CN114950590B

CN114950590B - Microfluidic pouch, fluid sample processing device and nucleic acid extraction method

Info

Publication number: CN114950590B
Application number: CN202210924960.1A
Authority: CN
Inventors: 周朋; 李晓宁; 马俊杰; 陈国礼; 黄真光; 海兴伟; 郭琦
Original assignee: Yixin Diagnostic Technology Suzhou Co ltd
Current assignee: Yixin Diagnostic Technology Suzhou Co ltd
Priority date: 2022-08-03
Filing date: 2022-08-03
Publication date: 2022-12-20
Anticipated expiration: 2042-08-03
Also published as: CN116037228A; CN114950590A

Abstract

The application discloses a microfluidic pouch, a fluid sample processing device and a nucleic acid extraction method. The microfluidic pouch comprises a laminated film, and a fixed combination area, a functional chamber, a valve area, a liquid inlet, a liquid outlet and the like which are distributed in the laminated film. The size and shape of each functional compartment is defined by the boundaries of the fixed binding area. The laminated film comprises a first film and a second film which are laminated, the first film and the second film are irreversibly combined in the fixed combination area, and the first film and the second film can be switched between a mutually separated state and a mutually attached state under the action of pressure in the functional chamber; the valve area is used for connecting adjacent functional chambers, and an irreversible disposable valve structure is arranged in the valve area; each liquid inlet is connected with a corresponding functional cavity; the liquid outlet is connected with a selected one of the functional chambers. The microfluidic pouch has the advantages of function integration, simple structure, convenience in use, large-scale production and the like, and can be conveniently and efficiently applied to scenes such as nucleic acid extraction.

Description

Microfluidic pouch, fluid sample processing device and nucleic acid extraction method

Technical Field

The application particularly relates to a microfluidic bag, a fluid sample processing device and a nucleic acid extraction method, and belongs to the technical field of microfluidics.

Background

Nucleic acid is a carrier of genetic information, is the most important biological information molecule, and is the main object of molecular biology research, therefore, the extraction of nucleic acid is the most important and basic operation in molecular biology experimental technology.

The traditional full-automatic nucleic acid extraction technology is almost completely realized on the basis of a large XYZ three-dimensional platform and on the basis of the design of additionally arranging a liquid transfer gun capable of accurately and quantitatively transferring liquid on a Z axis. The technical advantages of the mode are that the mechanical motion part is mature in design, the manufactured work station has large flux, the use cost is relatively low and controllable, and therefore the mode is widely applied.

In the nucleic acid extraction process, the sample needs to undergo several steps of cell lysis, adsorption and purification of nucleic acid, and elution and collection of nucleic acid. For most nucleic acid detection samples, these steps are performed under liquid reaction environment conditions. In order to complete a multi-step reaction scheme, the nucleic acid-adsorbing carriers, such as magnetic beads, may undergo several transfers from one reactor to another, or the same reactor may be used continuously but the reagents used need to be transferred out of the reactor after each step. When the nucleic acid extraction is performed by using the fully automatic nucleic acid extraction system, the transfer is performed in an exposed environment. No matter how elaborate the design of the system is, when a large number of detection samples are adjacently placed in a development space and liquid transfer is carried out through the movement of an XYZ three-dimensional platform, cross contamination among the samples, especially cross contamination caused by aerosol formation of high-concentration samples, can be almost impossible to completely eliminate. At the very least, the controllability of aerosol contamination is difficult to prove, especially in view of the conditions of use of the instrumentation and the uncontrollable factors of the environment of use.

Another problem with using XYZ three-dimensional platforms is that their flexibility of use is highly limited. For most medical units such as hospitals, the daily detection amount of each detection item is usually counted by several or dozens, and when the sample collection time and the detection time have uncertainty, the technical characteristics of the XYZ three-dimensional platform make the arrangement and use thereof extremely inapplicable to the small-flux and multi-batch use mode.

The advent of microfluidic technology has made possible continuous, highly integrated experimental manipulations in small, enclosed spaces. Such products are generally called POCT (Point Of Care Test) detection products because Of their portability and simplicity Of operation. Among them, cepheid GeneXpert in the United states and FilmArray in Merriere, france are most well known. On the other hand, the functional requirements of microfluidic products and the characteristics of disposable consumables thereof often result in that the production cost cannot be fused with the current price system in the market. Detection products which are more and more popular but have large-scale application appear in the microfluidic technology.

Microfluidic technology has been characterized from birth as a "Sample to result" (Sample to Answer) target, emphasizing the completeness of the flow and simplicity of operation. However, this pursuit of flow integrity neglects the current situation of application, that is, the existing large number of testing requirements in the medical diagnostic industry cannot be changed from complete laboratory manual operation to full-automatic operation from sample to result due to system and management, user habits, or limitations of testing reagent compliance suppliers. What is further needed for these existing assays is an automated nucleic acid extraction product that is simple to operate, flexible to use, and capable of addressing the most time-consuming and labor-intensive "sample-to-nucleic acid" process in molecular assays. In addition, the worldwide "Laboratory Developed Test" (LDT) cannot improve efficiency by means of integrated microfluidic "sample-to-result" automation technology, but only "sample-to-nucleic acid". The need to extend the traditional microfluidic products of "sample to result" to fully automated "sample to nucleic acid" that include fewer handling steps but are more flexible to apply is clear and urgent.

On the other hand, different cell types require different cell disruption methods for this link of cell lysis. Some processes require only mild processing conditions, while others require drastic methods. For example, ultrasound generators can be used to open the cell membranes of most animals, but are not suitable for certain plant cells because the cell walls are very resistant to ultrasound. Lysis of animal cells can usually be achieved chemically, but bacteria, fungi etc. are much more difficult. These microorganisms often require cell lysis by methods of strongly disrupting cell walls such as cavitation (sound) by high frequency sound, beating (bead milling) by transmission of glass microspheres, and mechanical disruption by a blender. Although these cell lysis methods are widely used in laboratory operations, it is difficult and important to apply them to tiny devices, especially to highly integrated devices for POCT (point-of-care testing), such as microfluidic chips. In addition, in the field of microchemical engineering and the like, there is a demand for sufficient mixing of reaction raw materials in a micro reaction system, but there has been no method which is particularly effective for achieving the object.

Disclosure of Invention

The present disclosure provides a microfluidic pouch, a fluid sample processing device and a method for extracting nucleic acid, which overcome the drawbacks of the prior art.

In order to achieve the above purpose, the present application adopts a technical solution comprising:

one aspect of the present application provides a microfluidic pouch comprising a laminated film, at least one fixed binding region, a plurality of functional chambers, at least one valve region and at least one liquid inlet, the fixed binding region, the functional chambers and the valve region being distributed within the laminated film, and the functional chambers having a size and a shape defined by boundaries of the fixed binding regions, one liquid inlet being connected to a corresponding one of the functional chambers;

the laminated film comprises a first film and a second film which are arranged in a laminated mode, wherein at least one of the first film and the second film is a flexible film;

the first film is irreversibly bonded to the second film in the immobilization bonding zone;

the first film and the second film in the functional cavity can be switched between two states of mutual separation and mutual attachment under the action of pressure, and when the first film and the second film in the functional cavity are separated from each other, the functional cavity can contain fluid;

an irreversible disposable valve structure is distributed in the valve area, the disposable valve structure is connected between two adjacent functional chambers, and when the disposable valve structure is changed from a closed state to an open state, fluid can flow between the two adjacent functional chambers;

the plurality of functional chambers comprise a central chamber and a plurality of auxiliary chambers, and each auxiliary chamber is respectively connected with the central chamber through one disposable valve structure;

and when the first film and the second film are separated from each other in the central cavity, the distance between the first film and the second film is controllable, so that the central cavity at least has two states of limiting opening and completely opening.

Another aspect of the present application also provides a fluid sample processing device comprising:

the micro-fluidic capsule bag is characterized in that,

and each first extrusion mechanism is arranged corresponding to one functional chamber of the microfluidic bag and used for selectively applying pressure to the corresponding functional chamber so as to drive fluid to flow between the mutually connected functional chambers.

Yet another aspect of the present application also provides a use of the fluid sample processing device, for example in the extraction of nucleic acids.

Compared with the prior art, the application has at least the following beneficial effects:

(1) The provided microfluidic bag has the advantages of function integration, simple structure, convenience in use, large-scale production and the like, and can well meet the requirements of POCT detection and the like.

(2) When the microfluidic bag is used, the whole experimental process is completely sealed in the bag, so that the possibility of cross contamination among a plurality of adjacent or non-adjacent liquid samples in the experimental process (such as a nucleic acid extraction operation process) due to rapid vibration or movement of a sample adding gun is avoided. The experimental process realized by the liquid flowing in the closed system also avoids the possibility of pollution caused by the contact of pollutants such as aerosol in the laboratory environment and the like with the sample.

(3) When the provided microfluidic bag is used, liquid flows between two adjacent functional chambers through a microfluidic channel due to pressure difference, the energy in the liquid is increased by virtue of the cavitation effect, and the cell wall breaking efficiency is improved. This increase in efficiency can be modulated by the aspect ratio of the microfluidic channels, by the pressure applied, and by the ratio of gas to liquid in the functional chamber in which the liquid is stored.

(4) After the completion of the experimental process (e.g., nucleic acid extraction process) using the microfluidic pouch, the obtained product (e.g., nucleic acid template extracted from the biological sample) is introduced into the sample tube with the sealing gland, which is hermetically connected to the microfluidic pouch, through the sample outlet connected to a closed microchannel. Such closed transfer ensures that the operation of complete closure during the experiment further continues to the transfer of the nucleic acid template.

Drawings

Exemplary embodiments of the present application are described below with reference to the accompanying drawings. It should be understood that the detailed description is only intended to teach one skilled in the art how to practice the present application, and is not intended to be exhaustive or to limit the scope of the application.

FIG. 1 is a schematic diagram of a microfluidic pouch according to an embodiment of the present disclosure;

FIG. 2 is a schematic layout of a plurality of functional chambers and valve zones in a microfluidic pouch according to an embodiment of the present disclosure;

FIGS. 3 a-3 j are schematic structural views of various first microfluidic channels in one embodiment of the present application;

FIGS. 4 a-4 g are schematic views of various crash features in an embodiment of the present application;

FIG. 5 is a schematic view of the connection of the liquid outlet to the eluent pool in an embodiment of the present application;

FIG. 6 is a schematic diagram of a fluid sample processing device according to an embodiment of the present application.

Detailed Description

Some embodiments of the present disclosure provide a microfluidic pouch comprising a laminated film, at least one fixed binding region, a plurality of functional chambers, at least one valve region, and at least one inlet port, the fixed binding region, the functional chambers, and the valve region all being distributed within the laminated film, and the functional chambers having a size and a shape defined by boundaries of the fixed binding regions, one of the inlet ports being connected to a corresponding one of the functional chambers;

the first film and the second film in the functional cavity can be switched between a state of mutually separating and a state of mutually attaching under the action of pressure, wherein when the first film and the second film in the functional cavity are in the state of mutually attaching, no physical space capable of containing fluid exists in the functional cavity, and when the first film and the second film in the functional cavity are in the state of mutually separating, particularly when the two films are separated and the flexible film in the functional cavity is deformed, the functional cavity can contain fluid, namely form a liquid storage cavity which is mainly bag-shaped and can be also called a liquid storage bag or a micro-bag, and the fluid comprises liquid and/or gas;

and when the first film and the second film are separated from each other in the central cavity, the distance between the first film and the second film is controllable, so that the central cavity has at least two states of limiting opening and complete opening.

According to the micro-fluidic bag, the micro-fluidic bag is formed by compounding the plurality of films, the liquid storage chambers and the like in the micro-fluidic bag correspondingly generate and disappear along with the liquid entering and flowing out, the problem of 'dead volume' of the fluid in different areas in the micro-fluidic bag during transfer can be avoided, the utilization rate of the fluid is obviously improved, and the defects that the fluid in the existing micro-fluidic bag cannot be completely transferred from a certain liquid storage chamber, but is slightly or slightly remained to cause pollution and the like can be effectively avoided. Meanwhile, the microfluidic pouch has the advantages of easiness in preparation, low cost, short development period and the like.

In one embodiment, when the central chamber is in a limit opening state and the magnetic field intensity in the central chamber is above a set value, magnetic particles contained in the fluid flowing through the central chamber can be efficiently captured in the central chamber by the magnetic field;

and when the central chamber is in a fully open state and the magnetic field intensity in the central chamber is less than the set value, the magnetic particles can freely flow into or out of the central chamber along with the fluid.

The set value can be adjusted according to the size of the central cavity, the material and the specification of the magnetic particles and the like.

The magnetic Particles may be magnetic microbeads, microspheres, etc., such as Superparamagnetic Particles (Superparamagnetic Particles).

In one embodiment, fluid can be caused to flow between two of said auxiliary chambers containing fluid through the central chamber when a set pressure is alternately applied to the two auxiliary chambers.

Have the central chamber who opens completely, partially open and close three kinds of states completely through the setting in this application to cooperate it with two at least auxiliary chamber, and combine to exert the magnetic field intensity in central chamber and regulate and control, can conveniently, realize the seizure to the magnetic bead high-efficiently.

In one embodiment, when a set pressure is applied to one of the auxiliary chambers containing fluid, the disposable valve structure connected between the auxiliary chamber and the central chamber can be opened by the fluid pressure, thereby allowing fluid to flow into the central chamber.

The disposable valve structure may employ a variety of weak link structures known in the art for isolating two functional chambers from each other and which may be broken from a closed state to an open state under certain heat, electromagnetic radiation or force.

Preferably, the first film and the second film are detachably combined in the valve area to form the disposable valve structure, and the disposable valve structure can be broken under the impact of a certain pressure of fluid, so that the valve structure is changed from a closed state to an open state. For example, when a set pressure is applied to one functional chamber containing a fluid, the fluid may be caused to be impact-broken against an irreversible disposable valve structure disposed between the functional chamber and another functional chamber, thereby allowing the fluid to enter the other functional chamber. The disposable valve structure can physically constitute "valves" between different reservoir chambers, which can be simply and quickly closed by selective squeezing, and which have no physical space after closing, are easier to operate and do not result in fluid being wasted compared to conventional valves.

In one embodiment, a plurality of the auxiliary chambers are disposed around a central chamber in a direction coplanar with the laminated film. In this way, fluid can be allowed to pass directly from the auxiliary chamber into the central chamber without flowing across the chambers, loss of fluid or some substance in the fluid can be reduced, and contamination of other chambers can be avoided.

In one embodiment, the microfluidic pouch further comprises at least a first connection region distributed within the laminated film, the first connection region also being sized and shaped by the boundary of the fixation bond region; the first connecting area is connected between two adjacent functional chambers, for example two adjacent auxiliary chambers, and the first film and the second film are detachably combined in the first connecting area, wherein when the first film and the second film are separated from each other, a first narrow microfluidic channel is formed in the first connecting area, and when a set pressure is applied to one functional chamber containing fluid, the fluid can pass through the first microfluidic channel at a high speed (for example, the flow speed is 5-20 m/s) and enter the other functional chamber. The pressure difference between the two functional chambers and the high velocity outflow of fluid may create a cavitation-like effect (cavitation) that causes bubbles in the fluid to undergo growth and collapse, which may in turn cause more efficient lysis of thick-walled cell walls and the like in the fluid or some microchemical reactions to proceed more rapidly and thoroughly. In the present application, the pressure, the length and the width of the first microfluidic channel, etc. can be adjusted to change the flow rate of the fluid flowing through the first microfluidic channel, etc. to achieve a more desirable cavitation effect.

Preferably, the first film and the second film in the first connection region can be switched between two states of mutual separation and mutual attachment under the action of pressure, when the first film and the second film in the first connection region are separated from each other, the first microfluidic channel is formed in the first connection region, and when the first film and the second film in the first connection region are attached to each other, the first microfluidic channel disappears, so that the first connection region no longer has a physical space capable of accommodating liquid and gas.

In addition, after the first microfluidic channel is opened, the communication part between the two functional chambers is only the first microfluidic channel, and the first microfluidic channel can also be regarded as an opened valve.

In one embodiment, the shape of the first microfluidic channel within the stacked membrane comprises a polygon or a curve. Wherein the polygon may be regular or irregular, and may be, for example, a rectangle, triangle, diamond, trapezoid, or the like. The curved shape may be an arc shape, a zigzag shape, a wavy shape, etc., without being limited thereto.

In one embodiment, a quantity of solid particles, particularly solid particles composed of inert materials that do not react with the fluid, such as glass microspheres, hard resin microspheres, magnetic microspheres, ceramic particles, metal particles, and the like, may be added to the fluid. When flowing with the fluid and through the first microfluidic channel, the solid particles may rapidly and repeatedly collide with some substances in the fluid, such as cells or certain chemicals, causing effective lysis of difficult-to-lyse cellular structures or thorough mixing of the chemicals.

In one embodiment, the microfluidic pouch further comprises a fluid blocking mechanism distributed in the first microfluidic channel and spaced from at least one side of the inner wall of the first microfluidic channel. By providing these fluid blocking structures, the flow direction, flow rate, etc. of the fluid in the first microfluidic channel can be regulated. In some cases, the fluid blocking mechanism may also collide with the high velocity fluid passing through the first microfluidic channel at high velocity and further facilitate more rapid, frequent contact of chemicals or destructive lysis of thick walled cells and the like in the fluid. Wherein the fluid barrier means may be formed by a partial area of the fixed joining zone extending into the first joining zone.

In one embodiment, the microfluidic pouch further comprises a collision structure disposed in front of the outlet of the first microfluidic channel and configured to collide with the fluid output from the first microfluidic channel. By providing the collision structure, the fluid as it exits the first microfluidic channel may impinge on the collision structure at a very high velocity, and these violent collisions further promote destructive cleavage of certain hard-to-break structures in the fluid or more rapid, frequent contact of chemicals. Wherein the collision structure may be regular or irregular shaped with a certain distance to the outlet of the first microfluidic channel.

In one embodiment, one of said auxiliary chambers is connected to another adjacent one of said auxiliary chambers by one of said disposable valve structures and one of said first connection regions.

In one embodiment, the microfluidic pouch further comprises a liquid outlet and a second connection region, wherein the liquid outlet is connected with a functional chamber (e.g. an auxiliary chamber) through a second connection region, the second connection region is also distributed in the laminated film, the first film and the second film in the second connection region can be switched between a state of mutually separating and mutually jointing under the action of pressure, when the first film and the second film in the second connection region are in a mutually separated state, a second microfluidic channel for allowing a fluid to pass through is formed in the second connection region, and when the first film and the second film in the second connection region are mutually jointed, a physical space capable of containing the fluid and a gas disappears. Through setting up the liquid outlet, can derive the fluid after micro-fluidic bag is handled. The second connecting area is arranged, so that a valve which is easy to open or close and has no physical space after being closed can be formed between the functional cavity and the liquid outlet, the fluid can be output almost without residues, and pollutants in the external environment can be prevented from entering the micro-fluidic bag.

In one embodiment, the microfluidic pouch further includes a third connection region, wherein a functional chamber (e.g., an auxiliary chamber) is connected to the liquid outlet sequentially through the disposable valve structure, the third connection region and the second connection region, the third connection region is also distributed in the laminated film, the first film and the second film in the third connection region can be switched between a separated state and a bonded state under pressure, when the first film and the second film in the third connection region are in the separated state, a third microfluidic channel for fluid to pass through is formed in the third connection region, the diameter of the third microfluidic channel decreases along the fluid flow direction, and when the first film and the second film in the third connection region are bonded, the third microfluidic channel disappears. By arranging the irreversible disposable valve structure and the third connecting area, a structure similar to a one-way valve can be formed, and the interior of the microfluidic bag can be better isolated from the external environment.

In the present application, the pressure action may be a pressure provided by a force application mechanism (e.g., a pressing mechanism) cooperating with each of the function chambers, the first connection region, the second connection region, and the third connection region. In some cases, the pressure effect may further include stress generated by the flexible film of the first film, the second film after deformation, and the like.

In one embodiment, the plurality of auxiliary chambers are at least one sample cell, at least one lysis cell, at least one binding solution cell, at least one washing solution cell and at least one elution solution cell, respectively, wherein one sample cell is connected with an adjacent lysis cell through one of the disposable valve structures and one of the first connection regions, and one elution solution cell is connected with a liquid outlet through one of the second connection regions.

Further, in the coplanar direction of the laminated films, the sample cell, the cracking cell, the binding solution cell, the washing solution cell and the elution solution cell are distributed around the central chamber, and any one of the sample cell, the cracking cell, the binding solution cell, the washing solution cell and the elution solution cell is independently and directly connected with the central chamber through an irreversible disposable valve structure.

Furthermore, the sample cell, the cracking cell, the binding liquid cell, the washing liquid cell and the elution liquid cell are respectively provided with a respective liquid inlet, and one elution liquid cell is also connected with the liquid outlet.

Furthermore, one eluent pool is connected with the liquid outlet sequentially through a disposable valve structure, a second connecting area and a third connecting area.

Furthermore, one or more of the functional chambers may be provided to form a spare reservoir to meet some additional requirements.

In the present application, the first film and the second film are irreversibly bonded at least by thermocompression bonding, ultrasonic bonding, or chemical bonding within the fixing and bonding region, and is not limited thereto.

In the present application, the laminate film is further formed with at least one positioning hole. By arranging the positioning holes, workers can quickly and accurately arrange the microfluidic bag into corresponding detection equipment, and selective extrusion and other operations on different functional chambers in the microfluidic bag can be more favorably and accurately executed.

In the present application, the first film and the second film include any one of a PET film, a PE film, a PP film, a PA film, a PS film, and a PI film, an aluminum-plated film of any one of the films, a composite film of any one of the films and an aluminum foil, a combination of a plurality of the films, a composite film of a combination of a plurality of the films and an aluminum foil, and the like, without being limited thereto. Preferably, the first film and the second film are both flexible films.

Some embodiments of the present application provide a fluid sample processing device comprising:

the micro-fluidic bag is characterized in that,

and more than one first extrusion mechanism, wherein each first extrusion mechanism is arranged corresponding to one functional chamber of the microfluidic bag and used for selectively applying pressure to the corresponding functional chamber so as to drive the fluid to flow between the mutually connected functional chambers.

In one embodiment, the fluid sample processing device further comprises more than one second pressing mechanism, each second pressing mechanism is disposed corresponding to one valve region of the microfluidic pouch and is used for selectively applying pressure to the corresponding valve region to open or close the corresponding valve.

In one embodiment, the first pressing mechanism comprises a magnetic pressing mechanism which is at least movable to a first station, a second station and a third station; at the first station, the magnetic extrusion mechanism extrudes the corresponding functional chamber, and the first film and the second film are tightly attached in the functional chamber; at the second station, the magnetic extrusion mechanism is not in contact with the microfluidic bag, and the magnetic field intensity applied to the corresponding functional chamber by the magnetic extrusion mechanism is below a set value; the third station is distributed between the first station and the second station, and the magnetic field intensity applied to the corresponding functional chamber by the magnetic extrusion mechanism is greater than the set value at the third station. The value of the set value is determined by the magnetic strength, the particle size and the like of the magnetic beads. Generally, when the magnetic field intensity applied to the corresponding functional chamber by the magnetic squeezing mechanism is greater than the set value, it can apply sufficient magnetic attraction force to the magnetic beads in the fluid and fix them, otherwise, if it is less than the set value, the magnetic beads in the fluid will not be attracted and fixed.

In one embodiment, the fluid sample processing device further comprises a control unit, wherein the control unit is connected with at least the first pressing mechanism and is used for regulating and controlling the working state of the first pressing mechanism. Furthermore, the second extrusion mechanism can also be connected with the control unit, and the working state is regulated and controlled by the control unit. The control unit can be selected from but not limited to a PLC, a MCU or a computer.

Further, the fluid sample processing device may further comprise other auxiliary mechanisms, such as the driving mechanism of the aforementioned pressing mechanism, a base, a cover plate, etc., which may be arranged in a manner well known in the mechanical art. The driving mechanism can be selected from, but not limited to, a pneumatic telescopic mechanism, an electromagnetic actuating telescopic mechanism, a hydraulic driving telescopic mechanism, a linear motor and the like or other mechanical driving mechanisms.

Some embodiments of the present application provide a nucleic acid extraction method that is implemented based on the fluid sample processing device, and the method includes:

s1, respectively injecting a sample, a lysis solution, a binding solution, a first washing solution and a second washing solution into a sample pool, a lysis pool, a binding solution pool, a first washing solution pool, a second washing solution pool and an eluent pool of the microfluidic bag, wherein magnetic beads are dispersed in the binding solution;

s2, extruding the sample cell or the cracking cell by set extrusion force to mix the liquid sample with the cracking liquid through the first microfluidic channel, and then alternately extruding the sample cell and the cracking cell by the set extrusion force until the liquid sample is fully mixed with the cracking liquid to obtain a cracked sample;

s3, closing a first microfluidic channel between the sample cell and the lysis cell, and extruding the sample cell and/or the lysis cell and the binding solution cell by set extrusion force to enable the lysed sample and the binding solution to form a liquid passage through the central chamber;

s4, enabling the magnetic extrusion mechanism corresponding to the central chamber to reach a second station, and alternately extruding the sample pool, the lysis pool and the binding solution pool with set extrusion force, so that the reaction liquid flows between two functional chambers through the central chamber and magnetic beads are sufficiently suspended, and the magnetic beads capture nucleic acid in the sample;

s5, after the capture of the nucleic acid is finished, enabling the magnetic extrusion mechanism corresponding to the central chamber to reach a third station, and continuously and alternately extruding the sample pool, the lysis pool and the binding liquid pool with set extrusion force to enable reaction liquid to flow between two functional chambers through the central chamber, so that the magnetic beads with the captured nucleic acid are captured in the central chamber;

s6, after the magnetic beads are captured, enabling the magnetic extrusion mechanism corresponding to the central chamber to reach a first station, and discharging the fluid in the central chamber;

s7, alternately extruding the first washing liquid pool and the second washing liquid pool with a set extrusion force to enable the first washing liquid and the second washing liquid to form a liquid passage through the central chamber, and then finishing the cleaning of the magnetic beads with the captured nucleic acids by referring to the operations of the steps S4-S6;

s8, extruding the eluent pool with a set extrusion force to enable the eluent to enter the central cavity to elute the nucleic acid combined on the magnetic beads, then extruding the central cavity with the set extrusion force to transfer the eluent into the eluent pool, and then extruding the eluent pool with the set extrusion force to enable the eluent to be output through the liquid outlet.

In this application, the set pressure refers to a pressure exceeding a threshold value, and the threshold value varies with the specification, material and the like of the microfluidic capsule.

In addition, the fluid sample processing device of the present application can also be applied to other biological and chemical sample processing, such as PCR reaction, microchemical reaction or other microchemical reaction.

The technical solutions of the present application will be explained in more detail below with reference to several embodiments, but these detailed descriptions are only used to teach those skilled in the art how to implement the present application, and are not intended to exhaust all feasible ways of the present application and are not intended to limit the scope of the present application.

Referring to fig. 1-2, the microfluidic pouch provided in this embodiment can be used for extracting nucleic acid, and mainly comprises a laminated film 1000, a liquid inlet port 2000 and a liquid outlet port 3000. The laminated film 1000, the liquid inlet port 2000 and the liquid outlet port 3000 can be combined into a whole through hot-press welding, ultrasonic welding, chemical bonding and the like.

The laminated film 1000 may also be referred to as a pouch body, and is mainly formed by laminating a first film 1001 and a second film 1002. At least one of the first film 1001 and the second film 1002 is preferably a flexible film having a longitudinal stretching property. For example, the first film includes, but is not limited to, a composite film formed by combining one or more of a PET film, a PE film, a PP film, a PA film, a PS film, and a PI film, and is preferably a composite film. The second film includes, but is not limited to, a composite film made by co-extrusion or composite bonding of one or more of a PET film, a PE film, a PP film, a PA film, a PS film, and a PI film, such as an aluminum-plated film or an aluminum foil film, and is preferably a composite film.

In this embodiment, one or more fixed bonding areas and a plurality of functional chambers may be defined in the laminated film 1000 along the film plane direction. Further, the fixed bonding area divides the entire laminated film 1000 into a plurality of functional chambers in the film plane direction, i.e., the size and shape of the functional chambers are defined by the boundaries of the fixed bonding area. The first film and the second film are irreversibly combined in the fixed combination area through ultrasonic welding, bonding, hot pressing and the like, and the first film and the second film can be switched between two states of mutual separation and mutual attachment in the functional cavity under the action of external force. Specifically, when fluid with a certain pressure is injected into the functional chamber, the first film and the second film in the functional chamber are deformed and separated from each other under the action of the fluid pressure, so that the functional chamber is in a bag structure and can contain liquid and gas. When sufficient pressure is applied to the functional chamber by a pressing mechanism and the like, the first film and the second film in the functional chamber are attached to each other, and the physical space for containing fluid and gas in the functional chamber disappears.

In this embodiment, the functional chambers are mainly used as reservoir chambers, which are respectively a sample cell 1101, a lysis cell 1103, a binding solution cell 1104, a first washing solution cell 1105, a second washing solution cell 1106, an elution solution cell 1107, a central chamber 1108, a first spare cell 1109, a second spare cell 1110, and the like. And the liquid storage chambers respectively have corresponding liquid inlets, such as a sample liquid inlet 1301, a lysis solution liquid inlet 1302, a binding solution liquid inlet 1303, a first washing solution liquid inlet 1304, a second washing solution liquid inlet 1305, a washing solution liquid inlet 1306 and the like. The sample inlet 1301 may be connected to an inlet port 2000.

The sample cell 1101, the lysis cell 1103, the binding solution cell 1104, the first washing solution cell 1105, the second washing solution cell 1106, the elution solution cell 1107, the first spare cell 1109, the second spare cell 1110, and the like are distributed in two dimensions as auxiliary chambers, and surround the central chamber 1108 in the center. Under the scenes of needing magnetic beads to assist in capturing nucleic acid and the like, an auxiliary chamber (which can be named as a magnetic bead capturing chamber) needing a magnetic bead capturing function is directly connected with a central chamber through a valve, so that the magnetic beads are prevented from flowing across the chambers, the loss of the magnetic beads is reduced, and the pollution of reagents brought into other chambers is avoided. Furthermore, when a large-volume solution magnetic bead cleaning and capturing function needs to be executed, the reaction solution containing the magnetic beads can flow back and forth through the central chamber, and the design structure ensures that the central chamber does not need to drive liquid through the compressed volume, so that the flowing speed of the liquid in the central chamber is reduced to the maximum extent, and the magnetic bead capturing efficiency is improved.

Before liquid and gas are not injected, the first film and the second film are tightly attached together in the functional chambers, and no physical space exists. After corresponding liquid and gas are injected into the corresponding functional chambers through the sample injection holes, the liquid generates internal pressure due to the longitudinal tensile property of the flexible film, so that the first film and the second film are deformed and separated to form a liquid storage chamber. When the liquid in the liquid storage cavity is squeezed out due to the pressure provided by the squeezing mechanism and the like and the stress of the flexible film, the first film and the second film are attached to each other in the corresponding functional cavity again, and the physical space disappears.

In this embodiment, one or more valve regions may be defined in the laminated film 1000 along the film plane direction. The valve area is distributed with an irreversible disposable valve structure, the disposable valve structure is connected between two adjacent functional chambers, when the disposable valve structure is in a closed state, the two adjacent functional chambers are isolated from each other, and when the disposable valve structure is changed into an open state, fluid can flow between the two adjacent functional chambers. In this embodiment, the first film and the second film are detachably bonded in the valve area to form the disposable valve structure. The term "detachably coupled" as used herein means that the coupling relationship can be broken under certain conditions, such as a certain external force, so that the first film and the second film in the valve region are separated from each other. Preferably, the first film and the second film in the valve area can be bonded by ultrasonic welding, bonding, thermocompression bonding, and the like, and the bonding can be broken under the impact of a fluid under a certain pressure. The disposable valve structure may be regular or irregular in shape, such as, but not limited to, chevron, V-shaped, zigzag, wave, etc. Generally, the width of the disposable valve structure is much smaller than the diameters of the two adjacent functional chambers. After two adjacent functional chambers are communicated, the communication port is only the torn valve. When the liquid storage bag formed by the two functional cavities is extruded oppositely, the extruded liquid passes through the communication port at a high speed and generates strong impact force. This impact force can realize the effect of vortex oscillation for liquid mixing is more abundant.

In this embodiment, there may be a plurality of valve regions, wherein the plurality of irreversible disposable valve structures may be named as a sample valve 1201, a sample lysis valve 1202, a lysis valve 1203, a binding solution valve 1204, a washing solution valve 1205, a washing solution valve 1206, an eluent valve 1207, a liquid outlet valve 1208, and the like.

In this embodiment, a first pressing mechanism may be disposed above or below each liquid storage chamber, and when liquid flow is required, the first pressing mechanism is pushed to make the liquid in the corresponding functional chamber bear pressure. Each reservoir has a respective valve section, and the valve in each valve section (i.e. the irreversible disposable valve structure) tears open when subjected to a fluid pressure exceeding its rupture threshold, thereby allowing the two reservoirs interconnected by the valve to communicate with each other, so that fluid can flow between the two reservoirs.

In this embodiment, if the extraction of nucleic acid needs to be performed by using an auxiliary medium such as magnetic beads, the first squeezing mechanism may be a structural member having a magnetic bead capture function, such as a magnet having a flat surface, and the magnetic squeezing mechanism may be relatively movable at a first station, a second station, and a third station at different distances from the magnetic bead capture chamber; at the first station, the magnetic extrusion mechanism extrudes the magnetic bead capture chamber by the flat surface of the magnetic extrusion mechanism, so that the first film and the second film are tightly attached in the functional chamber, and the dead volume in the magnetic bead capture chamber is minimum; at a second station, the magnetic extrusion mechanism is far away from the magnetic bead capture chamber, and the magnetic force formed by the magnet embedded in the magnetic extrusion machine on the magnetic bead capture chamber is very weak, so that the magnetic beads in the liquid flowing through the magnetic bead capture chamber cannot be captured; the third station is positioned between the first station and the second station, and at the third station, the magnetic extrusion mechanism is closely contacted with one of the two layers of films forming the magnetic bead capture chamber but separated from the other film, namely the magnetic bead capture chamber forms a micro-thin liquid flow cylindrical structure. When the liquid with magnetic beads passes through the thin liquid flow column structure, all the magnetic beads are influenced by the magnet on the magnetic pressing mechanism and may be adsorbed in the magnetic bead capturing chamber.

For example, the central chamber 1108 may have a movable magnet disposed at its corresponding location for magnetic bead capture. When the magnet is completely extended out, the magnet is attached to the central cavity area, liquid in the central cavity is discharged, the magnet returns for a small distance, the central cavity is changed into a tiny liquid flowing cylindrical structure, and magnetic beads in the liquid flowing through the central cavity can be grabbed. The magnet is fully retracted and the magnetic force is not felt by the central chamber, and the magnetic beads can flow through the central chamber along with the liquid. The position of the magnet away from the central chamber is divided into a first station, a second station and a third station. The first station can enable the magnet to be completely popped out, the magnet is directly attached to the bag and is completely pressed against the central cavity, the upper layer of film and the lower layer of film of the central cavity are attached together, and the function can discharge liquid in the central cavity out of the central cavity; the second station completely withdraws the magnet, the magnet is far away from the central chamber, and then magnetic beads captured in liquid in the central chamber can be released and can flow along with the liquid; the third station is in a position between fully ejected and fully retracted, which keeps the upper and lower membranes in the central chamber at a distance, which is mainly used for magnetic bead capture.

Further, a corresponding second pressing mechanism can be arranged above or below each valve area to serve as a physical valve. When the valves in each valve area are broken under the extrusion of liquid, the blocking function can be realized by the corresponding physical valves. In particular, a second squeezing mechanism may be used to squeeze the corresponding valve area and form a seal, thereby blocking the flow of liquid.

In this embodiment, at least one first connection region may be defined in the laminated film 1000 along the film plane direction. A first attachment area is attached between two adjacent functional compartments, preferably having a width that is substantially smaller than the diameter of the two functional compartments. The first film and the second film are detachably combined in the first connection area, so that the first connection area can form a first microfluidic channel, and when a set extrusion force is applied to one functional chamber containing fluid to enable the fluid to pass through the first microfluidic channel and enter the other functional chamber, the fluid passes through the narrow first microfluidic channel at a high speed due to the action of the pressure and the geometric size limitation of the microfluidic channel. The pressure difference between the two functional chambers and the high velocity of the outgoing fluid create a cavitation-like effect such that bubbles in the fluid undergo growth and collapse.

In this embodiment, an irreversible disposable valve structure and a first connection region to form the first microfluidic channel 1102 may be provided between the functional chamber for forming the sample cell 1101 and the functional chamber for forming the lysis cell 1103. When a squeezing force is applied to sample cell 1101, the liquid therein will escape through first microfluidic channel 1102. Due to the narrow structural design of the first microfluidic channel 1102, the liquid flow rate in the channel rises rapidly. This results in a drop in the pressure of the liquid therein. When the local absolute pressure of the high-speed flowing liquid is reduced to be lower than the saturated vapor pressure of the current environment, air dissolved in water is released, and a plurality of tiny vacuoles are formed. When these bubbles flow into the cracking cell 1103 with the liquid, they burst in an implosion form due to the change of external factors such as the release of pressure. In the process of rupture, the vacuole can generate instantaneous local high temperature and high pressure and form shock waves and micro jet, and the process can even cause chemical reactions which cannot be realized at normal temperature and normal pressure. These popping vacuoles will facilitate the destruction of difficult-to-lyse structures (e.g., bacteria, fungi, cells containing cell wall structures, etc.) and optimize lysis.

The first microfluidic channel 1102 may be in various regular or irregular shapes, such as triangle, trapezoid, waist drum, wave, interdigital, etc., and the efficiency of forming the vacuole may be improved by changing the shape of the first microfluidic channel 1102, and the length, width, etc. of the first microfluidic channel 1102 may be adjusted to further enhance the flow rate of the liquid flowing through the channel, so as to achieve the desired lysis effect. For example, the width of the first microfluidic channel 1102 may be set to 0.1-10mm and the length may be set to 0.1-50 mm in this embodiment.

In addition, a fluid blocking mechanism may be disposed in the first microfluidic channel, and in the coplanar direction of the laminated film, the fluid blocking mechanism may be in various regular or irregular forms, such as a rectangle, a triangle, a diamond, a dot matrix, and the like, and may be connected to an inner wall of one side of the first microfluidic channel and leave a gap with an inner wall of the other side of the first microfluidic channel, or may also leave a gap with inner walls of both sides of the first microfluidic channel, so as to regulate and control the fluidics parameters of the fluid in the first microfluidic channel, and may also collide with the high-speed fluid passing through the first microfluidic channel at a high speed.

In this embodiment, a typical first microfluidic channel 110201 shape and configuration can be seen in fig. 3a, with high pressure region 110202 and low pressure region 110203 formed on either side of it, and cavitation bubbles 110204 formed as fluid passes from high pressure region 110202 through first microfluidic channel 110201 into low pressure region 110203.

Illustratively, the first microfluidic channel may also be any one of the first microfluidic channel 11020101 shown in fig. 3b, the first microfluidic channel 11020102 shown in fig. 3c, the first microfluidic channel 11020103 shown in fig. 3d, the first microfluidic channel 11020104 shown in fig. 3e, the first microfluidic channel 11020105 shown in fig. 3f, the first microfluidic channel 11020106 shown in fig. 3g, the first microfluidic channel 11020107 shown in fig. 3h, the first microfluidic channel 11020108 shown in fig. 3i, or the first microfluidic channel 11020109 shown in fig. 3 j. The fluid blocking mechanisms may be rectangular fluid blocking mechanisms 11020501 in FIG. 3d, interdigitated fluid blocking mechanisms 11020502 in FIG. 3f, diamond fluid blocking mechanisms 11020503 in FIG. 3g, dot matrix fluid blocking mechanisms 11020504 in FIG. 3j, and the like.

In this embodiment, a collision structure may be further provided in front of the outlet of the first microfluidic channel, and the collision structure may be various regular or irregular shapes, such as a triangle, a rectangle, an arc, a fold, a circle, etc., in the coplanar direction of the laminated film, and is configured to collide strongly with the high-speed fluid output from the first microfluidic channel. The collision structure should be spaced from the outlet of the first microfluidic channel to allow fluid to smoothly enter one functional chamber from another functional chamber.

In this embodiment, an exemplary collision structure 110207 can be seen in fig. 4a, when the liquid flows out of the first microfluidic channel 1102, it hits the inner wall of the lysis cell 1103 or hits the collision structure 110207 at a very high velocity. These violent collisions further contribute to destructive cracking of the refractory structure. For example, the impact structure may be any one of impact structure 11020701 shown in fig. 4b, impact structure 11020702 shown in fig. 4c, impact structure 11020703 shown in fig. 4d, impact structure 11020704 shown in fig. 4e, impact structure 11020705 shown in fig. 4f or impact structure 11020706 shown in fig. 4 g.

In this embodiment, a certain amount of glass microspheres 110206, etc. may also be optionally added to the lysis solution. When the glass microspheres flow with the liquid, the sample cells rapidly and repeatedly collide with the glass microspheres when passing through any of first microfluidic channel 11020101-first microfluidic channel 11020108, more efficiently promoting efficient lysis of difficult-to-lyse structures.

Referring to fig. 1 and 5, in the present embodiment, a liquid outlet 1307 is further formed in the laminated film 1000, and the liquid outlet 1307 is connected to a liquid outlet interface 3000. The functional chambers forming the bath 1107 may be connected to the outlet port 1307 by a second junction 1111. The second attachment zone is also disposed within the laminate film where the first film is releasably attached to the second film, and a second microfluidic channel 13072 is formed in the second attachment zone for fluid passage when the first film is detached from the second film in the second attachment zone. The second microfluidic channel 13072 is preferably curved, for example S-shaped. Specifically, when no liquid passes through, the first film and the second film are tightly attached in the second connecting area, and no physical space exists. When the corresponding chambers are squeezed to create internal pressure in the liquid therein, the pressure exerted by the fluid entering the second connection region causes the first film to separate from the second film, forming a second microfluidic channel 13072 having physical space such that the liquid can be transferred within second microfluidic channel 13072.

Preferably, a valve area may be provided between the functional chamber forming the eluent pool 1107 and the effluent port 1307, wherein the irreversible disposable valve structure may be named effluent valve 1208. And, a third connection region may be further provided between the functional chamber for forming the eluent reservoir 1107 and the liquid outlet 1307, the third connection region also being distributed in the laminated film, the first film and the second film being separably combined in the third connection region, and when the first film and the second film in the third connection region are separated, a third microfluidic channel 13071 through which a fluid can pass is formed in the third connection region, and the third microfluidic channel may adopt a variable diameter structure, particularly, a diameter thereof becomes smaller in a liquid flow direction.

The functional chamber for forming the eluent reservoir 1107 may be connected to the liquid outlet 1307 sequentially through the liquid outlet valve 1208, the third microfluidic channel 13071 and the second microfluidic channel 13072.

In addition, referring to fig. 1-2 again, a plurality of positioning holes, such as a first positioning hole 1401, a second positioning hole 1402, etc., may be formed on the laminated film, especially on the edge portion thereof, to guide the positioning of the microfluidic pouch and to reduce the occupied space on the microfluidic pouch.

A method of nucleic acid extraction using the microfluidic pouch of this example may include the steps of:

s1, adding a sample to be extracted into a sample pool 1101 through a sample inlet 2000 to finish a sample adding action;

s2, placing the microfluidic bag into corresponding extraction equipment.

And S3, closing all the physical valves.

S4, opening a physical valve above the sample cracking valve 1202, applying pressure to the sample cell 1101, and breaking the sample cracking valve 1202. The liquid in the sample cell flows into the lysis cell 1103 through the sample lysis valve 1202, so that the sample and the lysis solution are mixed. The sample cell 1101, the release sample cell 1101, the extrusion lysis cell 1103 and the release lysis cell 1103 are sequentially extruded, so that the liquid flows back and forth between the sample cell and the lysis cell, and the sample liquid and the lysis solution are fully mixed.

S5, closing a physical valve at the sample cracking valve 1202 and cutting off the liquid flow between the sample cell and the cracking cell. Physical valves at sample valve 1201, lysis valve 1203, binding fluid valve 1204 are opened. And (3) destroying the sample valve 1201, the lysis valve 1203 and the binding solution valve 1204 to mix the three liquids of the sample, the lysis solution and the binding solution, so that the magnetic beads capture nucleic acid from the sample.

S6, lifting the magnet below the central chamber 1108 to a third station, wherein the magnet is tightly close to the lower surface of the microfluidic pouch but keeps a certain distance from the upper surface of the microfluidic pouch. The liquid in the sample pool, the cracking pool and the combining liquid pool flows through the central cavity in sequence, and the capture of magnetic beads is realized in the central cavity.

And S7, lifting the magnet below the central cavity 1108 to the first station, and discharging residual waste liquid in the central cavity.

S8, after the magnetic beads are captured, discharging waste liquid into the sample cell 1101 and the lysis cell 1103, closing the corresponding physical valves below the sample valve 1201 and the lysis valve 1203, descending the magnet below the central chamber 1108 to a second station, and releasing the magnetic beads.

S9, the liquid in the first washing reagent reservoir 1105 is used in two times to wash the magnetic beads in the central chamber 1108.

S10, discharging the waste liquid after washing to a combined liquid tank 1104 and a first washing liquid tank 1105.

S11, the magnetic beads in the central chamber are washed with the liquid in the second washing reagent reservoir 1106. After completion, the waste liquid is discharged into the second washing liquid tank 1106.

S12, eluting the nucleic acid in the central chamber 1108 by using the liquid in the eluent pool 1107, and transferring the eluent into the eluent pool 1107.

S13, opening a physical valve above the liquid outlet valve 1208, and closing a physical valve above the eluent valve 1207. And extruding the eluent pool 1107 to discharge the liquid into the PCR tube below through the liquid outlet, thereby completing the nucleic acid extraction process.

The aforementioned extraction device may also be considered a fluid sample processing device, which may have a variety of configurations. In the present embodiment, as shown in fig. 6, a fluid sample processing device may include a bladder gland 4001, a nucleic acid extraction bladder 4002, an actuator 4003, a physical valve 4004, a seal ring 4005, a magnet actuator 4006, a bladder support block 4007, an air inlet 4008, and the like. The nucleic acid extraction pouch 4002 employs the microfluidic pouch described above, the actuator 4003 and the magnet actuator 4006 serve as the first pressing mechanism, and the physical valve 4004 serves as the second pressing mechanism. The actuator 4003, the magnet actuator 4006, the physical valve 4004, and the like may be driven by pneumatic, electric, or the like. In the embodiment, a gas driving mode is mainly adopted, and the gas pressure ranges from 0.1 to 0.6MPa. Illustratively, the magnet actuator 4006 may be driven by a magnet movement cylinder 4009.

In this embodiment, at least one set of actuators 4003 is placed in correspondence to each functional chamber of the microfluidic pouch to facilitate the flow of liquid in the corresponding chamber;

a set of physical valves 4004 is provided corresponding to each valve zone of the microfluidic pouch. When the valve is broken, the physical valve is used to block liquid.

A seal 4005 may be placed at the rear end of the actuator 4003 and the valve 4004 and form a seal with a groove on the bladder support block 4007. When gas enters the groove of the bladder support block 4007 through the gas inlet 4008, the actuator or valve is pushed to move forward. When the actuator 4003 moves forwards and meets the bag 4002, the corresponding chamber is pushed to move forwards, but due to the blocking of the bag gland 4001, the actuator 4003 finally clings to the bag gland 4001, liquid in the corresponding chamber in the bag 4002 is discharged, and primary liquid extrusion is realized. When the air pressure of the air inlet is released, the actuator does not act on the extrusion force, and the corresponding chamber on the bag is not extruded any more. When valve 4004 moves forward, it will separate the flow of liquid between the two chambers. A rebound spring, a limit screw and other devices can be selectively added behind the actuator and the valve to limit the extending distance of the actuator and the active rebound.

In the embodiment, the air intake amount of the air inlet can be controlled by using a PWM (pulse width modulation) mode and the like, and the air intake time in a single control period can be controlled by adjusting the PWM high-level time, so that the aim of controlling the single air intake amount is fulfilled. Thus, slow air supply to the actuator 4003 and the magnet movement cylinder 4009 can be realized. In this way, slow extension and retraction of the actuator or cylinder can be achieved. Meanwhile, the extending distance of the actuator and the cylinder can be controlled by controlling the air inflow. The following are exemplary: the magnet moving cylinder 4009 has a first intake port and a second intake port. When the first air inlet is always supplied with positive pressure, the air cylinder extends out, and when the second air inlet is always supplied with positive pressure, the air cylinder retracts. When a fixed number of PWM waves are supplied to the first intake port, the cylinder may not fully extend. By adjusting the number of PWM waves, the operating position of the magnet actuator 4006 is switched.

Although the present application has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the application without departing from the scope thereof. Therefore, it is intended that the present application not be limited to the particular embodiments disclosed for carrying out the present application, but that the present application will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.

Claims

1. A nucleic acid extraction method is implemented based on a fluid sample processing device, wherein the fluid sample processing device comprises a microfluidic bag, more than one first pressing mechanism, and each first pressing mechanism is arranged corresponding to one functional chamber of the microfluidic bag and used for selectively applying pressure to the corresponding functional chamber so as to drive fluid to flow between the mutually connected functional chambers; the first extrusion mechanism comprises a magnetic extrusion mechanism, and the magnetic extrusion mechanism can move at least in a first station, a second station and a third station; at the first station, the magnetic extrusion mechanism extrudes the corresponding functional chamber, and the first film and the second film are tightly attached in the functional chamber; at the second station, the magnetic extrusion mechanism is not in contact with the microfluidic bag, and the magnetic field intensity applied to the corresponding functional chamber by the magnetic extrusion mechanism is below a set value; the third station is distributed between the first station and the second station, and the magnetic field intensity applied to the corresponding functional chamber by the magnetic extrusion mechanism at the third station is greater than the set value;

the microfluidic pouch comprises a laminated film, at least one fixed combination area, a plurality of functional chambers, at least one first connection area, at least one valve area, at least one liquid inlet and a collision structure, wherein the fixed combination area, the functional chambers, the first connection area and the valve area are all distributed in the laminated film, the sizes and the shapes of the functional chambers and the first connection area are defined by the boundaries of the fixed combination areas, and one liquid inlet is connected with one corresponding functional chamber;

the first film and the second film in the functional cavity can be switched between a mutually separated state and a mutually attached state under the action of pressure, and when the first film and the second film in the functional cavity are separated from each other, the functional cavity can contain fluid;

a first thin film and a second thin film are detachably combined in the first connection area, wherein when the first thin film and the second thin film are separated from each other, a first narrow microfluidic channel is formed in the first connection area, and when a set pressure is applied to one of the functional chambers containing fluid, the fluid can pass through the first microfluidic channel at a high speed and enter the other functional chamber, and the collision structure is arranged in front of an outlet of the first microfluidic channel and is used for colliding with the fluid output from the first microfluidic channel;

the plurality of functional chambers are respectively a central chamber and a plurality of auxiliary chambers, the plurality of auxiliary chambers are arranged around the central chamber in the direction coplanar with the laminated film, each auxiliary chamber is directly connected with the central chamber through one disposable valve structure, and one auxiliary chamber is connected with the other adjacent auxiliary chamber through one disposable valve structure and one first connecting area;

when the first film and the second film are separated from each other in the central cavity, the distance between the first film and the second film is controllable, so that the central cavity at least has two states of limiting opening and completely opening;

characterized in that the method comprises:

s2, extruding the sample cell or the cracking cell by using set extrusion force to mix the liquid sample with the cracking solution through the first microfluidic channel, and then alternately extruding the sample cell and the cracking cell by using the set extrusion force until the liquid sample is fully mixed with the cracking solution to obtain a cracked sample;

s5, after the capture of the nucleic acid is finished, enabling a magnetic extrusion mechanism corresponding to the central chamber to reach a third station, and continuously and alternately extruding the sample pool, the lysis pool and the binding solution pool with set extrusion force to enable reaction liquid to flow between two functional chambers through the central chamber, so that magnetic beads with the captured nucleic acid are captured in the central chamber;

s7, alternately squeezing the first washing liquid pool and the second washing liquid pool with a set squeezing force to enable the first washing liquid and the second washing liquid to form a liquid passage through the central chamber, and then finishing the cleaning of the magnetic beads with the captured nucleic acids by referring to the operations of the steps S4-S6;

2. The method for extracting nucleic acid according to claim 1, wherein: when the central chamber is in a limit opening state and the magnetic field intensity in the central chamber is above a set value, magnetic particles contained in the fluid flowing through the central chamber can be captured in the central chamber by the magnetic field;

and when the central chamber is in a fully opened state and the magnetic field intensity in the central chamber is less than the set value, the magnetic particles can freely flow into or out of the central chamber along with the fluid.

3. The method for extracting nucleic acid according to claim 1, wherein: when a set pressure is applied to one of the auxiliary chambers containing fluid, the disposable valve structure connected between the auxiliary chamber and the central chamber can be opened by the fluid pressure, and fluid can flow into the central chamber.

4. The method for extracting nucleic acid according to claim 3, wherein: when a set pressure is alternately applied to two of the auxiliary chambers containing a fluid, the fluid can be caused to flow between the two auxiliary chambers through the central chamber.

5. The method for extracting nucleic acid according to claim 1, wherein: the first film is detachably joined to the second film in the valve area and forms the disposable valve structure.

6. The method for extracting nucleic acid according to claim 1, wherein: the high speed means a flow velocity of 5 to 20m/s.

7. The method for extracting nucleic acid according to claim 1, wherein: the first film and the second film in the first connection area can be switched between two states of mutual separation and mutual attachment under the action of pressure, when the first film and the second film in the first connection area are mutually separated, the first microfluid channel is formed in the first connection area, and when the first film and the second film in the first connection area are mutually attached, the first microfluid channel disappears, and the fluid comprises liquid and gas.

8. The method for extracting nucleic acid according to claim 1, wherein: the width of the first microfluidic channel is 0.1-10mm, and the length is 0.1-50 mm.

9. The method for extracting nucleic acid according to claim 1, wherein: the shape of the first microfluidic channel within the laminated membrane comprises a polygon or a curve.

10. The method for extracting nucleic acid according to claim 1, wherein: the microfluidic pouch further comprises a fluid blocking mechanism, wherein the fluid blocking mechanism is distributed in the first microfluidic channel, and a gap is reserved between the fluid blocking mechanism and the inner wall of at least one side of the first microfluidic channel.

11. The method for extracting nucleic acid according to claim 1, wherein: the microfluidic pouch further comprises a liquid outlet and a second connection area, the liquid outlet is connected with one auxiliary chamber through the second connection area, the second connection area is also distributed in the laminated film, the first film and the second film in the second connection area can be switched between two states of mutual separation and mutual attachment under the action of pressure, when the first film and the second film in the second connection area are separated from each other, a second microfluidic channel for fluid to pass through is formed in the second connection area, and when the first film and the second film in the second connection area are attached to each other, the second microfluidic channel disappears.

12. The method for extracting nucleic acid according to claim 11, wherein: the microfluidic bag further comprises a third connecting area, the auxiliary chamber is connected with the liquid outlet sequentially through the disposable valve structure, the third connecting area and the second connecting area, the third connecting area is also distributed in the laminated film, the first film and the second film in the third connecting area can be switched between a mutually separated state and a mutually attached state under the action of pressure, when the first film and the second film in the third connecting area are in a mutually separated state, a third microfluidic channel for fluid to pass through is formed in the third connecting area, the diameter of the third microfluidic channel is reduced along the fluid flowing direction, and when the first film and the second film in the third connecting area are attached to each other, the third microfluidic channel disappears.

13. The method for extracting nucleic acid according to claim 11, wherein: the plurality of auxiliary chambers are respectively at least one sample cell, at least one cracking cell, at least one binding liquid cell, at least one washing liquid cell and at least one elution liquid cell, wherein one sample cell is connected with one adjacent cracking cell through one disposable valve structure and one first connecting area, and one elution liquid cell is connected with the liquid outlet through one second connecting area.

14. The method for extracting nucleic acid according to claim 1, wherein: the first film and the second film comprise any one of a PET film, a PE film, a PP film, a PA film, a PS film and a PI film, an aluminizer of any one of the films, a composite film of any one of the films and an aluminum foil, a combination of multiple films or a composite film of the combination of the multiple films and the aluminum foil.

15. The method for extracting nucleic acid according to claim 1, wherein: and in the fixed combination area, the first film and the second film are combined irreversibly at least by means of hot-press welding, ultrasonic welding or chemical bonding.

16. The method for extracting nucleic acid according to claim 1, wherein: at least one positioning hole is also formed in the laminated film.

17. The method for extracting nucleic acid according to claim 1, wherein: further comprising:

and each second pressing mechanism is arranged corresponding to one valve area of the microfluidic bag and used for selectively applying pressure to the corresponding valve area so as to open or close the corresponding valve.

18. The method for extracting nucleic acid according to any one of claims 1 to 17, wherein the fluid sample processing device further comprises a control unit connected to at least the first pressing mechanism and configured to control an operating state of the first pressing mechanism.