CN110938523A - Centrifugal microfluidic chip, system and detection method for SAT - Google Patents
Centrifugal microfluidic chip, system and detection method for SAT Download PDFInfo
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- CN110938523A CN110938523A CN201911328682.8A CN201911328682A CN110938523A CN 110938523 A CN110938523 A CN 110938523A CN 201911328682 A CN201911328682 A CN 201911328682A CN 110938523 A CN110938523 A CN 110938523A
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- 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
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- C12Q1/6844—Nucleic acid amplification reactions
Abstract
A centrifugal micro-fluidic chip, a system and a detection method for SAT are disclosed, the chip comprises a basal layer and a cover plate layer which are arranged together in a stacking way, the basal layer is provided with a lower sample inlet, a lower storage chamber, a lower gas outlet, a clockwise flow channel, a lower part of a constant temperature amplification chamber, a first anticlockwise flow channel and a liquid outlet, the cover plate layer is provided with an upper liquid inlet, an upper storage chamber, an upper gas outlet, a second anticlockwise flow channel and an upper part of the constant temperature amplification chamber, the lower storage chamber stores reaction liquid, the lower storage chamber is respectively connected with the lower sample inlet and the lower gas outlet and is connected with the lower part of the constant temperature amplification chamber through the clockwise flow channel, the lower part of the constant temperature amplification chamber is connected with the liquid outlet through the first anticlockwise flow channel, the upper storage chamber stores cleaning liquid, the upper storage chamber is respectively connected with the upper liquid inlet and the, the isothermal amplification chamber has a transparent region. The invention can complete the amplification and detection of nucleic acid rapidly, stably and reliably.
Description
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to a centrifugal microfluidic chip for SAT (real-time fluorescent nucleic acid isothermal amplification detection) and a detection method.
Background
The concept of micro total analysis system is first proposed in 90 s of 20 th century, and then the micro-fluidic technology is rapidly developed on the basis of micro-electronics, micro-mechanics, bioengineering and nano technology, and becomes one of the leading-edge scientific and technological fields in the world at present. The core technology of the prior art is a microfluidic chip based on the microfluidic technology, which is also called a Lab-on-a-chip (Lab on chip). The microfluidic chip has the advantages of low consumption, low cost, high throughput, automatic operation and the like, and is widely applied to the field of biomedicine, wherein an important application is the molecular diagnosis technology based on the microfluidic chip.
Molecular diagnosis is a fine detection method involving multiple amplification technologies, wherein, real-time isothermal amplification and testing (SAT) of fluorescent nucleic acids is a novel nucleic acid detection technology combining a new generation of isothermal amplification technology and real-time fluorescent detection technology. The technology has the advantages of rapid reaction, high sensitivity, high specificity, low pollution, stable reaction and the like. However, as with conventional nucleic acid detection techniques, existing devices and methods for performing SAT do not suffer from the disadvantages of long sample preparation time, cumbersome operation, etc., and do not meet the need for rapid, low-cost clinical diagnosis.
Disclosure of Invention
It is a primary object of the present invention to overcome at least one of the above-mentioned deficiencies of the prior art and to provide a centrifugal microfluidic chip, system and method of testing for SAT.
In order to achieve the purpose, the invention adopts the following technical scheme:
a centrifugal micro-fluidic chip for SAT comprises a basal layer and a cover plate layer which are arranged together in a stacking mode, wherein a lower sample inlet, a lower storage chamber, a lower gas outlet, a clockwise flow channel, a constant-temperature amplification chamber lower part, a first anticlockwise flow channel and a liquid outlet are arranged on the basal layer, an upper sample inlet, an upper storage chamber, an upper gas outlet, a second anticlockwise flow channel and a constant-temperature amplification chamber upper part are arranged on the cover plate layer, the lower storage chamber is used for storing reaction liquid, the lower storage chamber is respectively connected with the lower sample inlet and the lower gas outlet and is connected with the constant-temperature amplification chamber lower part through the clockwise flow channel, the constant-temperature amplification chamber lower part is connected with the liquid outlet through the first anticlockwise flow channel, the upper storage chamber is used for storing cleaning liquid, and the upper sample inlet and the upper gas outlet are respectively connected with the upper sample inlet, the upper part of the constant-temperature amplification chamber and the lower part of the constant-temperature amplification chamber are combined together to form the constant-temperature amplification chamber, and the constant-temperature amplification chamber is provided with a transparent area, wherein when the chip is centrifugally rotated anticlockwise, the cleaning solution in the upper storage chamber flows into the constant-temperature amplification chamber along the second anticlockwise flow channel for cleaning and flows out of the constant-temperature amplification chamber along the first anticlockwise flow channel; when the chip is rotated centrifugally clockwise, the reaction liquid in the lower storage chamber flows into the constant-temperature amplification chamber along the clockwise flow channel to carry out the constant-temperature amplification of the nucleic acid.
Further:
and a waste liquid pool connected between the first anticlockwise flow channel and the liquid outlet is also arranged on the substrate layer.
The basal layer is provided with a plurality of groups of lower storage chambers, clockwise runners, lower parts of the constant-temperature amplification chambers and first anticlockwise runners, and the cover plate layer is provided with a plurality of groups of upper storage chambers, second anticlockwise runners and upper parts of the constant-temperature amplification chambers.
And filter paper with a nucleic acid extraction function is arranged in the constant-temperature amplification chamber.
The material of the substrate layer and the cover plate layer comprises one or more of polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC) and ABS.
And the substrate layer and the cover plate layer are packaged by adopting a pressing process.
The lower sample inlet and the upper liquid inlet are externally connected with a fluid driving device.
A system for SAT comprising the centrifugal microfluidic chip.
A SAT detection method for performing real-time fluorescent nucleic acid amplification detection using the microfluidic chip, the method comprising the steps of:
(1) placing the filter paper adsorbed with the nucleic acid into the isothermal amplification chamber;
(2) pouring a proper amount of reaction liquid into the lower storage chamber from the lower sample inlet, and pouring a proper amount of cleaning liquid into the upper storage chamber from the upper liquid inlet;
(3) placing the chip on a centrifuge for anticlockwise centrifugal rotation, enabling the cleaning solution in the upper storage chamber to enter the constant-temperature amplification chamber along the second anticlockwise flow channel, and enabling the cleaning solution to flow out of the constant-temperature amplification chamber along the first anticlockwise flow channel after the filter paper is cleaned;
(4) performing clockwise centrifugal rotation to enable the reaction liquid in the lower storage chamber to completely enter the constant-temperature amplification chamber along the clockwise flow channel to react with the liquid in the filter paper;
(5) controlling the temperature of the constant temperature amplification chamber to be 37 ℃, observing the fluorescence intensity of the reactant under a microscope, and analyzing the concentration of the nucleic acid.
Further, the isothermal amplification chamber maintains the reaction conditions at 37 ℃ by contacting an external isothermal control plate.
The invention has the following beneficial effects:
the invention provides a centrifugal microfluidic chip for real-time fluorescent nucleic acid isothermal amplification detection and a detection method, which are used for carrying out fluid storage, isothermal amplification and real-time fluorescent detection on nucleic acid. The chip is designed in a centrifugal mode to realize the inflow and outflow of fluid, can quickly, stably and reliably finish the amplification and detection of nucleic acid, and further quickly, stably and reliably obtain the detection results of various target objects. The chip can realize centrifugal sample adding, reaction and detection, has simple structure and convenient control, and can be processed in batches at low cost. In the process of detecting nucleic acid on the chip, the dosage of the reagent can be accurately controlled, the reagent consumption is reduced, multiple indexes can be detected, and the detection result is reliable, stable and can be quickly obtained.
According to the invention, the real-time fluorescent nucleic acid isothermal amplification and detection are realized on the microfluidic chip by the technical scheme, the advantages of low cost, low reagent consumption, high flux 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 isothermal amplification and detection technology are fully exerted, and the requirements of rapid detection in clinic, epidemic disease detection and the like 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 schematic diagram of a centrifugal nucleic acid detection chip for real-time isothermal amplification of fluorescent nucleic acids according to an embodiment of the present invention;
FIG. 2 is a schematic structural view of the substrate layer shown in FIG. 1;
fig. 3 is a schematic structural view of the cover sheet layer shown in fig. 1.
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. In the present invention, the base layer and the cover sheet layer are relative to each other, and the upper and lower positional relationship between the base layer and the cover sheet layer is not limited.
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 to 3, an embodiment of the present invention provides a centrifugal microfluidic chip for real-time fluorescent nucleic acid isothermal amplification detection (SAT), including a substrate layer 1 and a cover plate layer 2 stacked together, where the substrate layer 1 is provided with a lower sample inlet 3, a lower storage chamber 4, a lower gas outlet 5, a clockwise flow channel 6, a lower part of a isothermal amplification chamber, a first counterclockwise flow channel 8 and a liquid outlet 9, the cover plate layer 2 is provided with an upper liquid inlet 11, an upper storage chamber 12, an upper gas outlet 13, a second counterclockwise flow channel 14 and an upper part of the isothermal amplification chamber, the lower storage chamber 4 is used for storing a reaction liquid, the lower storage chamber 4 is connected to the lower sample inlet 3 and the lower gas outlet 5 respectively and is connected to the lower part of the isothermal amplification chamber through the clockwise flow channel 6, the lower part of the isothermal amplification chamber is connected to the liquid outlet 9 through the first flow channel 8 counterclockwise, the upper storage chamber 12 is used for storing cleaning solution, the upper storage chamber 12 is respectively connected with the upper inlet 11 and the upper outlet 13, and is connected with the upper part of the constant temperature amplification chamber through the second counterclockwise flow channel 14, wherein the upper part of the constant temperature amplification chamber and the lower part of the constant temperature amplification chamber are combined together to form the constant temperature amplification chamber 7 for performing constant temperature amplification of nucleic acid. The area of the substrate layer 1 or the cover plate layer 2 corresponding to the isothermal amplification chamber 7 is made of transparent materials so as to facilitate detection. Wherein, when the chip is rotated centrifugally counterclockwise, the cleaning solution in the upper storage chamber 12 flows into the isothermal amplification chamber 7 along the second counterclockwise flow path 14 for cleaning, and flows out from the isothermal amplification chamber 7 along the first counterclockwise flow path 8, and when the chip is rotated centrifugally clockwise, the cleaning solution cannot flow into the second counterclockwise flow path 14 from the upper storage chamber 12; when the chip is rotated centrifugally clockwise, the reaction solution in the lower storage chamber 4 flows into the isothermal amplification chamber 7 along the clockwise flow channel 6 for isothermal amplification of nucleic acids, and the reaction solution does not flow out of the isothermal amplification chamber 7 along the first counterclockwise flow channel 8, whereas when the chip is rotated centrifugally counterclockwise, the reaction solution cannot flow into the clockwise flow channel 6 from the lower storage chamber 4.
The centrifugal microfluidic chip for real-time fluorescent nucleic acid isothermal amplification detection provided by the embodiment of the invention is used for carrying out fluid storage, isothermal amplification and real-time fluorescent detection on nucleic acid. The chip is designed in a centrifugal mode to realize the inflow and outflow of fluid, can quickly, stably and reliably finish the amplification and detection of nucleic acid, and further quickly, stably and reliably obtain the detection results of various target objects. The chip can realize centrifugal sample adding, reaction and detection, has simple structure and convenient control, and can be processed in batches at low cost. In the process of detecting nucleic acid on the chip, the dosage of the reagent can be accurately controlled, the reagent consumption is reduced, multiple indexes can be detected, and the detection result is reliable, stable and can be quickly obtained.
In a preferred embodiment, the substrate layer 1 is further provided with a waste liquid pool 10 connected between the first counterclockwise flow channel 8 and the liquid outlet 9.
In a preferred embodiment, the substrate layer 1 is provided with a plurality of sets of the lower storage chamber 4, the clockwise flow channel 6, the isothermal amplification chamber lower portion and the first counterclockwise flow channel 8, and the cover plate layer 2 is provided with a plurality of sets of the upper storage chamber 12, the second counterclockwise flow channel 14 and the isothermal amplification chamber upper portion.
In a preferred embodiment, a filter paper (not shown) with a nucleic acid extraction function is disposed in the isothermal amplification chamber 7.
In some embodiments, the material of the substrate layer 1 and the cover sheet layer 2 may comprise one or more of polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC), ABS.
In a preferred embodiment, the substrate layer 1 and the cover plate layer 2 are encapsulated by a press-fit process.
In a preferred embodiment, the lower sample inlet 3 and the upper sample inlet 11 are externally connected with a fluid driving device to realize the injection of the cleaning solution and the sample.
The embodiment of the invention also provides a system for real-time fluorescent nucleic acid isothermal amplification detection, which comprises the centrifugal microfluidic chip in any one of the embodiments.
The embodiment of the invention also provides a real-time fluorescent nucleic acid isothermal amplification detection method, which is used for carrying out real-time fluorescent nucleic acid amplification detection by using the centrifugal microfluidic chip of any one of the embodiments, and comprises the following steps:
(1) placing the filter paper adsorbed with the nucleic acid into the isothermal amplification chamber 7;
(2) pouring a proper amount of reaction liquid from the lower sample inlet 3 to the lower storage chamber 4, and pouring a proper amount of cleaning liquid from the upper liquid inlet 11 to the upper storage chamber 12;
(3) placing the chip on a centrifuge for anticlockwise centrifugal rotation, enabling the cleaning solution in the upper storage chamber 12 to enter the constant-temperature amplification chamber 7 along the second anticlockwise flow channel 14, and after the filter paper is cleaned, flowing out of the constant-temperature amplification chamber 7 along the first anticlockwise flow channel 8;
(4) performing clockwise centrifugal rotation, so that the reaction liquid in the lower storage chamber 4 completely enters the constant-temperature amplification chamber 7 along the clockwise flow channel 6 to react with the liquid in the filter paper;
(5) the temperature of the isothermal amplification chamber 7 was controlled to 37 ℃, the fluorescence intensity of the reactant was observed under a microscope, and the nucleic acid concentration was analyzed.
In a preferred embodiment, the isothermal amplification chamber 7 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, a centrifugal microfluidic chip for nucleic acid purification and concentration and real-time fluorescent isothermal nucleic acid amplification comprises a fluid storage unit and a nucleic acid isothermal amplification and detection unit according to functions; the fluid storage unit comprises a plurality of chambers and flow channels and is used for adding and storing a plurality of fluids; the nucleic acid constant temperature amplification and detection unit comprises a plurality of constant temperature amplification chambers 7, paper bases are placed in the chambers, and external links such as constant temperature control, fluorescence detection and the like are involved; the units on the chip are communicated through the flow channels, the fluid injected into the chip sequentially flows through the units in a centrifugal mode, and the inflow and outflow of the fluid are controlled by controlling the centrifugal direction and matching with the specific flow channels. The inflow and outflow of fluid can be realized on the chip by a centrifugal mode, so that the amplification and detection of nucleic acid are completed, and the detection results of various target objects are quickly obtained.
The specific structure of the chip comprises a substrate layer 1 and a cover plate layer 2. The base layer 1 and the cover plate layer 2 are made of common medical plastics such as polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC), ABS, and the like, and can be molded by various plastic molding methods such as mold pressing, thermoplastic molding, injection molding, and the like. The substrate layer 1 and the cover sheet layer 2 may be encapsulated together using a press-fit process. A storage chamber, a sample inlet/liquid inlet, an air outlet and a plurality of flow channels are respectively distributed on the cover plate layer 2 and the substrate layer 1.
The fluid storage unit comprises a sample inlet, a liquid inlet, two air outlets, two storage chambers and two flow channels communicated with the nucleic acid constant-temperature amplification and detection unit; the sample inlet and the liquid inlet are externally connected with a fluid driving device for sample injection, the lower storage chamber 4 of the substrate layer 1 stores reaction liquid, the upper storage chamber 12 of the cover plate layer 2 stores cleaning liquid, and the gas outlet can discharge gas in the storage chamber during sample injection; the flow channel of the upper storage chamber 12 of the cover plate layer 2 communicated with the nucleic acid constant-temperature amplification and detection unit is a specific anticlockwise flow channel, fluid can flow out along the anticlockwise flow channel when the chip is centrifuged anticlockwise, and fluid cannot enter the flow channel when the chip is centrifuged clockwise; the flow channel of the upper storage chamber 12 of the substrate layer 1 communicated with the nucleic acid constant temperature amplification and detection unit is a specific clockwise flow channel 6, when the chip is centrifuged clockwise, fluid can flow out along the clockwise flow channel 6, and when the chip is centrifuged anticlockwise, fluid cannot enter the flow channel.
Wherein, the nucleic acid constant temperature amplification and detection unit comprises a plurality of constant temperature amplification chambers 7 and a plurality of flow channels; the isothermal amplification chambers 7 are distributed between the substrate layer 1 and the cover plate layer 2; the isothermal amplification chamber is provided with a filter paper (e.g., Whatman No.1 filter paper) having a nucleic acid extraction function. The temperature of the constant temperature amplification chamber 7 is maintained at 37 ℃ by contacting with an external constant temperature control plate, and the change of fluorescence intensity is detected in real time to reflect the cyclic amplification condition by placing the chip in a fluorescence detection platform.
Wherein the waste liquid pools 10 are distributed on the substrate layer 1; the flow channel between the nucleic acid constant temperature amplification and detection unit and the waste liquid pool 10 is also a counterclockwise flow channel and is distributed on the substrate layer 1.
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:
(1) the filter paper absorbed with the nucleic acid is put into the isothermal amplification chamber 7 of the nucleic acid isothermal amplification and detection unit.
(2) Using an injection gun to pour a proper amount of reaction liquid into the storage cavity from the sample inlet of the base layer 1 fluid storage unit, and using the injection gun to pour a proper amount of cleaning liquid into the storage cavity from the sample inlet of the cover plate layer 2 fluid storage unit;
(3) and placing the chip on a centrifuge, performing anticlockwise centrifugation, enabling all cleaning liquid in the storage chamber of the cover plate layer 2 to enter the constant-temperature amplification chamber 7 along an anticlockwise flow channel, cleaning the filter paper, and enabling all cleaning liquid to flow to the waste liquid pool 10 along the anticlockwise flow channel on the base layer 1.
(4) And then clockwise centrifugation is carried out, so that the reaction liquid in the storage chamber of the substrate layer 1 completely enters the constant-temperature amplification chamber 7 along the clockwise flow channel 6 to react with the liquid in the paper base. Further, the reaction solution is retained in the constant temperature amplification chamber 7 by the clockwise centrifugation, and does not flow into the waste solution tank 10 along the counterclockwise flow path.
(5) 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 a nucleic acid detecting chip according to one embodiment is shown in FIGS. 1 to 3, and the chip includes a substrate layer 1 and a cover plate layer 2. As shown in fig. 2, the substrate layer 1 includes a lower sample inlet 3, a lower storage chamber 4, a lower gas outlet 5, a plurality of sets of clockwise flow channels 6, a constant temperature amplification chamber 7, a liquid outlet 9, and a plurality of counter-clockwise flow channels 8 communicated with a waste liquid pool 10. As shown in fig. 3, the cover plate layer 2 includes an upper inlet port 11, an upper storage chamber 12, an upper outlet port 13, a plurality of sets of counterclockwise flow channels 14, and a thermostatic amplification chamber 7. The base layer and the cover plate layer are made of common medical plastics, such as polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), Polycarbonate (PC) and ABS, and are molded by various plastic molding modes, such as mold pressing thermoplastic molding, injection molding and the like; for example, an injection molding method is adopted to pre-process a mold, then a polypropylene (PP) material is melted in a constant temperature cylinder (220-.
In one embodiment, the nucleic acid detection step comprises:
(1) the filter paper absorbed with the nucleic acid is put into a constant temperature amplification chamber (7) of the nucleic acid constant temperature amplification and detection unit.
(2) 500uL of the reaction solution was poured from the lower inlet port 3 of the base layer 1 to the lower storage chamber 4 at a rate of 200 uL/min using an injection gun, and 100uL of the washing solution was poured from the upper inlet port 11 of the cover plate layer 2 to the upper storage chamber 12 at a rate of 200 uL/min using an injection gun.
(3) And placing the microfluidic chip on a centrifuge, performing anticlockwise centrifugation, enabling the cleaning liquid in the upper storage chamber 12 to completely enter the constant-temperature amplification chamber 7 along the multiple groups of anticlockwise flow channels 14, cleaning the filter paper, and enabling the cleaning liquid to completely flow to the waste liquid pool 10 along the anticlockwise flow channels 8.
(4) And then clockwise centrifugation is carried out, so that the reaction liquid in the lower storage chamber 4 completely enters the isothermal amplification chamber 7 along the plurality of groups of clockwise flow channels 6 to react with the liquid in the paper base. Further, the reaction solution is retained in the isothermal amplification chamber 7 by the clockwise centrifugation and does not flow into the waste solution tank 10 along the counterclockwise flow path 8.
(5) Controlling the temperature of the nucleic acid isothermal amplification and detection unit to be 37 ℃, observing the fluorescence intensity of the reactants under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
According to the invention, the real-time fluorescent nucleic acid isothermal amplification and detection are realized on the microfluidic chip by the technical scheme, the advantages of low cost, low reagent consumption, high flux 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 isothermal amplification and detection technology are fully exerted, and the requirements of rapid detection in clinic, epidemic disease detection and the like 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 include background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, what is included in the background section is not an admission of prior art by the applicants.
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 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. A centrifugal micro-fluidic chip for SAT is characterized by comprising a basal layer and a cover plate layer which are stacked together, wherein the basal layer is provided with a lower sample inlet, a lower storage chamber, a lower gas outlet, a clockwise flow channel, a constant-temperature amplification chamber lower part, a first anticlockwise flow channel and a liquid outlet, the cover plate layer is provided with an upper liquid inlet, an upper storage chamber, an upper gas outlet, a second anticlockwise flow channel and a constant-temperature amplification chamber upper part, the lower storage chamber is used for storing reaction liquid, the lower storage chamber is respectively connected with the lower sample inlet and the lower gas outlet and is connected with the constant-temperature amplification chamber lower part through the clockwise flow channel, the constant-temperature amplification chamber lower part is connected with the liquid outlet through the first anticlockwise flow channel, the upper storage chamber is used for storing cleaning liquid, and the upper storage chamber is respectively connected with the upper liquid inlet and the upper gas outlet, the upper part of the constant-temperature amplification chamber and the lower part of the constant-temperature amplification chamber are combined together to form the constant-temperature amplification chamber, and the constant-temperature amplification chamber is provided with a transparent area, wherein when the chip is centrifugally rotated anticlockwise, the cleaning solution in the upper storage chamber flows into the constant-temperature amplification chamber along the second anticlockwise flow channel for cleaning and flows out of the constant-temperature amplification chamber along the first anticlockwise flow channel; when the chip is rotated centrifugally clockwise, the reaction liquid in the lower storage chamber flows into the constant-temperature amplification chamber along the clockwise flow channel to carry out the constant-temperature amplification of the nucleic acid.
2. The centrifugal microfluidic chip of claim 1, wherein said substrate layer further comprises a waste reservoir connected between said first counter-clockwise channel and said liquid outlet.
3. The centrifugal microfluidic chip according to claim 1, wherein said base layer has a plurality of sets of said lower storage chamber, said clockwise flow channel, said isothermal amplification chamber lower portion and said first counterclockwise flow channel disposed thereon, and said cover plate layer has a plurality of sets of said upper storage chamber, said second counterclockwise flow channel and said isothermal amplification chamber upper portion disposed thereon.
4. The centrifugal microfluidic chip according to any one of claims 1 to 3, wherein a filter paper with a nucleic acid extraction function is disposed in the isothermal amplification chamber.
5. The centrifugal microfluidic chip of any one of claims 1 to 3, wherein the material of the substrate layer and the cover plate layer comprises one or more of polyvinyl chloride, polyethylene, polypropylene, polystyrene, polycarbonate, ABS.
6. The microfluidic centrifugal chip of any one of claims 1 to 3, wherein the substrate layer and the cover plate layer are encapsulated using a lamination process.
7. The microfluidic centrifugal chip of any one of claims 1 to 3, wherein the lower inlet and the upper inlet are externally connected to a fluid driving device.
8. A system for SAT comprising a centrifugal microfluidic chip according to any one of claims 1 to 7.
9. A method for detecting SAT, wherein a real-time fluorescent nucleic acid amplification detection is performed using the microfluidic chip according to any one of claims 1 to 7, the method comprising the steps of:
(1) placing the filter paper adsorbed with the nucleic acid into the isothermal amplification chamber;
(2) pouring a proper amount of reaction liquid into the lower storage chamber from the lower sample inlet, and pouring a proper amount of cleaning liquid into the upper storage chamber from the upper liquid inlet;
(3) placing the chip on a centrifuge for anticlockwise centrifugal rotation, enabling the cleaning solution in the upper storage chamber to enter the constant-temperature amplification chamber along the second anticlockwise flow channel, and enabling the cleaning solution to flow out of the constant-temperature amplification chamber along the first anticlockwise flow channel after the filter paper is cleaned;
(4) performing clockwise centrifugal rotation to enable the reaction liquid in the lower storage chamber to completely enter the constant-temperature amplification chamber along the clockwise flow channel to react with the liquid in the filter paper;
(5) controlling the temperature of the constant temperature amplification chamber to be 37 ℃, observing the fluorescence intensity of the reactant under a microscope, and 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111482206A (en) * | 2020-04-30 | 2020-08-04 | 苏州博福生物医药科技有限公司 | Disc type microfluidic chip and use method thereof |
| CN113009136A (en) * | 2020-08-21 | 2021-06-22 | 东莞东阳光医疗智能器件研发有限公司 | Small-sized multi-index detection sample analysis device |
| CN111482206B (en) * | 2020-04-30 | 2024-04-26 | 苏州博福生物医药科技有限公司 | Disk-type microfluidic chip and use method thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014005969A1 (en) * | 2012-07-04 | 2014-01-09 | Coris Bioconcept Sprl | Method and device for rapid detection of amplified nucleotide sequences |
| CN107893026A (en) * | 2017-12-29 | 2018-04-10 | 苏州绘真医学检验所有限公司 | Detect micro-fluidic chip, detection method and the detecting system of human virus' nucleic acid |
| CN108424850A (en) * | 2018-01-21 | 2018-08-21 | 南京大学 | A kind of centrifugal force micro-fluidic chip for nucleic acid extraction |
| CN109207475A (en) * | 2018-10-22 | 2019-01-15 | 博迪泰（厦门）生物科技有限公司 | A kind of Rapid nucleic acid extracting method |
| CN208869582U (en) * | 2018-08-21 | 2019-05-17 | 苏州德思普生物科技有限公司 | The micro-fluidic chip of bacterium total nucleic acid in a kind of extraction human whole blood |
| CN110045102A (en) * | 2019-04-08 | 2019-07-23 | 深圳市刚竹医疗科技有限公司 | Reagent sequence loading device, centrifugal microfluidic control device and analysis system |
| CN110283940A (en) * | 2019-06-27 | 2019-09-27 | 深圳市刚竹医疗科技有限公司 | Nucleic acid compositions, the detection kit of influenza virus and micro-fluidic chip |
-
2019
- 2019-12-20 CN CN201911328682.8A patent/CN110938523A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014005969A1 (en) * | 2012-07-04 | 2014-01-09 | Coris Bioconcept Sprl | Method and device for rapid detection of amplified nucleotide sequences |
| CN107893026A (en) * | 2017-12-29 | 2018-04-10 | 苏州绘真医学检验所有限公司 | Detect micro-fluidic chip, detection method and the detecting system of human virus' nucleic acid |
| CN108424850A (en) * | 2018-01-21 | 2018-08-21 | 南京大学 | A kind of centrifugal force micro-fluidic chip for nucleic acid extraction |
| CN208869582U (en) * | 2018-08-21 | 2019-05-17 | 苏州德思普生物科技有限公司 | The micro-fluidic chip of bacterium total nucleic acid in a kind of extraction human whole blood |
| CN109207475A (en) * | 2018-10-22 | 2019-01-15 | 博迪泰（厦门）生物科技有限公司 | A kind of Rapid nucleic acid extracting method |
| CN110045102A (en) * | 2019-04-08 | 2019-07-23 | 深圳市刚竹医疗科技有限公司 | Reagent sequence loading device, centrifugal microfluidic control device and analysis system |
| CN110283940A (en) * | 2019-06-27 | 2019-09-27 | 深圳市刚竹医疗科技有限公司 | Nucleic acid compositions, the detection kit of influenza virus and micro-fluidic chip |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111482206A (en) * | 2020-04-30 | 2020-08-04 | 苏州博福生物医药科技有限公司 | Disc type microfluidic chip and use method thereof |
| CN111482206B (en) * | 2020-04-30 | 2024-04-26 | 苏州博福生物医药科技有限公司 | Disk-type microfluidic chip and use method thereof |
| CN113009136A (en) * | 2020-08-21 | 2021-06-22 | 东莞东阳光医疗智能器件研发有限公司 | Small-sized multi-index detection sample analysis device |
| CN113009136B (en) * | 2020-08-21 | 2024-04-05 | 东莞东阳光医疗智能器件研发有限公司 | Small multi-index detection sample analysis device |
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