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

Abstract

The present invention discloses a flexible thin film microfluidic chip, which includes a laminated membrane, at least one fixed binding area, multiple functional chambers, a fluid channel, at least one valve area and at least one liquid inlet interface, wherein the fixed binding area, the functional chamber, the fluid channel and the valve area are all distributed in the laminated membrane, and the size and shape of the functional chamber and the fluid channel are defined by the boundary of the fixed binding area, the liquid inlet interface is connected to at least one functional chamber and is closed after liquid is introduced; the flexible thin film microfluidic chip is divided into at least three functional areas: a nucleic acid extraction area, a nucleic acid quantification area and a nucleic acid detection area, wherein the nucleic acid quantification area is located between the nucleic acid extraction and capture area and the nucleic acid detection area; any functional area includes at least one functional chamber; the functional chamber in the nucleic acid quantification area includes at least a nucleic acid quantification chamber, and during the process of liquid flowing from the nucleic acid extraction area to the nucleic acid detection area, it needs to be temporarily stored in the nucleic acid quantification chamber and filled with the nucleic acid quantification chamber. The present invention can realize the liquid quantification function of non-fixed volume without changing the physical size of the nucleic acid quantification chamber, thereby meeting the actual use requirements under different conditions.

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—including 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 beads can be enriched and released 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 flow method, the adsorption gradient of the silicon membrane is inversely proportional to its thickness. Since the membrane can usually be very thin, the adsorption working surface is large, the gradient drop is minimal, and the adsorption efficiency is very high. However, achieving nucleic acid adsorption, rinsing, and elution based on the silicon membrane method in a closed two-dimensional flexible thin-film microfluidic chip, and completing nucleic acid detection, is difficult. However, if the silicon membrane is fixed in the channel of the flexible thin-film microfluidic chip and a sample solution with an appropriate salt ion concentration, a rinse solution, and an eluent are sequentially passed through it, it is theoretically possible to extract and purify nucleic acids from complex samples. However, during implementation, on the one hand, because the flexible thin film microfluidic chip has a two-dimensional planar structure, the microfluidic channel embedded in the silicon membrane has a certain thickness, but its cross-sectional dimensions are much smaller than the planar cross-sectional dimensions of the silicon membrane centrifugal column when conventionally used. In this case, in order to fully adsorb the nucleic acid in the sample on the silicon membrane, when the fluid flows along the microfluidic channel through the nucleic acid extraction silicon membrane, the length of the flow channel loaded with the silicon membrane must be much greater than the thickness of the silicon membrane through which the liquid flows when using the centrifugal column method. The liquid flow resistance generated during the process will also increase with the increase in the amount of liquid flowing through the silicon membrane. On the other hand, the flexibility of the thin film microfluidic chip itself is determined to be limited by both the materials used and the preparation method. Under the action of continuous and increasing pressure, the microchannel may form destructive deformation, resulting in failed experimental results. Therefore, the existing centrifugal column silicon membrane method cannot be directly applied to thin film microfluidic chips.

[0013] On the other hand, during the nucleic acid extraction process, saliva samples need to go through several steps such as cell lysis and binding, nucleic acid adsorption and purification, and nucleic acid elution and collection; during the nucleic acid detection process, the extracted nucleic acid samples need to go through steps such as mixing with PCR reaction solution and qPCR amplification.

[0014] For example, the patent document CN202211170669.6 disclosed in the prior art proposes an integrated nucleic acid extraction microfluidic chip box and a nucleic acid extraction and detection method, wherein the integrated nucleic acid extraction microfluidic chip box includes a microfluidic chip body, a driving component and a reaction component. The microfluidic chip body includes a plurality of cavities that can be interconnected, a valve body for controlling the conduction or cutoff between the cavities, and a driving component for driving the liquid to flow in each chamber; the method for nucleic acid extraction and detection using the integrated nucleic acid extraction microfluidic chip box includes: sample addition, lysis, nucleic acid capture, cleaning, elution, PCR mixing, PCR amplification and other steps.

[0015] However, in this existing technology, in the steps of elution, PCR mixing, and PCR amplification, the nucleic acid is first eluted and mixed with the eluent, the eluent mixed with the nucleic acid is then mixed with the PCR reaction reagent, and then the PCR reaction chamber is heated, amplified, and optically detected, thereby completing the nucleic acid detection; and in the process of mixing the eluent mixed with the nucleic acid with the PCR reaction reagent, the eluent mixed with the nucleic acid cannot be quantified, which can easily lead to failure of the nucleic acid detection or inaccurate results of the nucleic acid detection; for example, in theory, the measurement of the eluent containing the nucleic acid corresponding to the measurement of the PCR reaction reagent is defined as a predetermined measurement. If the measurement of the eluent that reacts with the PCR reaction reagent is greater than the predetermined measurement, it is easy to cause the nucleic acid quantitative result to be higher than the actual result. Conversely, if the measurement of the eluent that reacts with the PCR reaction reagent is less than the predetermined measurement, it is easy to cause the nucleic acid quantitative result to be lower than the actual result. Summary of the Invention

[0016] In response to the above problems in the prior art, the main purpose of the invention is to provide a flexible thin film microfluidic chip and a fully integrated quantitative nucleic acid detection method. This method can realize nucleic acid extraction based on the silicon membrane method, and can simply and efficiently realize the quantification of nucleic acid eluate, as well as real-time PCR amplification detection to realize the entire process of nucleic acid extraction, nucleic acid quantification, PCR amplification, and fluorescence detection on the chip, thereby expanding the application of existing microfluidic technology.

[0017] The present invention is achieved through the following technical solutions:

[0018] A flexible thin film microfluidic chip comprises a laminated membrane, at least one fixed bonding area, multiple functional chambers, a fluid channel, at least one valve area, and at least one liquid inlet interface. The fixed bonding area, functional chamber, fluid channel, and valve area are all distributed within the laminated membrane, and the size and shape of the functional chamber and fluid channel are defined by the boundaries of the fixed bonding area. The liquid inlet interface is connected to at least one functional chamber and is sealed after liquid is introduced. The valve area is optionally disposed between the functional chamber and the fluid channel, and an irreversible disposable valve structure is distributed within the valve area.

[0019] The laminated film includes a first film and a second film stacked together, at least one of the first film and the second film being a flexible film; the first film and the second film are irreversibly bonded in a fixed bonding area;

[0020] The first film and the second film of the functional chamber can switch between two states of separation and mutual adhesion under the action of 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 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 through the fluid channel;

[0022] The flexible thin film microfluidic chip comprises at least three sequentially connected functional areas: a nucleic acid extraction area, a nucleic acid quantification area, and a nucleic acid detection area, wherein the nucleic acid quantification area is located between the nucleic acid extraction area and the nucleic acid detection area; each functional area comprises at least one functional chamber;

[0023] The functional chambers in the nucleic acid quantification area include at least a nucleic acid quantification chamber and a nucleic acid quantification auxiliary chamber. The two ends of the nucleic acid quantification chamber are respectively connected to the functional chambers of the nucleic acid extraction area and the nucleic acid detection area. During the flow of the nucleic acid extract from the nucleic acid extraction area to the nucleic acid detection area, it flows into the nucleic acid quantification chamber and the nucleic acid quantification auxiliary chamber in sequence. After the nucleic acid quantification chamber is filled, the extract exceeding the capacity of the nucleic acid quantification chamber flows into the nucleic acid quantification auxiliary chamber.

[0024] The principle of nucleic acid quantification in the present invention is as follows: the pressure difference between the inlet and outlet of the nucleic acid quantification cell is set to ΔP, which determines the volume separated by the first and second membranes of the nucleic acid quantification cell. When ΔP is a fixed value and greater than the longitudinal elastic deformation force of the flexible membrane, the first and second flexible membranes are separated, and the separated volume is a fixed volume. This volume is the quantitative volume under these conditions.

[0025] The magnitude of ΔP is determined by the pressure difference between the inlet and outlet. The pressure difference at the outlet is determined by the outlet channel structure and is a fixed value. The pressure at the inlet can be adjusted by adjusting the force used to squeeze the second eluent pool. When a greater force is used to squeeze the second eluent pool, the pressure at the inlet of the nucleic acid quantification pool increases, resulting in a larger ΔP value and a larger quantitative volume. Conversely, when a smaller force is used to squeeze the second eluent pool, the pressure at the inlet of the nucleic acid quantification pool decreases, resulting in a smaller ΔP value and a smaller quantitative volume.

[0026] In this way, the application requirements of different quantitative volumes can be achieved without changing the structure of the quantitative pool.

[0027] Furthermore, the nucleic acid extraction area includes at least one silicon membrane chamber; the silicon membrane chamber is used to capture nucleic acids in the liquid;

[0028] 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;

[0029] The functional chamber of the nucleic acid extraction zone is connected to the silicon membrane chamber via a fluid channel, and the fluid channel allows fluid to pass therethrough at a low speed and in a controllable manner.

[0030] Furthermore, the functional chambers in the nucleic acid extraction area include at least one sample chamber, at least one rinse liquid chamber, at least one waste liquid chamber and at least two eluent chambers, the two eluent chambers being a first eluent chamber and a second eluent chamber, respectively, and the nucleic acid quantification chamber being connected to the second eluent chamber via a disposable valve.

[0031] Furthermore, the functional chambers in the nucleic acid extraction area include a sample chamber, two rinsing liquid chambers, a waste liquid chamber and two elution liquid chambers, and the two rinsing liquid chambers are respectively a first rinsing liquid chamber and a second rinsing liquid chamber.

[0032] Furthermore, the fluid channel in the nucleic acid extraction area is divided into multiple sections with the silicon membrane chamber as the dividing point according to the functional chambers connected thereto, and 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.

[0033] Furthermore, the functional chambers in the nucleic acid extraction area also include at least one lysis liquid chamber and at least one binding liquid chamber. The lysis liquid chamber is located between the sample chamber and the binding liquid chamber, and is connected to the sample chamber and the binding liquid chamber respectively via disposable valves, and the liquid inlet interface is connected to the sample chamber.

[0034] 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.

[0035] Furthermore, the functional chambers in the nucleic acid extraction area are 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.

[0036] Furthermore, the functional chamber in the nucleic acid extraction area 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.

[0037] Furthermore, the sample chamber in the nucleic acid extraction area is connected to the silicon membrane chamber through a disposable valve 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.

[0038] Furthermore, the rinse liquid chamber is connected to the first fluid channel via a disposable valve; the first eluent chamber is connected to the second fluid channel via or without a disposable valve, and the second eluent chamber is connected to the first fluid channel via or without a disposable valve; or, the first eluent chamber is connected to the first fluid channel via or without a disposable valve, and the second eluent chamber is connected to the second fluid channel via or without a disposable valve.

[0039] Furthermore, the functional chambers in the nucleic acid detection area include at least one PCR reaction liquid chamber and at least one PCR amplification chamber, wherein the PCR reaction liquid chamber is connected to the PCR amplification chamber and the nucleic acid quantification chamber respectively via or without a disposable valve.

[0040] Furthermore, the PCR reaction liquid chamber and the nucleic acid quantification auxiliary chamber are connected to the nucleic acid quantification chamber by sharing a portion of the fluid channel.

[0041] Furthermore, there are two PCR reaction liquid chambers, namely a first PCR reaction liquid chamber and a second PCR reaction liquid chamber. The first PCR reaction liquid chamber is connected to the second PCR reaction liquid chamber via a disposable valve, and the second PCR reaction liquid chamber is connected to the PCR amplification chamber and the nucleic acid quantification chamber respectively via or without a disposable valve.

[0042] Furthermore, there is more than one PCR amplification chamber, which are connected to the PCR reaction liquid chamber through fluid channels respectively.

[0043] Furthermore, the lysate chamber and the sample chamber are directly connected, and / or the lysate chamber and the binding liquid chamber are directly connected, and / or the second elution liquid chamber and the nucleic acid quantification chamber are directly connected, and / or the nucleic acid quantification and the nucleic acid quantification auxiliary chamber and the second PCR reaction liquid chamber are directly connected, and / or the first PCR reaction liquid chamber and the second PCR reaction liquid chamber are directly connected, and / or the second PCR reaction liquid chamber and the PCR amplification chamber are directly connected.

[0044] 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 and the silicon membrane to have sufficient time for adsorption and elution, thereby minimizing the degree of gradient decline in adsorption and elution, allowing the silicon membrane to fully adsorb the nucleic acid in the liquid and the eluted liquid to fully elute, achieving an effect similar to that of the liquid passing vertically through the silicon membrane.

[0045] The low-speed controllable flow described in the present invention refers to the flow of liquid in the fluid channel at an average flow rate of less than 45u l / s, preferably less than 40u l / s, 30u l / s, 20u l / s, 15u l / s, 10u l / s, or 5u l / s.

[0046] 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:

[0047] a. The maximum angle between the fluid channel film and the microfluidic chip plane after the fluid channel is stretched open by the liquid;

[0048] b. The width of the fluid channel;

[0049] c. Length of the fluid channel.

[0050] 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:

[0051] 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.

[0052] 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.

[0053] Third, the longer the length of the fluid channel, the greater the resistance given by the entire fluid channel.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 provided 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.

[0060] The present invention also provides a fluid sample processing device, comprising:

[0061] Flexible thin film microfluidic chip as described above.

[0062] Furthermore, the fluid sample processing device further includes a plurality of physical valves, which are used to repeatedly and physically control the opening and closing of a fluid channel that does not have a disposable valve, or to repeatedly and physically continue to control the opening and closing of the fluid channel corresponding to the disposable valve after the disposable valve is opened.

[0063] Furthermore, it includes more than one first extrusion mechanism, the position and geometric shape of each first extrusion mechanism correspond to a functional chamber setting, and are used to selectively apply pressure to the corresponding functional chamber to open the disposable valve or drive the fluid into the fluid channel and flow to the desired position.

[0064] Furthermore, the physical valve includes more than one second squeezing mechanism, 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.

[0065] 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.

[0066] The present invention also provides use of the aforementioned fluid sample processing device in fluid sample processing.

[0067] Furthermore, the use is in nucleic acid extraction or nucleic acid PCR amplification detection.

[0068] The present invention also provides a nucleic acid PCR amplification detection method, which is implemented based on the fluid sample processing device as described above and includes the following steps:

[0069] S1. Nucleic acid extraction: After the nucleic acid sample passes through the nucleic acid extraction area, a nucleic acid eluate that meets the detection standards is obtained and stored in a functional chamber connected to the nucleic acid quantification area;

[0070] 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.

[0071] S2. Quantification of nucleic acid eluate:

[0072] Adjust the physical valve to open only the liquid passages between the nucleic acid quantification chamber and the chamber storing the nucleic acid eluent and the nucleic acid quantification auxiliary chamber, and squeeze the chamber storing the nucleic acid eluent with a set squeezing force, so that the nucleic acid eluent flows through the nucleic acid quantification chamber through the fluid channel and into the nucleic acid quantification auxiliary chamber. At this time, the nucleic acid quantification chamber is full and contains the required amount of nucleic acid eluent;

[0073] Adjust the physical valve to open only the chamber connecting the nucleic acid detection area and the nucleic acid quantification chamber and the liquid passage of the nucleic acid quantification chamber, and squeeze the nucleic acid quantification chamber filled with nucleic acid eluent with a set squeezing force, so that the nucleic acid eluent flows through the fluid passage into the chamber connecting the nucleic acid detection area and the nucleic acid quantification chamber;

[0074] S3. PCR amplification detection: In the nucleic acid detection area, the PCR reaction solution is amplified and detected to obtain the test results.

[0075] Furthermore, the step S1 includes the following steps:

[0076] S1a, reagent filling and sample addition steps:

[0077] Injecting a lysate into the lysate chamber of the microfluidic chip, injecting a nucleic acid binding solution into the binding solution chamber, injecting a protease into the sample chamber, 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 an unlysed nucleic acid sample selected from a tissue grinding solution, a sample preservation solution, plasma, or serum into the sample chamber;

[0078] S1b, sample lysis step:

[0079] Adjusting the physical valve to open only the passage between the lysate chamber and the sample chamber, squeezing the lysate chamber with a set squeezing force, opening the disposable valve to achieve liquid mixing between the lysate chamber and the sample chamber, and sample lysis, thereby obtaining a sample reaction solution;

[0080] After the sample lysis is completed, the passage between the binding liquid chamber and the lysis liquid chamber is opened and the nucleic acid binding liquid chamber is squeezed with a set squeezing force. The disposable valve is opened and the nucleic acid binding liquid is squeezed into the lysed sample reaction liquid to achieve mixing between the sample reaction liquid and the nucleic acid binding liquid.

[0081] S1 c, nucleic acid capture step: adjusting the physical valve to open only the passage from the sample chamber through the silicon membrane chamber to the waste liquid chamber, squeezing the sample chamber with a set squeezing force, so that the liquid sample opens the disposable valve and flows through the silicon membrane chamber through the fluid channel and then into the waste liquid chamber, completing nucleic acid capture;

[0082] S1 d, 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;

[0083] S1 e, nucleic acid elution step:

[0084] Adjust the physical valve to open only the passages of the first and second eluent chambers through the silicon membrane chamber, squeeze the eluent chamber filled with eluent with the set extrusion pressure, and allow the eluent to flow through the silicon membrane chamber through the fluid channel and 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 eluent chamber connected to the nucleic acid quantification area, completing the nucleic acid extraction.

[0085] Furthermore, the step S1 further includes the following steps:

[0086] S1 f, reagent filling and sample loading step: injecting PCR reaction solution into the first PCR reaction solution chamber;

[0087] The S3 step includes the following steps:

[0088] S3a, PCR amplification solution mixing: After a certain amount of nucleic acid eluate flows into the second PCR reaction solution chamber, the physical valve is adjusted to open only the passages between the first PCR reaction solution chamber and the second PCR reaction solution chamber, and the first PCR reaction solution chamber and the second PCR reaction solution chamber are alternately squeezed to achieve mixing of the nucleic acid eluate and the PCR reaction solution. The PCR mixture containing the nucleic acid eluate is stored in the second PCR reaction solution chamber;

[0089] S3b. PCR amplification detection: Adjust the physical valve to open only the passage between the second PCR reaction liquid chamber and the PCR reaction chamber, and squeeze the second PCR reaction liquid chamber with a set extrusion force to transfer the PCR mixed solution stored in the chamber to the PCR reaction chamber; load the PCR reaction chamber with a preset PCR heating and cooling program, and collect fluorescence at the set temperature point to complete nucleic acid detection.

[0090] Compared with the prior art, the advantages of the present invention are:

[0091] 1. In the present invention, the nucleic acid extraction and capture zone includes an eluent with an elution function. The nucleic acid detection zone is the detection zone for the PCR reaction. During the flow of the liquid from the nucleic acid extraction and capture zone to the nucleic acid detection zone, it is temporarily stored in the nucleic acid quantification chamber and filled with the nucleic acid quantification chamber to obtain a quantitative amount of liquid for mixing with the PCR reaction reagents. The present invention utilizes a pressure differential and a flexible membrane in the quantification chamber to achieve quantitative eluent quantification. The quantitative volume can be varied within a certain range according to needs.

[0092] 2. 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.

[0093] 3. The flexible thin-film microfluidic chip of the present invention is provided with a fluid channel. This fluid channel not only connects the functional chambers with the silicon membrane chamber to form a liquid passage, but also provides space for the silicon membrane chamber. The silicon membrane chamber is equivalent to being arranged on a fluid channel, and each of the other functional chambers is connected to the fluid channel containing the silicon membrane chamber via a fluid channel, or is directly connected to the silicon membrane chamber. The silicon membrane chamber requires minimal expansion deformation, and its projected area is much smaller than that of the functional chambers. In the silicon membrane method, a wide variety of liquids flow through the silicon membrane chamber in batches, so the silicon membrane chamber effectively becomes the core chamber. In the prior art, the functional chambers are connected directly or via very short connecting channels. These existing connection methods cannot reliably connect the functional chambers to the smaller silicon membrane chamber, nor can they arbitrarily switch the liquid passages through the silicon membrane chamber using physical valves or other means. Furthermore, they cannot prevent cross-contamination between the liquids after switching the liquid passages due to residual liquids, rapid vibration, or movement of the pipetting gun.

[0094] 4. A flexible, 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, 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

[0095] Figure 1 This is a schematic structural diagram of the flexible thin film microfluidic chip of Example 1;

[0096] Figure 2 This is a schematic diagram of the layout of the liquid inlet and valve area in the flexible thin film microfluidic core of Example 1;

[0097] Figure 3 for Figure 2 Schematic diagram of the structure of area A;

[0098] Figure 4 This is a partial diagram of another solution of the flexible thin film microfluidic chip in Example 1.

[0099] Markings in the figure: laminated membrane 1000, liquid inlet interface 2000, first film 1001, second film 1002, fluid channel 1110, silicon membrane chamber 1201, first fluid channel 1111, second fluid channel 1112, sample chamber 1301, lysate chamber 1302, binding liquid chamber 1303, PCR amplification chamber 1304, pressure relief chamber 1305, waste liquid chamber 1306, first rinsing liquid chamber 1307, second rinsing liquid chamber 1308, first elution liquid chamber 1309, second rinsing liquid chamber 1301 Deliquidation chamber 1310, first PCR reaction liquid chamber 1311, second PCR reaction liquid chamber 1312, nucleic acid quantification chamber 1313, nucleic acid quantification auxiliary chamber 1314, sample liquid inlet 1401, lysis liquid inlet 1402, binding liquid inlet 1403, first rinse liquid inlet 1407, second rinse liquid inlet 1408, first elution liquid inlet 1409, first PCR reaction liquid inlet 1411, second PCR reaction liquid inlet 1412, positioning hole 1003. DETAILED DESCRIPTION

[0100] The following is a further non-restrictive detailed description of the technical solution of the invention in conjunction with the preferred embodiments and the accompanying drawings. In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and cannot be understood as limiting the present invention.

[0101] Explanation of terms:

[0102] Fixed bonding area: In the present invention, the fixed bonding area divides the entire laminated membrane into a plurality of functional chambers and fluid channels along the membrane plane.

[0103] Functional chambers: Each functional chamber can be defined as a sample chamber, lysis solution chamber, nucleic acid capture chamber, rinse solution chamber, eluent chamber, PCR amplification chamber, PCR reaction solution chamber, etc. according to the function of the reagent it contains.

[0104] Fluid channel: The fluid channel of the present invention adopts a fluid channel that can switch between two states of mutual separation and mutual adhesion, and cooperates with the pressure applied to the liquid storage functional chamber to achieve liquid flow at a slow, uniform speed or in a wave band manner.

[0105] Valve Area: A valve area is provided between the functional chamber and the fluid channel as needed. This requirement includes the need for the functional chamber to be pre-filled with a liquid or solid, such as a lyophilized powder. The valve area contains an irreversible, disposable valve structure. 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. The disposable valve structure can utilize various weak link structures known in the art to isolate the functional chamber from the fluid channel and can be broken by heat, electromagnetic radiation, or force, thereby changing from a closed state to an open state. Preferably, a first film and a second film are detachably coupled within the valve area to form the disposable valve structure. This structure can be broken by a fluid impact 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 fluid, the fluid can be forced into the irreversible, disposable valve structure between the functional chamber and the fluid channel, causing the impact to break, 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 opened simply and quickly by selective squeezing, and are easier to implement in a two-dimensional plane space than traditional valves.

[0106] Laminated film: The laminated film is mainly composed of a first film and a second film; at least one of the first film and the second film is preferably a flexible film with longitudinal stretchability; one or more fixed bonding areas and multiple functional chambers can be defined in the laminated film along the film plane direction.

[0107] In the present invention, at least one positioning hole may be formed on the laminated membrane. By providing the positioning hole, the staff can more quickly and accurately place the flexible thin film microfluidic chip into the corresponding detection equipment, and more accurately perform operations such as selective extrusion of different functional chambers.

[0108] 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.

[0109] Pressure: In the present invention, the pressure effect can 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 can also include stress generated by deformation of the flexible film in the first or second film.

[0110] An example of controlling the opening and closing of a fluid channel and driving the fluid from one functional chamber to another functional chamber can be referred to Figure 3 : First, the fluid channel between the eluent chamber and the nucleic acid quantification chamber is opened, the fluid channel between the nucleic acid quantification chamber and the nucleic acid quantification auxiliary chamber is opened, and the fluid channel between the nucleic acid quantification chamber and the PCR reaction liquid chamber is cut off by a physical valve; the first squeezing mechanism is driven to squeeze the eluent chamber, so that the liquid in the eluent chamber enters the nucleic acid quantification chamber and fills the nucleic acid quantification chamber, and the excess liquid flows into the nucleic acid quantification auxiliary chamber; then, the fluid channel between the eluent chamber and the nucleic acid quantification chamber is cut off by a physical valve, the fluid channel between the nucleic acid quantification chamber and the nucleic acid quantification auxiliary chamber is cut off by the physical valve, the fluid channel between the nucleic acid quantification chamber and the PCR reaction liquid chamber is opened, and the first squeezing mechanism is driven to squeeze the nucleic acid quantification chamber, so that all the liquid in the nucleic acid quantification chamber flows into the PCR reaction liquid chamber.

[0111] Example 1

[0112] 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.

[0113] The laminated film 1000 is mainly 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 with longitudinal stretchability. In this embodiment, the first film 1001 and the second film 1002 are both composite flexible films.

[0114] In this embodiment, one or more fixed bonding areas and multiple functional chambers can be defined along the film plane in the stacked film 1000. Specifically, the fixed bonding areas divide the entire stacked film 1000 along the film plane into a plurality of functional chambers, fluid channels 1110, and silicon membrane chambers 1201. That is, the sizes and shapes of the functional chambers, fluid channels 1110, and silicon membrane chambers 1201 are defined by the boundaries of the fixed bonding areas.

[0115] In this embodiment, the functional chambers include: one sample chamber 1301, one lysis liquid chamber 1302, and one binding liquid chamber 1303; one PCR amplification chamber 1304, one pressure relief chamber 1305, and one waste liquid chamber 1306; two rinsing liquid chambers, namely, a first rinsing liquid chamber 1307 and a second rinsing liquid chamber 1308; two elution liquid chambers, namely, a first elution liquid chamber 1309 and a second elution liquid chamber 1310; two PCR reaction liquid chambers, namely, a first PCR reaction liquid chamber 1311 and a second PCR reaction liquid chamber 1312; and one nucleic acid quantification chamber 1313 and one nucleic acid quantification auxiliary chamber 1314.

[0116] In this embodiment, the fluid channel 1110 can be divided into multiple sections according to the functional chambers connected to it, and the multiple sections include at least a first fluid channel 1111 and a second fluid channel 1112, wherein the first fluid channel 1111 is connected to the silicon membrane chamber 1201, and the second fluid channel 1112 is connected to the waste liquid chamber 1314.

[0117] Among them, the sample chamber 1301, the lysate chamber 1302, and the binding liquid chamber 1303 are connected in sequence. The sample chamber 1301 is connected to the silicon membrane chamber through the first fluid channel 1111, the first rinse liquid chamber 1307, the second rinse liquid chamber 1308, and the first eluent chamber 1309 are connected to the silicon membrane chamber through their respective first fluid channels 1111, and the waste liquid chamber 1306 is connected to the silicon membrane chamber through the second fluid channel 1112; the remaining functional chambers are connected to the first or second fluid channel 1112 through their respective fluid channels; one end of the nucleic acid quantification chamber is connected to the second eluent chamber 1310, and the other end is connected to the second PCR reaction liquid chamber 1312 is connected; the second PCR reaction liquid chamber 1312 is located between the first PCR reaction liquid chamber 1311 and the PCR amplification chamber 1304, the PCR amplification chamber 1304 is located between the second PCR reaction liquid chamber 1312 and the pressure relief chamber 1305, the nucleic acid quantification chamber 1313 is located between the second elution liquid chamber 1310 and the second PCR reaction liquid chamber 1312, the nucleic acid quantification auxiliary chamber 1314 is only connected to the nucleic acid quantification chamber 1313, and the nucleic acid quantification auxiliary chamber 1314 and the second PCR reaction liquid chamber 1312 are connected to the nucleic acid quantification chamber 1313 by sharing a part of the fluid channel.

[0118] In another embodiment, see Figure 4 There are three PCR amplification chambers 1304 arranged in parallel, each of which is independently connected to the second PCR reaction liquid chamber 1312 through a fluid channel.

[0119] The above-mentioned liquid storage chambers respectively have corresponding liquid inlets, such as the sample liquid inlet 1401, the lysis liquid inlet 1402, the binding liquid inlet 1403, the first rinse liquid inlet 1407, the second rinse liquid inlet 1408, the first eluent liquid inlet 1409, the first PCR reaction liquid inlet 1411, and the second PCR reaction liquid inlet 1412; the above-mentioned liquid inlets can be closed after the liquid is filled, wherein the sample liquid inlet 1401 can be connected to the liquid inlet interface 2000.

[0120] Before liquids and solids 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 liquids and solids are injected into the corresponding functional chambers through the sample injection holes, the longitudinal stretchability of the flexible films generates internal pressure, 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.

[0121] In this embodiment, one or more valve regions are defined within the laminated film 1000 along the film plane. Distributed within each valve region are irreversible, disposable valve structures connected between a functional chamber and a fluid channel 1110, or between 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, a first film and a second film are releasably bonded within the valve region 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 laser welding, 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.

[0122] In this embodiment, there can be multiple valve areas, among which multiple irreversible disposable valve structures can be distributed at the liquid outlets of the sample chamber 1301, the lysis liquid chamber 1302, the binding liquid chamber 1303, the first rinsing liquid chamber 1307, the second rinsing liquid chamber 1308, the first elution liquid chamber 1309, and the first PCR reaction liquid chamber 1311 that need or may be pre-filled with liquid.

[0123] In this embodiment, three positioning holes 1003 may be formed on the stacked film 1000 .

[0124] Example 2

[0125] This embodiment provides a fluid sample processing device, which includes the flexible thin film microfluidic chip in embodiment 1.

[0126] 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 a corresponding valve area. The valve within each valve area (i.e., the aforementioned irreversible, disposable valve structure) tears open when subjected to fluid pressure exceeding its rupture threshold, allowing the liquid storage chamber and fluid channel connected by the valve to communicate with each other, allowing liquid to enter the fluid channel from the liquid storage chamber.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] The specific mechanical structure of the above-mentioned fluid sample processing device can be implemented using existing technologies, such as those described in CN2022110178734.

[0131] Example 3

[0132] A method for extracting nucleic acid using the microfluidic chip and fluid sample processing device of Examples 1-2 may include the following steps:

[0133] (1) Sample and liquid injection step: injecting a saliva sample into the sample chamber of the flexible thin film microfluidic chip, injecting a lysate into the lysate chamber, and injecting a binding solution into the binding solution chamber;

[0134] Injecting rinsing liquid into the first rinsing liquid chamber and the second rinsing liquid chamber respectively, and injecting eluent into the first eluent chamber;

[0135] Inject the reaction solution containing the primers, probes, enzymes, PCR buffer and other reagents required for PCR amplification into the first PCR reaction solution chamber; and seal the above-mentioned inlet.

[0136] Preferably, 200 uL of saliva sample, 300 uL of rinsing liquid in the first rinsing liquid chamber, 100 uL of rinsing liquid in the second rinsing liquid chamber, 40 uL of eluent in the first eluent chamber, and no restrictions on the reagents in the other chambers.

[0137] (2) Lysis step: Close the relevant physical valves, only open the passage between the sample chamber and the lysis solution chamber, set the extrusion force to squeeze the sample chamber and the lysis solution chamber in turn, achieve mixing of the sample and the lysis solution, and complete the sample lysis process; preferably, the reaction time between the sample and the lysis solution is 10 minutes.

[0138] (3) Binding step: extruding the liquid in the binding liquid chamber into the lysis liquid chamber;

[0139] Then, the relevant physical valves are closed, and only the passage between the binding liquid chamber and the lysis liquid chamber is opened. The extrusion force is set to sequentially press the binding liquid chamber and the lysis liquid chamber to achieve mixing of the binding liquid, sample and lysis liquid, thus completing the binding process.

[0140] (4) Nucleic acid capture step: close the relevant physical valves, leaving only the passage from the lysis chamber to the sample chamber open, and set the squeezing force to squeeze the lysis chamber so that all the liquid enters the sample chamber;

[0141] Then, the relevant physical valves were closed, leaving only the passage from the sample chamber through the silicon membrane chamber to the waste chamber open. The extrusion force was set to squeeze the sample chamber, allowing the liquid sample to flow through the fluid channel, through the silicon membrane chamber, and into the waste chamber at a flow rate of 20 μl / s, completing nucleic acid capture.

[0142] Preferably, the sample chamber is squeezed by a SmoothPump to control the flow rate of the liquid so that the liquid flows slowly through the silicon membrane chamber.

[0143] (5) Nucleic acid rinsing step: Close the relevant physical valves, leaving only the passage from the first rinsing liquid chamber through the silicon membrane chamber to the waste liquid chamber open. Set the extrusion force to squeeze the first rinsing liquid chamber, so that the rinsing liquid flows through the silicon membrane chamber through the fluid channel and then discharges into the waste liquid chamber, completing the first nucleic acid rinsing;

[0144] Close the relevant physical valves and only open the passage from the second rinsing liquid chamber through the silicon membrane chamber to the waste liquid chamber. Set the extrusion force to squeeze the second rinsing liquid chamber so that the rinsing liquid flows through the silicon membrane chamber through the fluid channel and then discharges into the waste liquid chamber to complete the second nucleic acid rinsing.

[0145] (6) Nucleic acid elution step: Close the relevant physical valves, leaving only the passages between the first eluent chamber, the silicon membrane chamber, and the second eluent chamber open, set the squeezing force, and squeeze the first eluent chamber and the second eluent chamber multiple times in sequence;

[0146] Finally, the first eluent chamber is squeezed so that all the eluent is stored in the second eluent chamber, completing the elution process; preferably, the first eluent chamber and the second eluent chamber are squeezed three times in sequence.

[0147] (7) Nucleic acid quantification step: Close the relevant physical valves, leaving only the passages between the second eluent chamber, the nucleic acid quantification chamber, and the nucleic acid quantification auxiliary chamber open. Set the squeezing force to squeeze the second eluent chamber, causing the liquid to flow out of the second eluent chamber until the nucleic acid quantification chamber is filled, and the excess liquid flows into the nucleic acid quantification auxiliary chamber.

[0148] Close the relevant physical valves, only open the passage between the nucleic acid quantification chamber and the second PCR reaction liquid chamber, set the extrusion force to squeeze the nucleic acid quantification chamber, and all the liquid in the nucleic acid quantification chamber flows into the second PCR reaction liquid chamber.

[0149] (8) PCR mixing step: close the relevant physical valves, only open the passage between the first PCR reaction liquid chamber and the second PCR reaction liquid chamber, set the extrusion force, squeeze the first PCR reaction liquid chamber and the second PCR reaction liquid chamber multiple times in sequence, and finally squeeze the first PCR reaction liquid chamber to allow all the liquid to flow into the second PCR reaction liquid chamber; preferably, squeeze the first PCR reaction liquid chamber and the second PCR reaction liquid chamber three times in sequence.

[0150] (9) Transfer of PCR reaction liquid: Close the relevant physical valves, leaving only the passage from the second PCR reaction liquid chamber through the PCR amplification chamber to the pressure relief chamber open. Set the extrusion force and slowly squeeze the second PCR reaction liquid chamber so that the liquid slowly fills the PCR amplification chamber, and the air originally in the PCR amplification chamber is squeezed into the pressure relief chamber.

[0151] Then, the relevant physical valves are closed to seal the liquid in the PCR amplification chamber.

[0152] (10) PCR amplification and fluorescence acquisition steps: Load the pre-set PCR amplification program on the computer and control the heating module to raise and lower the temperature of the PCR amplification chamber. When fluorescence acquisition is required, use a dedicated device to scan the PCR amplification chamber to complete the fluorescence acquisition. Calculate the Ct value using post-processing software and output the results.

[0153] In other optional embodiments, when the sample is a nucleic acid sample that has been cleaved and bound, the cleavage and binding steps are omitted based on this embodiment.

[0154] In other optional embodiments, based on this embodiment, the nucleic acid rinsing step is reduced to once.

[0155] In other optional embodiments, on the basis of the present embodiment, the sample addition and liquid injection steps are modified, freeze-dried powders of components such as primers, probes, and enzymes are added to the second PCR reaction liquid chamber, and a dissolving reagent is added to the first PCR reaction liquid chamber; and the PCR mixing step is modified, the relevant physical valves are closed, and the quantified liquid mixed with nucleic acids is first stored in the second PCR reaction liquid chamber, and then only the passage between the first PCR reaction liquid chamber and the second PCR reaction liquid chamber is opened, the extrusion pressure is set, and the first PCR reaction liquid chamber and the second PCR reaction liquid chamber are squeezed in turn multiple times to mix the dissolving reagent, freeze-dried powder, and the quantified nucleic acids to complete the mixing process.

[0156] In other optional embodiments, based on the present embodiment, the storage positions of the primers, probes, and enzymes are modified, and the positions of the primers, probes, and enzymes are set in the PCR amplification chamber. The quantified nucleic acid flows directly into the second PCR reaction liquid chamber, and then flows into the PCR amplification chamber, and is directly mixed with the reagents in the PCR amplification chamber to complete the mixing process. This step can be regarded as a variation of the combination of step S8 and step S9.

[0157] Example 4: Fluid channel flow rate and silicon membrane adsorption experiment

[0158] The experimental sample is 50ng / ul human genomic DNA, the sample volume is 200ul, and the total amount of DNA added is 10,000ng.

[0159] Repeat the nucleic acid capture steps: Close the physical valve, leaving only the passage from the sample chamber through the silicon membrane chamber to the waste chamber open. Apply a set pressure to squeeze the sample chamber, allowing 200 μl of liquid sample to flow through the first fluid channel, through the silicon membrane chamber, and then through the second fluid channel into the waste chamber, completing nucleic acid capture. When the sample chamber is completely drained, record the drain time. Following sequential rinsing and elution, measure the DNA content in the eluate (using a Qubit or Nanodrop device) and convert this into a DNA recovery rate.

[0160] In one set of experimental schemes, the total length of the fluid channel between the sample chamber and the waste liquid chamber (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.

[0161] The experimental results are shown in Table 1

[0162] Table 1

[0163]

[0164] 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.

[0165] 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 the art 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 elution process.

[0166] Example 5: Relationship between fluid channel length and flow rate

[0167] In order to study the relationship between the length of the fluid channel and the flow rate, the following experiment was designed:

[0168] The silicon membrane chamber was removed, the physical valve was closed, and only the passage from the sample chamber to the waste chamber was open. The sample chamber was squeezed with a set pressure, forcing 200 μl of liquid sample into the waste chamber through the fluid channel. When the sample chamber was completely drained, the drain time was recorded. The fluid channel between the sample chamber and the waste chamber 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.

[0169] Table 2

[0170]

[0171] Through experiments, we also found that in the range of more than 2mm in length, the sensitivity of changes in channel length to flow rate is significantly less than that of changes in width.

[0172] The above embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A flexible thin film microfluidic chip comprising a laminated membrane, at least one fixed bonding region, multiple functional chambers, a fluid channel, at least one valve region, and at least one liquid inlet port. The fixed bonding region, functional chamber, fluid channel, and valve region are all distributed within the laminated membrane, and the size and shape of the functional chamber and fluid channel are defined by the boundaries of the fixed bonding region. The liquid inlet port is connected to at least one functional chamber and is sealed after liquid is introduced. The valve region is optionally disposed between the functional chamber and the fluid channel, and an irreversible, disposable valve structure is distributed within the valve region. The laminated film includes a first film and a second film stacked together, at least one of the first film and the second film being a flexible film; the first film and the second film are irreversibly bonded in a fixed bonding area; The first film and the second film of the functional chamber can switch between two states of separation and mutual adhesion under the action of 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 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 through the fluid channel; The flexible thin film microfluidic chip comprises at least three sequentially connected functional areas: a nucleic acid extraction area, a nucleic acid quantification area, and a nucleic acid detection area, wherein the nucleic acid quantification area is located between the nucleic acid extraction area and the nucleic acid detection area; each functional area comprises at least one functional chamber; The functional chambers in the nucleic acid quantification area include at least a nucleic acid quantification chamber and a nucleic acid quantification auxiliary chamber. The two ends of the nucleic acid quantification chamber are respectively connected to the functional chambers of the nucleic acid extraction area and the nucleic acid detection area. During the flow of the nucleic acid extract from the nucleic acid extraction area to the nucleic acid detection area, it flows into the nucleic acid quantification chamber and the nucleic acid quantification auxiliary chamber in sequence. After the nucleic acid quantification chamber is filled, the extract exceeding the capacity of the nucleic acid quantification chamber flows into the nucleic acid quantification auxiliary chamber.

2. The flexible thin film microfluidic chip according to claim 1, characterized in that: The accommodation range of the nucleic acid quantification chamber is adjustable.

3. The flexible thin film microfluidic chip according to claim 1 or 2, characterized in that: The nucleic acid extraction area includes at least one silicon membrane chamber; 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 functional chamber of the nucleic acid extraction zone is connected to the silicon membrane chamber via a fluid channel, and the fluid channel allows fluid to pass therethrough at a low speed and in a controllable manner.

4. The flexible thin film microfluidic chip according to claim 3, characterized in that: The functional chambers in the nucleic acid extraction area include 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, and the nucleic acid quantification chamber is connected to the second eluent chamber via a disposable valve or directly connected to the second eluent chamber.

5. The flexible thin film microfluidic chip according to claim 4, characterized in that: The functional chambers in the nucleic acid extraction area include 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.

6. The flexible thin film microfluidic chip according to claim 4 or 5, characterized in that: The fluid channel in the nucleic acid extraction area is divided into multiple sections based on the functional chambers connected to the silicon membrane chamber. 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.

7. The flexible thin film microfluidic chip according to any one of claims 4 to 6, characterized in that: The functional chambers in the nucleic acid extraction area also include at least one lysis liquid chamber and at least one binding liquid chamber. The lysis liquid chamber is located between the sample chamber and the binding liquid chamber, and is connected to the sample chamber and the binding liquid chamber respectively via disposable valves, and the liquid inlet interface is connected to the sample chamber.

8. The flexible thin film microfluidic chip according to claim 3, characterized in that: The functional chambers in the nucleic acid extraction area are connected to the silicon membrane chamber by sharing a portion of the fluid channels, and the number of fluid channels directly connected to the silicon membrane chamber is no more than 5, 4, 3 or 2.

9. The flexible thin film microfluidic chip according to claim 6, characterized in that: The functional chamber in the nucleic acid extraction area 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.

10. The flexible thin film microfluidic chip according to claim 6, characterized in that: The sample chamber in the nucleic acid extraction area is connected to the silicon membrane chamber through a disposable valve 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.

11. The flexible thin film microfluidic chip according to claim 6, characterized in that: The rinse liquid chamber is connected to the first fluid channel via a disposable valve; the first eluent chamber is connected to the second fluid channel via or without a disposable valve, and the second eluent chamber is connected to the first fluid channel via or without a disposable valve, or the first eluent chamber is connected to the first fluid channel via or without a disposable valve, and the second eluent chamber is connected to the second fluid channel via or without a disposable valve.

12. The flexible thin film microfluidic chip according to any one of claims 1-2, 4-5, 8-10, characterized in that: The functional chambers in the nucleic acid detection area include at least one PCR reaction liquid chamber and at least one PCR amplification chamber, wherein the PCR reaction liquid chamber is connected to the PCR amplification chamber and the nucleic acid quantification chamber respectively via or without a disposable valve.

13. The flexible thin film microfluidic chip according to claim 12, characterized in that: The PCR reaction liquid chamber and the nucleic acid quantification auxiliary chamber are connected with the nucleic acid quantification chamber by sharing a part of the fluid channel.

14. The flexible thin film microfluidic chip according to claim 12, characterized in that: There are two PCR reaction liquid chambers, namely the first PCR reaction liquid chamber and the second PCR reaction liquid chamber. The first PCR reaction liquid chamber is connected to the second PCR reaction liquid chamber via a disposable valve, and the second PCR reaction liquid chamber is connected to the PCR amplification chamber and the nucleic acid quantification chamber respectively via or without a disposable valve.

15. The flexible thin film microfluidic chip according to claim 12, characterized in that: There are more than one PCR amplification chambers, which are connected to the PCR reaction liquid chamber through fluid channels respectively.

16. The flexible thin film microfluidic chip according to claim 7, characterized in that: The lysis liquid chamber and the sample chamber are directly connected, and / or the lysis liquid chamber and the binding liquid chamber are directly connected, and / or the second elution liquid chamber and the nucleic acid quantification chamber are directly connected, and / or the nucleic acid quantification and the nucleic acid quantification auxiliary chamber and the second PCR reaction liquid chamber are directly connected, and / or the first PCR reaction liquid chamber and the second PCR reaction liquid chamber are directly connected, and / or the second PCR reaction liquid chamber and the PCR amplification chamber are directly connected.

17. The flexible thin film microfluidic chip according to claim 14, characterized in that: The nucleic acid quantification and nucleic acid quantification auxiliary chambers are directly connected to the second PCR reaction liquid chamber, and / or the first PCR reaction liquid chamber is directly connected to the second PCR reaction liquid chamber, and / or the second PCR reaction liquid chamber is directly connected to the PCR amplification chamber.

18. A fluid sample processing device, comprising: The flexible thin film microfluidic chip according to any one of claims 1 to 17.

19. The fluid sample processing device according to claim 18, characterized in that: Also included are several physical valves, which are used to repeatedly and physically control the opening and closing of a fluid channel that does not have a disposable valve, or to repeatedly and physically continue to control the opening and closing of the fluid channel corresponding to the disposable valve after the disposable valve is opened; It also includes more than one first squeezing mechanism, each first squeezing mechanism is set corresponding to a functional chamber and is used to selectively apply pressure to the corresponding functional chamber to open the disposable valve or drive the fluid into the fluid channel and flow to the desired position.

20. Use of the fluid sample processing device according to claim 18 or 19 in fluid sample processing.

21. The use according to claim 20, characterized in that The use is in nucleic acid extraction or nucleic acid PCR amplification detection.

22. A nucleic acid PCR amplification detection method, said method being implemented based on the fluid sample processing device according to claim 17 or 18, characterized in that: S1. Nucleic acid extraction: After the nucleic acid sample passes through the nucleic acid extraction area, a nucleic acid eluate that meets the detection standards is obtained and stored in a functional chamber connected to the nucleic acid quantification area; S2. Quantification of nucleic acid eluate: Adjust the physical valve to open only the liquid passages between the nucleic acid quantification chamber and the chamber storing the nucleic acid eluent and the nucleic acid quantification auxiliary chamber, and squeeze the chamber storing the nucleic acid eluent with a set squeezing force, so that the nucleic acid eluent flows through the nucleic acid quantification chamber through the fluid channel and into the nucleic acid quantification auxiliary chamber. At this time, the nucleic acid quantification chamber is full and contains the required amount of nucleic acid eluent; Adjust the physical valve to open only the chamber connecting the nucleic acid detection area and the nucleic acid quantification chamber and the liquid passage of the nucleic acid quantification chamber, and squeeze the nucleic acid quantification chamber filled with nucleic acid eluent with a set squeezing force, so that the nucleic acid eluent flows through the fluid passage into the chamber connecting the nucleic acid detection area and the nucleic acid quantification chamber; S3. PCR amplification detection: In the nucleic acid detection area, a quantitative nucleic acid eluate is amplified and detected to obtain the test results.

23. The method of claim 22, wherein: The S1 step includes the following steps: S1a, reagent filling and sample addition steps: Injecting a lysate into the lysate chamber of the microfluidic chip, injecting a nucleic acid binding solution into the binding solution chamber, injecting a protease into the sample chamber, 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 an unlysed nucleic acid sample into the sample chamber, wherein the nucleic acid sample is selected from a tissue grinding solution, a sample preservation solution, plasma, or serum; S1b, sample lysis step: Adjusting the physical valve to open only the passage between the lysate chamber and the sample chamber, squeezing the lysate chamber with a set squeezing force, opening the disposable valve to achieve liquid mixing between the lysate chamber and the sample chamber, and sample lysis, thereby obtaining a sample reaction solution; After the sample lysis is completed, the passage between the binding liquid chamber and the lysis liquid chamber is opened, and the nucleic acid binding liquid chamber is squeezed with a set squeezing force. The disposable valve is opened to squeeze the nucleic acid binding liquid into the lysed sample reaction solution to achieve mixing between the sample reaction solution and the nucleic acid binding liquid; S1c, nucleic acid capture step: adjusting the physical valve to open only the passage from the sample chamber through the silicon membrane chamber to the waste chamber, squeezing the sample chamber with a set squeezing force, so that the liquid sample opens the disposable valve and flows through the silicon membrane chamber through the fluid channel and then into the waste chamber, completing nucleic acid capture; S1d, 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; S1e, nucleic acid elution step: Adjust the physical valve to open only the passages of the first and second eluent chambers through the silicon membrane chamber, squeeze the eluent chamber filled with eluent with the set extrusion pressure, and allow the eluent to flow through the silicon membrane chamber through the fluid channel and 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 eluent chamber connected to the nucleic acid quantification area, completing the nucleic acid extraction.

24. The method according to claim 22 or 23, wherein: The S1 step further includes the following steps: S1f, reagent filling and sample addition step: injecting PCR reaction solution into the first PCR reaction solution chamber; The S3 step includes the following steps: S3a, PCR amplification solution mixing: After the quantitative PCR eluate flows into the second PCR reaction solution chamber, the physical valve is adjusted to open only the passages between the first PCR reaction solution chamber and the second PCR reaction solution chamber, and the first PCR reaction solution chamber and the second PCR reaction solution chamber are alternately squeezed to achieve mixing of the nucleic acid eluate and the PCR reaction solution. The PCR mixture containing the nucleic acid eluate is stored in the second PCR reaction solution chamber; S3b, PCR amplification detection: Adjust the physical valve to open only the passage between the second PCR reaction liquid chamber and the PCR reaction chamber, and squeeze the second PCR reaction liquid chamber with a set squeezing force to transfer the PCR mixture stored in the chamber into the PCR reaction chamber; The PCR reaction chamber is loaded with a preset PCR temperature ramp program, and fluorescence is collected at the set temperature point to complete nucleic acid detection.

Images