CN113388513A - Micro-fluidic chip for nucleic acid extraction, amplification and detection - Google Patents
Micro-fluidic chip for nucleic acid extraction, amplification and detection Download PDFInfo
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- CN113388513A CN113388513A CN202110717406.1A CN202110717406A CN113388513A CN 113388513 A CN113388513 A CN 113388513A CN 202110717406 A CN202110717406 A CN 202110717406A CN 113388513 A CN113388513 A CN 113388513A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
A micro-fluidic chip for nucleic acid extraction amplification detection is provided with a sample cracking and purifying cavity, a nucleic acid extraction cavity, a transfer channel, a nucleic acid release cavity, a magnetic microsphere collection cavity, a liquid separation structure, a waste liquid cavity and an amplification reaction cavity; wherein, the nucleic acid solution purified in the sample cracking and purifying cavity flows into the nucleic acid extracting cavity, and the magnetic microspheres are specifically combined with the nucleic acid in the solution environment with lower pH value in the nucleic acid extracting cavity; transferring the magnetic microspheres to a nucleic acid release cavity through magnetic field control, and releasing nucleic acid in a solution environment with a higher pH value; and after removing the magnetic microspheres by magnetic adsorption of the amplification preparation solution containing nucleic acid in the magnetic microsphere collection cavity, distributing the amplification preparation solution to a plurality of amplification reaction cavities through a liquid separation structure, and carrying out constant-temperature amplification reaction and real-time fluorescence detection. The micro-fluidic chip can realize rapid and convenient nucleic acid extraction, simple and stable isothermal amplification and accurate and reliable real-time fluorescence detection, and provides a good application platform for high-flux and high-efficiency instant nucleic acid detection.
Description
Technical Field
The invention relates to the field of molecular diagnosis, in particular to a micro-fluidic chip for nucleic acid extraction, amplification and detection.
Background
The microfluidic technology is a technology for precisely controlling fluid on a micrometer scale. The microfluidic chip can utilize the characteristics of trace fluid and the micro-structural design of the chip to realize the extraction of samples, the addition and mixing of reagents, the reaction of mixed liquid and other complex steps which are originally required to be carried out by professional instrument equipment in a laboratory, and the reaction is integrated on a simple chip, thereby completing the required biochemical analysis project. Compared with the traditional biochemical diagnosis technology, the instant detection technology represented by the microfluidic chip technology has the following advantages: the micro-fluidic chip and the equipment thereof have the characteristic of miniaturization, are convenient to carry, and can be applied to scenes with incomplete medical facilities; the micro-fluidic chip is based on precise micro-channel design and standardized program control of a matched instrument, has strong controllability and high stability, and is not influenced by factors such as artificial errors; the microfluidic chip has high integration level, and can integrate complex steps of nucleic acid extraction, amplification, detection and the like into the chip and matched equipment; the nucleic acid detection based on the microfluidic chip is simple and convenient to operate, and operators do not need to master abundant professional knowledge and skills, so that the cost of human resources is greatly saved; the micro-fluidic-based nucleic acid detection requires small amount of reagents, and the possibility of reagent waste is avoided due to accurate quantification, so that the detection cost is reduced; the microfluidic nucleic acid detection can realize high-flux rapid detection, greatly improves the detection efficiency and creates great economic benefit; the micro-fluidic chip is an integral closed system, can effectively avoid the infection of medical detection personnel caused by aerosol formed by pathogens with strong infectivity, and has high safety.
However, the functions realized by the current microfluidic nucleic acid detection chip are basically limited to automatic amplification and detection after nucleic acid extraction, and how to integrate sample pretreatment, i.e. extraction of nucleic acid, into the chip becomes a big problem. The fully integrated microfluidic nucleic acid extraction amplification detection product still faces a great challenge due to the factors of high cost, difficulty in realization of industrial processing and manufacturing, excessively complex chip internal structure, requirement of professional operation, poor stability, incapability of guaranteeing specification unification and the like.
It is to be noted that the information disclosed in the above background section is only for understanding the background of the present application and thus may include information that does not constitute prior art known to a person of ordinary skill in the art.
Disclosure of Invention
The main purpose of the present invention is to overcome the above-mentioned drawbacks of the background art, and to provide a microfluidic chip for nucleic acid amplification detection, so as to achieve simple, fast, efficient and reliable nucleic acid detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a microfluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection comprises a substrate layer and a cover plate layer which are stacked together, wherein a sample cracking and purifying cavity, a nucleic acid extraction cavity, a transfer channel, a nucleic acid release cavity, a magnetic microsphere collecting cavity, a liquid separating structure, a waste liquid cavity and an amplification reaction cavity are arranged on the substrate layer; the lysis and purification cavity is connected with the nucleic acid extraction cavity, the nucleic acid extraction cavity is connected with the nucleic acid release cavity through the transfer channel, and the nucleic acid release cavity is connected with the waste liquid cavity and the amplification reaction cavity through the magnetic microsphere collection cavity and the liquid separation structure; wherein, a sample is injected into the sample cracking and purifying cavity, a nucleic acid extracting solution containing magnetic microspheres is injected into the nucleic acid extracting cavity, and an amplification preparation solution is injected into the nucleic acid releasing cavity; wherein the nucleic acid solution purified in the sample lysis and purification cavity flows into the nucleic acid extraction cavity, and the magnetic microspheres are specifically combined with the nucleic acid in the first lower pH value solution environment of the nucleic acid extraction cavity; the magnetic microspheres are transferred from the nucleic acid extraction cavity to the nucleic acid release cavity through the transfer channel under the control of a magnetic field, and release nucleic acids in the second higher pH value solution environment of the nucleic acid release cavity; and the amplification preparation liquid containing nucleic acid passes through the magnetic microsphere collecting cavity, is distributed to the plurality of amplification reaction cavities through the liquid separating structure after the magnetic microspheres are removed by magnetic adsorption, so that constant-temperature amplification reaction and real-time fluorescence detection are carried out in the amplification reaction cavities, and redundant reaction liquid enters the waste liquid cavity.
Further:
the device comprises a plurality of groups of sample cracking and purifying cavities, a nucleic acid extracting cavity, a transfer channel, a nucleic acid releasing cavity, a magnetic microsphere collecting cavity, a liquid separating structure, a waste liquid cavity and an amplification reaction cavity, wherein the groups are uniformly distributed along the circumferential direction.
The material of the substrate layer and/or the cover plate layer is selected from one or more of polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC) and ABS; the substrate layer and/or the cover plate layer are molded by mould pressing and thermoplastic molding or injection molding; and the substrate layer and the cover plate layer are packaged by adopting a pressing process.
The base layer and the cover plate layer are provided with central round holes matched with a rotating shaft of centrifugal equipment.
Through the first centrifugal control of the chip, the nucleic acid solution purified in the sample cracking and purifying cavity flows into the nucleic acid extracting cavity; through the second centrifugal control of the chip, the amplification preparation liquid is uniformly distributed to the plurality of amplification reaction cavities through the magnetic microsphere collection cavities and the liquid separation structure; preferably, the rotation speed and duration of the first and second centrifugal control are respectively: the optimal speed is 1200-1800 rpm, 1500rpm is preferred, and 25s is preferred in 20-30 s; preferably 3000rpm at 2500-3500 rpm, and preferably 55s at 50-60 s.
Chelating resin particles, preferably Chelex-100 weak cation chelating resin cations, are embedded in the cracking and purifying cavity in advance.
The magnetic microspheres comprise amino surface modified ferroferric oxide magnetic nano microspheres, and the preferred diameter is 100-200 nm.
And a powerful magnet is embedded in the magnetic microsphere collection cavity in advance, and a neodymium iron boron magnet is preferably used.
The freeze-drying primer with a sequence designed according to the specificity of the nucleic acid gene sequence to be detected is pre-embedded in the amplification reaction cavity.
A nucleic acid detecting apparatus comprising:
the microfluidic chip;
the micro-fluidic chip is arranged on the centrifugal device, and the motor is used for driving the centrifugal device to rotate so as to realize first centrifugal motion and second centrifugal motion through at least two stages of rotating speeds;
the temperature control device is used for heating the sample cracking and purifying cavity and the amplification reaction cavity to the temperature required by cracking and amplification;
the fluorescence signal detection device is used for collecting fluorescence signals in the chip amplification reaction cavity in real time and monitoring fluorescence values of nucleic acid isothermal amplification reaction so as to draw an amplification curve for follow-up research and analysis according to the collected fluorescence signals after the reaction is finished.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a micro-fluidic chip for nucleic acid extraction amplification detection, which realizes fully integrated rapid and convenient nucleic acid extraction, simple and stable isothermal amplification and accurate and reliable real-time fluorescence detection under the action of magnetic field control, provides a good application platform for high-flux and efficient instant nucleic acid detection, and is particularly suitable for high-flux and large-scale primary screening detection under the condition of limited medical resources. When the device is used, a sample is heated after being injected into the cracking and purifying cavity, so that the sample cracking and nucleic acid purification are realized; the purified nucleic acid solution flows into the nucleic acid extraction cavity; injecting nucleic acid extracting solution containing magnetic microspheres into a nucleic acid extracting cavity, wherein the magnetic microspheres are specifically combined with nucleic acid, and simultaneously injecting amplification preparation solution into a nucleic acid releasing cavity; controlling the magnetic microspheres through a magnetic field to pass through the transfer channel from the nucleic acid extraction cavity to the nucleic acid release cavity, wherein the nucleic acid bound on the magnetic microspheres is released; the amplification preparation solution is uniformly distributed to a plurality of amplification reaction cavities through a liquid separating structure, wherein the magnetic microspheres are collected in the magnetic microsphere collecting cavities through magnetic adsorption, and redundant reaction solution enters the waste liquid cavity without influencing the final nucleic acid amplification reaction; and then heating the amplification reaction cavity to the temperature required by the amplification reaction, immediately starting the constant-temperature amplification reaction and the real-time fluorescence detection, and automatically obtaining an amplification curve for subsequent research and analysis after the reaction is finished. In the preferred scheme, the chip adopts centrifugal force as a drive to control multi-stage rotating speed, combines simple magnetic field control, and realizes the control of liquid flowing, mixing, distribution and magnetic microspheres under the dual actions of centrifugal control and magnetic field control, thereby greatly reducing manual operation and shortening detection time.
The chip disclosed by the invention is simple in structure and convenient to control, does not need to use toxic and harmful reagents, greatly simplifies manual operation so as to improve the detection efficiency, ensures that the detection result is reliable and stable and can be quickly obtained, and can well meet the requirements of quick detection such as clinic detection and epidemic disease detection. The invention has wide application prospect in the instant nucleic acid detection, and is particularly suitable for being applied to remote areas with relatively deficient medical resources and limited quantity of professional medical staff or dealing with emergent environments such as epidemic outbreak and the like.
Drawings
Fig. 1 is a schematic diagram of an overall structure of a microfluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection according to an embodiment of the present invention.
Fig. 2 is a schematic structural diagram of a cover plate layer and a substrate layer of a microfluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection according to an embodiment of the present invention.
Fig. 3 is a schematic diagram of an operation flow of the microfluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection according to an embodiment of the present invention.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for either a fixed or coupled or communicating function.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of the present invention, "a plurality" means two or more unless specifically limited otherwise.
Referring to fig. 1 and 2, in an embodiment, a microfluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection includes a substrate layer 10 and a cover plate layer 9 stacked together, where the substrate layer 10 is provided with a sample lysis and purification chamber 1, a nucleic acid extraction chamber 2, a transfer channel 3, a nucleic acid release chamber 4, a magnetic microsphere collection chamber 5, a liquid separation structure 6, a waste liquid chamber 7 and an amplification reaction chamber 8. The cracking and purifying cavity 1 is connected with the nucleic acid extracting cavity 2, the nucleic acid extracting cavity 2 is connected with the nucleic acid releasing cavity 4 through the transfer channel 3, and the nucleic acid releasing cavity 4 is connected with the waste liquid cavity 7 and the amplification reaction cavity 8 through the magnetic microsphere collecting cavity 5 and the liquid separating structure 6. Wherein, the sample is heated after being injected into the sample cracking and purifying cavity 1; after the chip is controlled by first centrifugation, purified nucleic acid solution flows into the nucleic acid extraction cavity 2; a nucleic acid extracting solution containing magnetic microspheres is injected into the nucleic acid extracting cavity 2, and an amplification preparation solution is injected into the nucleic acid releasing cavity 4; after the chip is controlled by a magnetic field, magnetic microspheres pass through the transfer channel 3 from the nucleic acid extraction cavity 2 to the nucleic acid release cavity 4; after the chip is subjected to second centrifugal control, amplification preparation liquid is uniformly distributed to the plurality of amplification reaction cavities 8 through the magnetic microsphere collection cavities 5 and the liquid separation structures 6, and redundant reaction liquid enters the waste liquid cavity 7; then, isothermal amplification reaction and real-time fluorescence detection are performed in the amplification reaction chamber 8. The magnetic field control mode may be to control the magnetic microspheres by manually pulling the permanent magnet, or to control the magnetic microspheres by pulling an automatic permanent magnet rod disposed in an external device, which is not limited in the present invention.
In a preferred embodiment, the sample lysis and purification cavity 1, the nucleic acid extraction cavity 2, the transfer channel 3, the nucleic acid release cavity 4, the magnetic microsphere collection cavity 5, the liquid separation structure 6, the waste liquid cavity 7 and the amplification reaction cavity 8 are uniformly distributed in the circumferential direction.
In a preferred embodiment, the rotational speed and duration of the first and second centrifugal control are respectively: the optimal speed is 1200-1800 rpm, 1500rpm is preferred, and 25s is preferred in 20-30 s; preferably 3000rpm at 2500-3500 rpm, and preferably 55s at 50-60 s. The above grading of the rotation speed and duration allows a good control of the flow and distribution of the liquid in stages. In a particularly preferred embodiment, the rotational speed and the duration of the first and second centrifugal movements are, in this order: 1500rpm, 25 s; 3000rpm, 55 s.
In a preferred embodiment, the substrate layer 10 and the cover plate layer 9 are provided with a central circular hole for cooperating with a spindle of a centrifugal device.
In some embodiments, the material of the substrate layer 10 and the cover plate layer 9 may be, but not limited to, polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC), ABS, and other common medical plastics. The base layer 10 and the cover layer 9 can be formed by various plastic forming methods such as mould pressing thermoplastic forming, injection molding and the like. The base layer 10 and the cover layer 9 may be encapsulated using a press-fit process.
In some embodiments, the sample lysis purification chamber 1 is pre-embedded with a nucleic acid purification reagent by injection; the nucleic acid cracking and purifying reagent comprises but is not limited to weak cation chelating resin Chelex-100, can prevent nucleic acid from degrading while thermally cracking cells at 95 ℃, and can specifically adsorb non-nucleic acid organic matter impurities, wherein the impurities comprise cell fragments, lipid, protein and the like, so that the primary purification of nucleic acid is realized; the nucleic acid extraction cavity 2 contains nucleic acid extraction magnetic microspheres by means of injection; the nucleic acid extraction magnetic microspheres comprise but are not limited to amino surface modified ferroferric oxide magnetic nano microspheres, the diameter of the microspheres is 100-200nm, amino protonation can occur at a lower pH value, and electroneutrality is recovered at a higher pH value, so that specific charges adsorb or release nucleic acid with negative electricity; powerful magnets are pre-embedded in the magnetic microsphere collecting cavity 5, wherein the powerful magnets comprise but are not limited to neodymium iron boron magnets, and can efficiently adsorb the magnetic microspheres; the amplification reaction cavity 8 is pre-embedded with a freeze-drying primer, and the sequence of the freeze-drying primer is designed according to the specificity of a nucleic acid gene sequence to be detected, and can be rapidly dissolved after contacting with an amplification reaction liquid to form a uniform amplification reaction liquid system.
In some embodiments, the microfluidic chip is provided with a centrifugal device and a motor, so that centrifugal control of the chip can be realized. The micro-fluidic chip is arranged on the centrifugal equipment, and the motor is used for driving the centrifugal equipment to rotate so as to realize first centrifugal motion and second centrifugal motion through at least two stages of rotating speeds, so that liquid in the micro-fluidic chip is driven to flow.
In some embodiments, the microfluidic chip is equipped with a temperature control device, and the microfluidic chip is disposed on the temperature control device, and the temperature control device is used for heating and maintaining the sample lysis and purification chamber 1 and the amplification reaction chamber 8 at the temperature required for lysis and amplification. Preferably, the temperature control device comprises a heating plate capable of changing temperature rapidly.
In some embodiments, the microfluidic chip is provided with a fluorescence signal detection device, and the fluorescence signal detection device is configured to collect a fluorescence signal in the chip amplification reaction chamber 8 in real time, and monitor a fluorescence value of the nucleic acid isothermal amplification reaction, so that an amplification curve is drawn according to the collected fluorescence signal after the reaction is finished for subsequent research and analysis.
In some embodiments, a method for extracting, amplifying and detecting nucleic acid using the microfluidic chip comprises the following steps:
1) injecting the sample into the sample cracking and purifying cavity 1, heating the sample cracking and purifying cavity 1 to 95 ℃, and standing for ten to fifteen minutes so as to realize the cracking of the sample and the primary purification of the nucleic acid;
2) the chip is controlled by first centrifugation, purified nucleic acid solution flows into the nucleic acid extraction cavity 2 from the sample cracking and purifying cavity 1, and cracking and purifying reagents such as Chelex-100 in the sample cracking and purifying cavity 1 are left in place due to size limitation;
3) injecting the nucleic acid extracting solution into the nucleic acid extracting cavity 2, wherein the magnetic microspheres in the nucleic acid extracting solution are specifically combined with the nucleic acid in a solution environment with a lower pH value; injecting the amplification preparation solution into a nucleic acid release cavity 4, controlling the magnetic field of the chip to enable the magnetic microspheres to pass through a transfer channel 3 from a nucleic acid extraction cavity 2 to the nucleic acid release cavity 4, and releasing the magnetic microspheres in the nucleic acid release cavity 4 into nucleic acid in a solution environment with a higher pH value;
4) the chip is controlled by centrifugation for the second time, the amplification preparation liquid is uniformly distributed to a plurality of amplification reaction cavities 8 from the nucleic acid release cavity 4 through the liquid separating structure 6, the liquid separating structure 6 ensures the uniform distribution of the amplification preparation liquid, the magnetic microspheres are adsorbed by the strong magnet in the magnetic microsphere collecting cavity 5, and the redundant reaction liquid enters the waste liquid cavity 7 without influencing the final nucleic acid amplification reaction;
5) then the amplification reaction cavity 8 is heated to the temperature 65 ℃ required by the constant temperature amplification reaction, the amplification reaction and the real-time fluorescence detection immediately start, and an amplification curve can be automatically obtained for subsequent research and analysis after the reaction is finished.
Specific embodiments of the present invention are described further below with reference to the accompanying drawings.
Referring to fig. 1 and 2, an embodiment provides a microfluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection, including a substrate layer 10 and a cover plate layer 9 stacked together, where the substrate layer 10 is provided with a sample lysis and purification chamber 1, a nucleic acid extraction chamber 2, a transfer channel 3, a nucleic acid release chamber 4, a magnetic microsphere collection chamber 5, a liquid separation structure 6, a waste liquid chamber 7 and an amplification reaction chamber 8. The sample is firstly injected into the sample cracking and purifying cavity 1 and then heated; after the chip is controlled by first centrifugation, purified nucleic acid solution flows into the nucleic acid extraction cavity 2; a nucleic acid extracting solution containing magnetic microspheres is injected into the nucleic acid extracting cavity 2, and an amplification preparation solution is injected into the nucleic acid releasing cavity 4; after the chip is controlled by a magnetic field, the magnetic microspheres pass from the nucleic acid extraction cavity 2 to the nucleic acid release cavity 4 through the transfer channel 3; after the chip is subjected to second centrifugal control, the amplification preparation solution is uniformly distributed to a plurality of amplification reaction cavities 8 through the magnetic microsphere collection cavity 5 and the liquid separation structure 6, and redundant reaction solution enters the waste solution cavity 7; then, isothermal amplification reaction and real-time fluorescence detection are carried out in the amplification reaction chamber 8.
Referring to fig. 3, in an embodiment, the steps of performing nucleic acid extraction, isothermal amplification and real-time fluorescence detection using the microfluidic chip specifically include:
1) taking out the microfluidic chip (11 in fig. 3) pre-embedded with lysis and purification reagents such as weakly chelating resin particles, strong magnets and freeze-drying primers;
2) injecting 40 mu L of a sample to be lysed into the sample lysis and purification cavity 1 by using a pipette gun, heating the lysis and purification cavity 1 to 95 ℃ by using a temperature control device, and standing for ten to fifteen minutes to realize sample lysis and primary nucleic acid purification (12 in FIG. 3);
3) fixing the microfluidic chip on a matched centrifugal device, and turning on a switch to start a preset centrifugal mode;
4) the chip is controlled by first centrifugation, the purified nucleic acid solution flows into the nucleic acid extraction cavity 2 from the sample lysis and purification cavity 1, and the lysis and purification reagent such as weak chelating resin particles are remained in the sample lysis and purification cavity 1 due to size limitation (13 in FIG. 3);
5) injecting 40. mu.L of a nucleic acid extracting solution into the nucleic acid extracting chamber 2 using a pipette gun, and injecting 70. mu.L of an amplification preparation solution into the nucleic acid releasing chamber 4 using a pipette gun (14 in FIG. 3);
6) the chip controls the magnetic microspheres to pass through the transfer channel 3 from the nucleic acid extraction cavity 2 to the nucleic acid release cavity 4 through a magnetic field, and the magnetic microspheres in the nucleic acid release cavity 4 release nucleic acid in a solution environment with higher pH value (15 and 16 in the figure 3);
7) the chip is controlled by centrifugation for the second time, the amplification preparation liquid is accurately and uniformly distributed to a plurality of amplification reaction cavities 8 from the nucleic acid release cavity 4 through the liquid separating structure 6, wherein the liquid separating structure 6 ensures the uniform distribution of the amplification preparation liquid, the magnetic microspheres are adsorbed by the strong magnet in the magnetic microsphere collecting cavity 5, and the redundant reaction liquid enters the waste liquid cavity 7 (17 and 18 in the picture 3);
8) the temperature control device heats the amplification reaction cavity 8 to 65 ℃ required by isothermal amplification, the fluorescence signal detection device collects fluorescence signals in the amplification reaction cavity 8 when the amplification reaction starts, monitors the fluorescence value of the nucleic acid isothermal amplification reaction, and draws an amplification curve for subsequent research and analysis according to the collected fluorescence signals after the reaction is finished.
The micro-fluidic chip integrating nucleic acid extraction, isothermal amplification and real-time fluorescence detection provided by the invention can realize rapid and convenient nucleic acid extraction, simple and stable isothermal amplification and accurate and reliable real-time fluorescence detection, and provides a good application platform for high-throughput and efficient real-time nucleic acid detection. The preferred embodiment adopts centrifugal force as drive control for multi-stage rotating speed, and simultaneously combines simple magnetic field control to realize the flow, mixing, distribution and control of the magnetic microspheres of the liquid, thereby greatly reducing manual operation, shortening detection time, simultaneously playing the advantages of low cost, automation, high flux, simple operation and low cost of the microfluidic chip technology, and being particularly suitable for large-scale primary screening detection under the condition of limited medical resources. The fully integrated microfluidic chip can provide a reliable and efficient research platform for the field of biochemical analysis represented by nucleic acid detection in the future, and has great market potential.
The background of the present invention may contain background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the claims.
Claims (10)
1. The microfluidic chip for nucleic acid extraction amplification detection is characterized by comprising a substrate layer and a cover plate layer which are stacked together, wherein a sample cracking and purifying cavity, a nucleic acid extraction cavity, a transfer channel, a nucleic acid release cavity, a magnetic microsphere collecting cavity, a liquid separating structure, a waste liquid cavity and an amplification reaction cavity are arranged on the substrate layer; the lysis and purification cavity is connected with the nucleic acid extraction cavity, the nucleic acid extraction cavity is connected with the nucleic acid release cavity through the transfer channel, and the nucleic acid release cavity is connected with the waste liquid cavity and the amplification reaction cavity through the magnetic microsphere collection cavity and the liquid separation structure; wherein, a sample is injected into the sample cracking and purifying cavity, a nucleic acid extracting solution containing magnetic microspheres is injected into the nucleic acid extracting cavity, and an amplification preparation solution is injected into the nucleic acid releasing cavity; wherein the nucleic acid solution purified in the sample lysis and purification cavity flows into the nucleic acid extraction cavity, and the magnetic microspheres are specifically combined with the nucleic acid in the first lower pH value solution environment of the nucleic acid extraction cavity; the magnetic microspheres are transferred from the nucleic acid extraction cavity to the nucleic acid release cavity through the transfer channel under the control of a magnetic field, and release nucleic acids in the second higher pH value solution environment of the nucleic acid release cavity; and the amplification preparation liquid containing nucleic acid passes through the magnetic microsphere collecting cavity, is distributed to the plurality of amplification reaction cavities through the liquid separating structure after the magnetic microspheres are removed by magnetic adsorption, so that constant-temperature amplification reaction and real-time fluorescence detection are carried out in the amplification reaction cavities, and redundant reaction liquid enters the waste liquid cavity.
2. The microfluidic chip according to claim 1, comprising a plurality of groups of the sample lysis and purification chamber, the nucleic acid extraction chamber, the transfer channel, the nucleic acid release chamber, the magnetic microsphere collection chamber, the liquid separation structure, the waste liquid chamber, and the amplification reaction chamber, wherein each group is uniformly distributed along a circumferential direction.
3. The microfluidic chip according to any of claims 1 to 2, wherein the material of the substrate layer and/or the cover plate layer is selected from one or more of polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC), ABS; the substrate layer and/or the cover plate layer are molded by mould pressing and thermoplastic molding or injection molding; and the substrate layer and the cover plate layer are packaged by adopting a pressing process.
4. The microfluidic chip according to any of claims 1 to 3, wherein the substrate layer and the cover plate layer are provided with a central circular hole for cooperating with a rotation shaft of a centrifugal device.
5. The microfluidic chip according to any one of claims 1 to 4, wherein the nucleic acid solution purified in the sample lysis and purification chamber flows into the nucleic acid extraction chamber through a first centrifugal control of the chip; through the second centrifugal control of the chip, the amplification preparation liquid is uniformly distributed to the plurality of amplification reaction cavities through the magnetic microsphere collection cavities and the liquid separation structure; preferably, the rotation speed and duration of the first and second centrifugal control are respectively: the optimal speed is 1200-1800 rpm, 1500rpm is preferred, and 25s is preferred in 20-30 s; preferably 3000rpm at 2500-3500 rpm, and preferably 55s at 50-60 s.
6. The microfluidic chip according to any of claims 1 to 5, wherein chelating resin particles, preferably Chelex-100 weak cation chelating resin cations, are pre-embedded in the lysis purification chamber.
7. The microfluidic chip according to any of claims 1 to 6, wherein the magnetic microspheres comprise amino surface modified ferroferric oxide magnetic nanospheres, preferably with a diameter of 100-200 nm.
8. The microfluidic chip according to any of claims 1 to 7, wherein a strong magnet, preferably a neodymium iron boron magnet, is pre-embedded in the magnetic microsphere collection cavity.
9. The microfluidic chip according to any one of claims 1 to 8, wherein the amplification reaction chamber is pre-embedded with a lyophilized primer having a sequence designed according to the specificity of the nucleic acid gene sequence to be detected.
10. A nucleic acid detecting apparatus characterized by comprising:
the microfluidic chip of any one of claims 1 to 9;
the micro-fluidic chip is arranged on the centrifugal device, and the motor is used for driving the centrifugal device to rotate so as to realize first centrifugal motion and second centrifugal motion through at least two stages of rotating speeds;
the temperature control device is used for heating the sample cracking and purifying cavity and the amplification reaction cavity to the temperature required by cracking and amplification;
the fluorescence signal detection device is used for collecting fluorescence signals in the chip amplification reaction cavity in real time and monitoring fluorescence values of nucleic acid isothermal amplification reaction so as to draw an amplification curve for follow-up research and analysis according to the collected fluorescence signals after the reaction is finished.
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