: The microfluidic chip further comprises one or more first squeezing mechanisms, each of which is provided corresponding to a functional chamber of the microfluidic chip and is used to selectively apply pressure to the corresponding functional chamber to open a disposable valve or drive the fluid into the fluid channel and flow to a desired location; One or more physical valves that selectively cooperate with disposable valves on a microfluidic chip to repeatedly physically control the opening and closing of a fluid channel that does not have a disposable valve or, after a disposable valve is opened, to repeatedly physically continue to control the opening and closing of the fluid channel corresponding to the disposable valve.

13. Use of the fluid sample processing device according to claim 11 or 12 in fluid sample processing.

14. The use according to claim 13, characterized in that The use is in nucleic acid extraction or nucleic acid PCR amplification detection.

15. A method for extracting nucleic acid, the method being implemented based on the fluid sample processing device according to claim 11 or 12, wherein: The functional chamber of the fluid sample processing device further includes a PCR amplification detection chamber, the PCR amplification detection chamber includes a PCR reaction liquid chamber, and the PCR reaction liquid chamber is connected to the second eluent chamber. The method includes: S1, reagent filling step: injecting a nucleic acid sample into the sample chamber of the microfluidic chip, injecting a rinse solution into the rinse solution chamber, injecting an eluent into at least one of the first eluent chamber and the second eluent chamber, and injecting a PCR reaction solution into the PCR reaction solution chamber; S2. Nucleic acid capture step: Adjust the physical valve to open only the passage from the sample chamber through the silicon membrane chamber to the waste chamber. Squeeze the sample chamber with a set extrusion force, causing the liquid sample to break the disposable valve and flow through the silicon membrane chamber through the fluid channel and into the waste chamber, thereby completing nucleic acid capture. S3, nucleic acid rinsing step: adjusting the physical valve to open only the passage from the rinsing liquid chamber through the silicon membrane chamber to the waste liquid chamber, and squeezing the rinsing liquid chamber with a set squeezing force, so that the rinsing liquid flows through the fluid channel through the silicon membrane chamber and then into the waste liquid chamber, completing the nucleic acid rinsing; S4, nucleic acid elution step: Adjust the physical valve to open only the passages of the first eluent chamber and the second eluent chamber through the silicon membrane chamber, and squeeze the eluent chamber filled with eluent with the set extrusion pressure, so that the eluent flows through the silicon membrane chamber through the fluid channel and flows into the other eluent chamber. Then, alternately squeeze the two eluent chambers with the set extrusion pressure until the nucleic acid adsorbed on the silicon membrane is completely eluted, and the eluent carrying the nucleic acid is finally stored in the first eluent chamber or the second eluent chamber, completing the nucleic acid extraction.

16. The method for extracting nucleic acid according to claim 15, wherein The rinsing liquid chamber is divided into a first rinsing liquid chamber and a second rinsing liquid chamber, and the rinsing liquids are the corresponding first rinsing liquid and the second rinsing liquid respectively.

17. The method for extracting nucleic acid according to claim 16, wherein When the rinsing liquid chamber is provided as the first rinsing liquid chamber and the second rinsing liquid chamber, The step S3 is as follows: first, only the passage from the first rinsing liquid chamber to the waste liquid chamber through the silicon membrane chamber is opened, and the first rinsing liquid chamber is squeezed with a set squeezing force, so that the first rinsing liquid flows through the silicon membrane chamber through the fluid channel and then is discharged into the waste liquid chamber; then, only the passage from the second rinsing liquid chamber to the waste liquid chamber through the silicon membrane chamber is opened, and the second rinsing liquid chamber is squeezed with a set squeezing force, so that the second rinsing liquid flows through the silicon membrane chamber through the fluid channel and then is discharged into the waste liquid chamber, thereby completing the nucleic acid rinsing.

18. A nucleic acid PCR amplification detection method, said method being implemented based on the fluid sample processing device according to claim 11 or 12, wherein: The functional chamber of the fluid sample processing device further includes a PCR amplification detection chamber, the PCR amplification detection chamber includes a PCR reaction liquid chamber, the PCR reaction liquid chamber is connected to the second eluent chamber, the PCR amplification detection chamber further includes a PCR reaction chamber, the PCR reaction chamber is connected to the second eluent chamber, and the method is characterized in that, after the nucleic acid extraction method according to any one of claims 15 to 17, further includes: S5. Quantification of nucleic acid eluate: S5a: Forward nucleic acid quantification step: When the nucleic acid is stored in the first eluent chamber, the physical valve is adjusted to open only the passages between the first eluent chamber and the second eluent chamber; The first eluent chamber is squeezed continuously or intermittently at a set pressure, so that the liquid flows from the first eluent chamber into the second eluent chamber at a uniform speed or in a wave band. The squeezing time is controlled so that a fixed amount of nucleic acid eluent flows into the second eluent chamber and is stored; Alternatively, S5b: a reverse nucleic acid quantification step: when the nucleic acid is stored in the second eluent chamber, adjusting the physical valve to open only the passage to the second eluent chamber and one other chamber, wherein the other chamber is selected from the group consisting of: the sample chamber, the first rinse chamber, the second rinse chamber, the waste chamber, and the first eluent chamber; continuously or intermittently squeezing the second eluent chamber at a set pressure to allow liquid to flow from the second eluent chamber into the other chamber at a uniform speed or in a wave pattern, controlling the squeezing time to quantitatively discharge excess eluent, and obtaining a quantitative nucleic acid eluate in the second eluent chamber and storing it; S6. Amplification solution mixing: Adjust the physical valves to open only the second eluent chamber and the PCR reaction solution chamber, and sequentially squeeze the second eluent chamber and the PCR reaction solution chamber to achieve mixing of the nucleic acid and the PCR reaction solution. The nucleic acid mixture is stored in the second eluent chamber; S7: PCR amplification: The nucleic acid mixture is transferred from the second elution liquid chamber to the PCR reaction chamber, or not transferred, to complete nucleic acid amplification and detection of the nucleic acid mixture.

19. The nucleic acid PCR amplification detection method according to claim 18, wherein: In step S7, the chamber loaded with the nucleic acid mixture is loaded with a preset PCR temperature ramp program, and fluorescence is collected at the set temperature point, a PCR fluorescence curve is drawn, and a Ct value is calculated to complete the nucleic acid detection.

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