CN111073810A - Microfluidic chip, system and method integrating nucleic acid extraction, amplification and detection - Google Patents
Microfluidic chip, system and method integrating nucleic acid extraction, amplification and detection Download PDFInfo
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- CN111073810A CN111073810A CN201911328740.7A CN201911328740A CN111073810A CN 111073810 A CN111073810 A CN 111073810A CN 201911328740 A CN201911328740 A CN 201911328740A CN 111073810 A CN111073810 A CN 111073810A
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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
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
Abstract
A micro-fluidic chip, a system and a method for integrating nucleic acid extraction, amplification and detection are provided, the chip comprises a sample inlet, a washing liquid storage cavity, an eluent storage cavity, a nucleic acid extraction cavity, a washing waste liquid receiving part and a constant temperature amplification cavity, the nucleic acid extraction cavity is provided with nucleic acid extraction filter paper, the washing liquid storage cavity and the eluent storage cavity are respectively connected with the nucleic acid extraction cavity through a one-way valve, the nucleic acid extraction cavity is connected with the washing waste liquid receiving part and the constant temperature amplification cavity, washing liquid is pre-stored in the washing liquid storage cavity, eluent is pre-stored in the eluent storage cavity, the washing liquid storage cavity is used for enabling the washing liquid to break through the one-way valve to flow through the nucleic acid extraction cavity for washing under external force driving, the waste liquid flows into the washing waste liquid receiving part, the eluent storage cavity is used for enabling the break-through eluent, the waste liquid flows into the waste liquid eluting cavity. The chip can rapidly, simply, stably and reliably complete nucleic acid extraction, amplification and detection.
Description
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to a micro-fluidic chip, a system and a method for integrating nucleic acid extraction, amplification and detection.
Background
Micro-fluidic chips, also known as Lab-on-a-Chip (LOC), have rapidly grown up after Micro-Electro-mechanical systems (MEMS) technology was introduced in the 90 s of the 20 th century, and received much attention from the scientific community. The microfluidic chip technology is an experimental technology for performing precise control on microfluid in a microchannel, has the advantages of low consumption, low cost, high flux, automatic operation and the like, is widely applied to the field of biomedicine, and one important application is a molecular diagnosis technology based on the microfluidic chip.
Nucleic acid is a molecular biomarker widely applied to aspects of molecular diagnosis, food safety control, environmental monitoring and the like. Nucleic acid detection, which mainly involves extraction, amplification, detection steps, is a labor intensive, expensive, time consuming and largely instrument dependent method. With the increasing incidence of infectious diseases, food and water borne diseases, instant detection is becoming a focus of more and more attention.
Clinically, molecular diagnostics is widely used for the detection of infectious diseases, tumors, and genetic diseases. With the breakthrough of life science, biotechnology and micromachining technology, the related industries of high-end nucleic acid molecule detection become new global future competitive fields.
Molecular diagnosis is a fine detection method involving multiple amplification technologies, wherein a real-time isothermal fluorescence nucleic acid amplification and detection technology (SAT for short) is a novel nucleic acid detection technology combining a new generation of isothermal nucleic acid amplification technology and a real-time fluorescence detection technology. The technology has the advantages of rapid reaction, high sensitivity, high specificity, low pollution, stable reaction and the like. However, like the conventional nucleic acid detection technology, the existing detection device using the technology does not get rid of the disadvantages of long sample preparation time, complicated operation and the like, and does not meet the requirements of quick and low-cost clinical diagnosis.
Disclosure of Invention
The main object of the present invention is to overcome at least one of the above drawbacks and to provide a microfluidic chip, system and method for integrated nucleic acid extraction, amplification and detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the utility model provides a micro-fluidic chip of integrated nucleic acid extraction, amplification and detection, includes introduction port, washing liquid storage chamber, eluent storage chamber, nucleic acid extraction chamber, washing waste liquid receiving part and constant temperature amplification chamber, the introduction port is connected the nucleic acid extraction chamber, the nucleic acid extraction chamber is provided with the filter paper that possesses the nucleic acid extraction function, washing liquid storage chamber with eluent storage chamber respectively through the runner that is provided with the check valve with nucleic acid extraction chamber links to each other, nucleic acid extraction chamber connects washing waste liquid receiving part with constant temperature amplification chamber, washing waste liquid receiving part with constant temperature amplification chamber connects respectively and accesss to the controllable hole of external switching, wherein, washing liquid prestore of washing liquid in washing liquid storage chamber, eluent in eluent storage chamber, washing liquid storage chamber is used for prestore washing liquid under the external force drive and breaks through the check valve flow path the nucleic acid extraction chamber washes, the waste liquid flows to the washing waste liquid receiving part, the eluent storage cavity is used for enabling the eluent to break through the one-way valve to enter the nucleic acid extraction cavity for elution under the driving of external force, the eluted nucleic acid solution flows into the constant-temperature amplification cavity, and the constant-temperature amplification cavity is used for carrying out nucleic acid constant-temperature amplification and is provided with a transparent area.
Further:
the micro-fluidic chip comprises a base layer and a working layer which are stacked, wherein the sample inlet is provided with a washing liquid storage cavity, the eluent storage cavity is provided with a nucleic acid extraction cavity, the washing waste liquid receiving part and the constant-temperature amplification cavity are arranged on the working layer.
Washing waste liquid receiving unit includes washing waste liquid chamber, the controllable hole of switching includes washing waste liquid chamber exhaust hole and constant temperature amplification chamber exhaust hole, the nucleic acid draws the chamber and connects washing waste liquid chamber, washing waste liquid chamber exhaust hole is connected to washing waste liquid chamber, constant temperature amplification chamber connects constant temperature amplification chamber exhaust hole washing waste liquid chamber exhaust hole is opened and under the state that constant temperature amplification chamber exhaust hole closed, washing waste liquid flows in washing waste liquid chamber the washing waste liquid chamber exhaust hole is closed and under the state that constant temperature amplification chamber exhaust hole was opened, the eluant flows in constant temperature amplification chamber.
The micro-fluidic chip also comprises an elution waste liquid cavity, the nucleic acid extraction cavity is connected with the elution waste liquid cavity, and elution waste liquid flows into the elution waste liquid cavity.
The upper portion of the wash solution storage chamber and/or the eluent storage chamber is closed by a flexible membrane, and the stored liquid is released by applying external pressure to the flexible membrane.
The base layer and the working layer are made of one or more of polyvinyl chloride, polyethylene, polypropylene, polystyrene, polycarbonate or ABS, and are packaged together through a pressing process.
The microfluidic chip comprises a plurality of constant-temperature amplification cavities which are arranged in parallel.
A system for integrating nucleic acid extraction, amplification and detection comprises the centrifugal microfluidic chip.
A real-time fluorescent nucleic acid amplification detection method is used for carrying out real-time fluorescent nucleic acid amplification detection by using the microfluidic chip, and comprises the following steps:
(1) injecting a sample to be detected into the nucleic acid extraction cavity from the sample inlet;
(2) opening a hole connected with the washing waste liquid receiving part, closing a hole connected with the constant-temperature amplification cavity, pressurizing the washing liquid storage cavity, enabling the washing liquid to flow through the nucleic acid extraction cavity for washing, and enabling waste liquid to flow to the washing waste liquid receiving part;
(3) closing the hole connected with the washing waste liquid receiving part, opening the hole connected with the constant-temperature amplification cavity, pressurizing the eluent storage cavity, enabling eluent to slowly flow through the nucleic acid extraction cavity, and enabling the eluted nucleic acid solution to flow to the constant-temperature amplification cavity;
(4) controlling the temperature of the constant-temperature amplification cavity to be 37 ℃, observing the fluorescence intensity of the reactant under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
Further, the isothermal amplification chamber maintains the reaction condition of 37 ℃ by contacting an external isothermal control plate.
The invention has the following beneficial effects:
the invention provides a micro-fluidic chip, a system and a method for integrating nucleic acid extraction, amplification and detection, which are used for extracting nucleic acid, performing constant-temperature amplification and performing real-time fluorescence detection. The invention realizes the extraction, amplification and detection of integrated nucleic acid on a microfluidic chip, can realize the automatic adsorption, extraction, purification, amplification and detection of nucleic acid on the chip, and shortens the amplification and detection time. The chip can be processed with low cost, produced in batch and convenient to store and transport. All reagents required by the reaction are pre-embedded in the chip, manual addition is not needed, operation steps are reduced, meanwhile, one chip can detect multiple indexes, detection paths are independent and cannot influence each other, and the reliable, stable and quick obtaining of detection results is sequentially guaranteed. The invention can reduce the complex instruments and reagents used in the processes of nucleic acid extraction, amplification and detection, effectively reduce the detection cost and improve the detection efficiency; in addition, the chip can be used as a disposable product, and the sample inlet is sealed after sample adding, so that potential positive pollution can be effectively avoided.
According to the invention, real-time fluorescent nucleic acid constant-temperature amplification and detection are realized on the microfluidic chip by the technical scheme, and the nucleic acid extraction process is integrated on the chip, so that the complicated manual operation is simplified, the advantages of low cost, low reagent consumption, high flux, automatic process and the like of the microfluidic chip technology can be fully exerted, the advantages of rapidness, simplicity, stability, reliability and the like of the fluorescent nucleic acid constant-temperature amplification and detection technology are fully exerted, and the requirements of clinical and epidemic disease detection and other rapid detection are met. The chip can be widely used in the field of rapid diagnosis of nucleic acid molecules, reduces the detection cost and improves the detection efficiency.
Drawings
FIG. 1 is a front perspective structural view of a working layer of a microfluidic chip integrating nucleic acid extraction, amplification and real-time fluorescence detection according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a reverse perspective view of a working layer of a microfluidic chip integrating nucleic acid extraction, 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 function or a circuit/signal communication 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 fig. 2, an embodiment of the present invention provides a microfluidic chip integrating nucleic acid extraction, amplification and detection, which has a sample inlet 4, a washing solution storage chamber 3, an eluent storage chamber 11, a nucleic acid extraction chamber 10, a washing waste liquid receiving portion and a constant temperature amplification chamber 10, wherein the sample inlet 4 is connected to the nucleic acid extraction chamber 10, the nucleic acid extraction chamber 10 is provided with filter paper having a nucleic acid extraction function, preferably, a washing solution inlet 2 and an eluent inlet 1 are further provided, the washing solution inlet 2 is connected to the washing solution storage chamber 3, the eluent inlet 1 is connected to the eluent storage chamber 11, the washing solution storage chamber 3 and the eluent storage chamber 11 are respectively connected to the nucleic acid extraction chamber 10 through a flow channel provided with a check valve, the nucleic acid extraction chamber 10 is connected to the washing waste liquid receiving portion and the constant temperature amplification chamber 10, the washing waste liquid receiving part and the constant temperature amplification chamber 10 are respectively connected with a hole (such as a waste liquid chamber exhaust hole 5 and a constant temperature amplification chamber exhaust hole 7) which is opened and closed and is controlled to the outside, wherein, the washing liquid storage cavity 3 is pre-stored with washing liquid, the eluent storage cavity 11 is pre-stored with eluent, the washing liquid storage cavity 3 is used for enabling the washing liquid to break through the one-way valve and flow through the nucleic acid extraction cavity 10 for washing under the driving of external force, the waste liquid flows to the washing waste liquid receiving part, the eluent storage cavity 11 is used for enabling the eluent to break through the one-way valve to enter the nucleic acid extraction cavity 10 for elution under the driving of external force, the eluted nucleic acid solution flows into the constant-temperature amplification cavity 10, the constant-temperature amplification cavity 10 is provided with a specific primer and DNA polymerase, for mixing with a nucleic acid solution to perform isothermal amplification of nucleic acids, the isothermal amplification chamber 10 has a transparent region for detection. Wherein the controllable opening and closing hole can be controlled by using a matched sealing plug.
The micro-fluidic chip integrating nucleic acid extraction, amplification and detection provided by the embodiment of the invention is used for extracting nucleic acid, performing constant-temperature amplification and performing real-time fluorescence detection. The embodiment of the invention realizes the extraction, amplification and detection of integrated nucleic acid on one microfluidic chip, can realize automatic nucleic acid adsorption, extraction, purification, amplification and detection on the chip, and shortens the amplification and detection time. The chip can be processed with low cost, produced in batch and convenient to store and transport. All reagents required by the reaction are pre-embedded in the chip, manual addition is not needed, operation steps are reduced, meanwhile, one chip can detect multiple indexes, detection paths are independent and cannot influence each other, and the reliable, stable and quick obtaining of detection results is sequentially guaranteed. The invention can reduce the complex instruments and reagents used in the processes of nucleic acid extraction, amplification and detection, effectively reduce the detection cost and improve the detection efficiency; in addition, the chip can be used as a disposable product, and the sample inlet 4 is sealed after sample adding, so that potential positive pollution can be effectively avoided.
In the preferred embodiment, the micro-fluidic chip includes stratum basale and the working layer of range upon range of setting, the introduction port 4, the washing liquid inlet 2, eluent inlet 1, washing liquid storage chamber 3, eluent storage chamber 11, nucleic acid extraction chamber 10, washing waste liquid receiving part, constant temperature amplification chamber 10 and corresponding runner set up on the working layer.
In a preferred embodiment, the washing waste liquid receiving unit includes a washing waste liquid chamber 6, the opening/closing controllable hole includes a washing waste liquid chamber vent hole 5 and a constant temperature amplification chamber vent hole 7, the nucleic acid extraction chamber 10 is connected to the washing waste liquid chamber 6, the washing waste liquid chamber 6 is connected to the washing waste liquid chamber vent hole 5, the constant temperature amplification chamber 10 is connected to the constant temperature amplification chamber vent hole 7, the washing waste liquid flows into the washing waste liquid chamber 6 in a state where the washing waste liquid chamber vent hole 5 is opened and the constant temperature amplification chamber vent hole 7 is closed, and the eluent flows into the constant temperature amplification chamber 10 in a state where the washing waste liquid chamber vent hole 5 is closed and the constant temperature amplification chamber vent hole 7 is opened.
In a preferred embodiment, the microfluidic chip further comprises an elution waste liquid cavity 9, the nucleic acid extraction cavity 10 is connected with the elution waste liquid cavity 9, and elution waste liquid flows into the elution waste liquid cavity 9.
In a preferred embodiment, the upper portion of the wash solution storage chamber 3 and/or the eluent storage chamber 11 is closed by a flexible membrane, and the stored liquid is released by applying external pressure to the flexible membrane.
In some embodiments, the material of the substrate layer and the working layer comprises one or more of polyvinyl chloride, polyethylene, polypropylene, polystyrene, polycarbonate, or ABS.
In some embodiments, the substrate layer and the working layer are encapsulated together by a lamination process.
In a preferred embodiment, the microfluidic chip comprises a plurality of isothermal amplification chambers 10 arranged in parallel, so that a plurality of detections can be performed simultaneously.
The embodiment of the invention also provides a system for integrating nucleic acid extraction, amplification and detection, which comprises the centrifugal microfluidic chip.
The embodiment of the invention also provides a real-time fluorescent nucleic acid amplification detection method, which is used for carrying out real-time fluorescent nucleic acid amplification detection by using the microfluidic chip and comprises the following steps:
(1) injecting a sample to be detected into the nucleic acid extraction cavity 10 from the sample inlet 4;
(2) opening a hole connected with the washing waste liquid receiving part, closing a hole connected with the constant temperature amplification chamber 10, pressurizing the washing liquid storage chamber 3, allowing the washing liquid to flow through the nucleic acid extraction chamber 10 for washing, and allowing the waste liquid to flow to the washing waste liquid receiving part;
(3) closing the hole connected with the washing waste liquid receiving part, opening the hole connected with the constant-temperature amplification cavity 10, pressurizing the eluent storage cavity 11, enabling eluent to slowly flow through the nucleic acid extraction cavity 10, and enabling the eluted nucleic acid solution to flow to the constant-temperature amplification cavity 10;
(4) the temperature of the isothermal amplification chamber 10 was controlled to 37 ℃, the fluorescence intensity of the reactants was observed under a microscope, and the nucleic acid concentration was quantitatively analyzed.
In a preferred embodiment, the isothermal amplification chamber 10 maintains 37 ℃ reaction conditions by contacting an external isothermal control plate.
Specific embodiments of the present invention are further described below with reference to the accompanying drawings.
In one embodiment, the microfluidic chip for nucleic acid extraction, amplification and detection can be functionally divided into a nucleic acid extraction unit and a nucleic acid isothermal amplification and detection unit; the chip body is provided with a sample inlet 4, a nucleic acid extraction cavity 10, a washing liquid storage cavity 3, an eluent storage cavity 11, a constant temperature amplification cavity 10, a washing waste liquid cavity 6 and an elution waste liquid cavity 9. The washing liquid stores up chamber 3 and the eluent and stores up and has had required reagent solution in advance in the chamber 11 on the chip, through the runner intercommunication between each cavity, is equipped with the check valve in the washing liquid stores up chamber 3 and the eluent and stores up the exit flow way of chamber 11, but washing liquid stores up chamber 3 and eluent and stores up 11 plus drive in chamber. The chip can be used as a carrier for realizing nucleic acid adsorption, extraction, purification, amplification and detection after a sample is manually added, and realizes the functions of integrating the extraction, amplification and detection of nucleic acid on the chip. The chip can be a disposable product, and the sample inlet 4 is sealed after sample adding, so that potential positive pollution can be effectively avoided.
One particular chip structure includes a base layer and an active layer. The base layer and the working layer are made of common medical plastics such as polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC), ABS and the like, and are molded by various plastic molding modes such as mold pressing thermoplastic molding, injection molding and the like. The substrate layer and the working layer may be encapsulated together using a press-fit process.
Wherein, the chip structure contains sample inlet 4, nucleic acid extraction chamber 10, washing liquid storage chamber 3, eluent storage chamber 11, constant temperature amplification chamber 10 (be reaction chamber), washing waste liquid chamber 6 and elution waste liquid chamber 9, communicates through the runner between the chamber. A filter paper (e.g., Whatman No.1 filter paper) having a nucleic acid extraction function is fixed in advance in the nucleic acid extraction chamber 10. The isothermal amplification cavity 10 is internally provided with a specific primer and DNA polymerase.
Wherein, the accessible the artifical injection sample of introduction port 4, and the accessible drive washing liquid storage chamber 3 with washing liquid and the eluant of prestoring in the eluant storage chamber 11 lets in proper order in nucleic acid extraction chamber 10 and wait to extract nucleic acid sample, washing liquid, eluant etc. and the waste liquid flows into the waste liquid chamber, and the nucleic acid solution under the elution flows into nucleic acid constant temperature amplification and detecting element.
Wherein, be provided with the check valve in advance in the intercommunication outlet flow channel with washing liquid storage chamber 3 and eluant storage chamber 11 to realize the stable storage of washing liquid and eluant. After the washing liquid storage cavity 3 and the eluent storage cavity 11 are pressurized by external drive, liquid in the cavities is released from the cavities and breaks through the one-way valve to enter the nucleic acid extraction cavity 10. The eluted nucleic acid solution enters the constant temperature amplification cavity 10 and dissolves the specific primer and DNA polymerase which are freeze-dried in advance in the cavity to form a mixed solution, and the nucleic acid is amplified at constant temperature. The temperature of the constant temperature amplification cavity 10 is maintained at 37 ℃ by a constant temperature control plate which is contacted with the outside, and the cyclic amplification condition is reflected by placing the chip in a fluorescence detection system and detecting the change of fluorescence intensity in real time.
The nucleic acid detection method of the embodiment of the invention is based on a real-time fluorescent nucleic acid amplification detection technology. The nucleic acid detection method of an embodiment may include the steps of:
injecting a sample to be detected into the nucleic acid extraction cavity 10 to the sample inlet 4 of the nucleic acid extraction unit by using an injector, wherein DNA is adsorbed by the filter paper, and the rest waste liquid flows to a waste liquid cavity;
pressurizing the washing liquid storage cavity 3, washing the washing liquid by flowing through the nucleic acid extraction cavity 10, and flowing the waste liquid to the waste liquid cavity;
closing the waste liquid cavity exhaust hole 5 and opening the reaction cavity exhaust hole;
pressurizing the eluent storage cavity 11, slowly flowing eluent through the nucleic acid extraction cavity 10, and flowing the eluted nucleic acid solution to the reaction cavity;
controlling the temperature of the nucleic acid isothermal amplification and detection unit to be 37 ℃, observing the fluorescence intensity of the reactant under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
The structure of the microfluidic chip integrating nucleic acid extraction, amplification and real-time fluorescence detection in one embodiment is shown in fig. 1-2, and the working layer of the chip comprises an eluent liquid inlet 1, a washing liquid inlet 2, a washing liquid storage cavity 3, a sample inlet 4, a washing waste liquid cavity exhaust hole 5, a washing waste liquid cavity 6, a constant temperature amplification cavity exhaust hole 7, a constant temperature amplification cavity 8, an elution waste liquid cavity 9, a nucleic acid extraction cavity 10 and a plurality of flow channels which are communicated with one another.
The material of the working layer can adopt common medical plastics, such as polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC) and ABS, and adopts various plastic forming modes such as mould pressing thermoplastic forming, injection molding and the like; for example, an injection molding method is adopted to process a mold in advance, then a polypropylene (PP) material is melted in a constant temperature cylinder (20-280 ℃), then the melted PP material is injected into the mold under pressure (800-. Before assembling the chip, the primers and the DNA polymerase need to be freeze-dried in the constant temperature amplification cavity 8 in advance, filter paper with a nucleic acid extraction function needs to be fixedly inlaid in the nucleic acid extraction cavity 10, and then the working layer is plastically packaged by a flexible thin film. The sealing plugs of the exhaust holes and the sampling holes have the same specification, and are also made of medical plastics which are the same as the working layer. After the chip is assembled, the eluent is required to be quantitatively injected through an eluent inlet 1; after quantitative injection of the washing solution through the washing solution inlet 2, the eluent inlet 1, the washing solution inlet 2 and the constant-temperature amplification cavity exhaust hole 7 are sealed by sealing plugs.
The steps of nucleic acid extraction, amplification and detection of one embodiment comprise:
1. injecting a sample to be detected to the nucleic acid extraction cavity 10 from the sample inlet 4 by using an injector, and adsorbing DNA in the sample by the filter paper;
2. a pressurizing device is used for slowly applying pressure to the flexible film above the washing liquid storage cavity 3, the washing liquid flows through the filter paper in the nucleic acid extraction cavity 10 to fully wash the DNA adsorbed on the filter paper, and the washed waste liquid flows to the washing waste liquid cavity 6;
3. taking down the sealing plug on the exhaust hole 7 of the constant-temperature amplification cavity, and sealing the exhaust hole 5 of the washing waste liquid cavity by using the sealing plug;
4. a pressurizing device is used for slowly applying pressure to the flexible film above the eluent storage cavity 11, the eluent flows through the filter paper in the nucleic acid extraction cavity 10 and elutes the DNA adsorbed on the filter paper, and the eluent containing the DNA uniformly flows to each constant temperature amplification cavity 8;
5. the primers and the enzyme which are pre-frozen and dried in the constant temperature amplification cavity 8 are fully dissolved by the eluent containing the DNA;
6. controlling the temperature of the nucleic acid isothermal amplification and detection unit to be 37 ℃, observing the fluorescence intensity of the reactant under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
According to the invention, real-time fluorescent nucleic acid constant-temperature amplification and detection are realized on the microfluidic chip by the technical scheme, and the nucleic acid extraction process is integrated on the chip, so that the complicated manual operation is simplified, the advantages of low cost, low reagent consumption, high flux, automatic process and the like of the microfluidic chip technology can be fully exerted, the advantages of rapidness, simplicity, stability, reliability and the like of the fluorescent nucleic acid constant-temperature amplification and detection technology are fully exerted, and the requirements of clinical and epidemic disease detection and other rapid detection are met. The chip can be widely used in the field of rapid diagnosis of nucleic acid molecules, reduces the detection cost and improves the detection efficiency.
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 utility model provides a micro-fluidic chip of integrated nucleic acid extraction, amplification and detection which characterized in that, includes introduction port, washing liquid storage chamber, eluent storage chamber, nucleic acid extraction chamber, washing waste liquid receiving part and constant temperature amplification chamber, the introduction port is connected the nucleic acid extraction chamber, the nucleic acid extraction chamber is provided with the filter paper that possesses the nucleic acid extraction function, washing liquid storage chamber with eluent storage chamber respectively through the check valve with the nucleic acid extraction chamber links to each other, the nucleic acid extraction chamber is connected washing waste liquid receiving part with the constant temperature amplification chamber, washing waste liquid receiving part with the constant temperature amplification chamber is connected respectively and is access to the controllable hole of external switching, wherein, washing liquid prestores in washing liquid storage chamber, eluent in eluent storage chamber, washing liquid storage chamber is used for making washing liquid break through under the external force drive the check valve flow through the nucleic acid extraction chamber washes, the waste liquid flows to the washing waste liquid receiving part, the eluent storage cavity is used for enabling the eluent to break through the one-way valve to enter the nucleic acid extraction cavity for elution under the driving of external force, the eluted nucleic acid solution flows into the constant-temperature amplification cavity, and the constant-temperature amplification cavity is used for carrying out nucleic acid constant-temperature amplification and is provided with a transparent area.
2. The microfluidic chip according to claim 1, comprising a substrate layer and a working layer stacked on each other, wherein the sample inlet, the washing solution storage chamber, the eluent storage chamber, the nucleic acid extraction chamber, the washing waste liquid receiving portion, and the constant temperature amplification chamber are disposed on the working layer.
3. The microfluidic chip according to claim 1 or 2, wherein the washing waste liquid receiving portion comprises a washing waste liquid chamber, the opening/closing controllable hole comprises a washing waste liquid chamber vent hole and a constant temperature amplification chamber vent hole, the nucleic acid extraction chamber is connected to the washing waste liquid chamber, the washing waste liquid chamber is connected to the washing waste liquid chamber vent hole, the constant temperature amplification chamber is connected to the constant temperature amplification chamber vent hole, the washing waste liquid flows into the washing waste liquid chamber in a state where the washing waste liquid chamber vent hole is opened and the constant temperature amplification chamber vent hole is closed, and the eluent flows into the constant temperature amplification chamber in a state where the washing waste liquid chamber vent hole is closed and the constant temperature amplification chamber vent hole is opened.
4. The microfluidic chip according to claim 3, further comprising an elution waste chamber, wherein the nucleic acid extraction chamber is connected to the elution waste chamber, and an elution waste flows into the elution waste chamber.
5. The microfluidic chip according to claim 1 or 2, wherein an upper portion of the wash solution storage chamber and/or the eluent storage chamber is closed by a flexible film, and the stored liquid is released by applying an external pressure to the flexible film.
6. The microfluidic chip according to claim 2, wherein the material of the substrate layer and the working layer comprises one or more of polyvinyl chloride, polyethylene, polypropylene, polystyrene, polycarbonate, or ABS, and the substrate layer and the working layer are encapsulated by a lamination process.
7. The microfluidic chip according to claim 1 or 2, comprising a plurality of said isothermal amplification chambers arranged in parallel.
8. A system for integrated nucleic acid extraction, amplification and detection comprising a centrifugal microfluidic chip according to any one of claims 1 to 7.
9. A real-time fluorescent nucleic acid amplification detection method using the microfluidic chip according to any one of claims 1 to 7, the method comprising the steps of:
(1) injecting a sample to be detected into the nucleic acid extraction cavity from the sample inlet;
(2) opening a hole connected with the washing waste liquid receiving part, closing a hole connected with the constant-temperature amplification cavity, pressurizing the washing liquid storage cavity, enabling the washing liquid to flow through the nucleic acid extraction cavity for washing, and enabling waste liquid to flow to the washing waste liquid receiving part;
(3) closing the hole connected with the washing waste liquid receiving part, opening the hole connected with the constant-temperature amplification cavity, pressurizing the eluent storage cavity, enabling eluent to slowly flow through the nucleic acid extraction cavity, and enabling the eluted nucleic acid solution to flow to the constant-temperature amplification cavity;
(4) controlling the temperature of the constant-temperature amplification cavity to be 37 ℃, observing the fluorescence intensity of the reactant under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
10. The real-time fluorescent nucleic acid amplification detection method of claim 9, wherein the isothermal amplification chamber maintains the reaction condition at 37 ℃ by contacting an external isothermal control plate.
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