CN116218653A - Centrifugal fully-integrated high-flux nucleic acid detection micro-fluidic chip and detection method - Google Patents
Centrifugal fully-integrated high-flux nucleic acid detection micro-fluidic chip and detection method Download PDFInfo
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Abstract
The invention relates to a centrifugal fully-integrated high-flux nucleic acid detection microfluidic chip and a detection method, wherein the centrifugal fully-integrated high-flux nucleic acid detection microfluidic chip comprises a nucleic acid extraction unit, a reactant mixing unit and a nucleic acid isothermal amplification and detection unit; the nucleic acid extraction unit uses a silica gel mold structure to perform nucleic acid adsorption; the reactant mixing unit comprises a plurality of chambers and mixing flow channels, and is used for adding and mixing a plurality of reactants; the nucleic acid isothermal amplification and detection unit comprises a plurality of isothermal amplification chambers, and relates to external equipment such as isothermal control and fluorescence detection. The invention can realize purification, concentration, amplification and detection of nucleic acid on a chip in one step, and can rapidly obtain detection results of various targets; the chip can be widely used in the field of rapid diagnosis of nucleic acid molecules, reduces detection cost, simplifies manual operation and further improves detection efficiency.
Description
Technical Field
The invention relates to the field of microfluidic technology and molecular diagnosis, in particular to a method for integrating nucleic acid purification, isothermal amplification and fluorescence detection based on the microfluidic technology.
Technical Field
The rapid and accurate detection of viral infectious agents or carriers through nucleic acid detection and isolation of the infectious agents or carriers, which have been outbreaked many times in human history, is an effective means of suppressing viral transmission. However, conventional nucleic acid detection methods rely on complex machines and specialized detectors, which cannot achieve rapid detection in large scale and high throughput. The microfluidic technology integrates multiple fields including electric, acoustic, optical, magnetic, mechanical and the like, is rapid in development, combines the advantages of the multiple technical fields, and has the advantages of high integration level, small consumption, portability in use and the like. Thus, biological detection techniques based on microfluidic principles have great potential.
Compared with the traditional laboratory operation process, the microfluidic technology has the advantages of high automation level, high flux, small reagent consumption, less pollution, integration compatibility with other equipment and the like. Therefore, the microfluidic technology has great development prospect in the instant detection, is a novel tool for later clinical diagnosis, and can be used as an emerging technology to provide a new direction and an updated space for the development of the fields of high-throughput biological index determination, compound screening and biochemical synthesis, organ chip, multiphase droplet generation and the like.
Microfluidic chips for nucleic acid detection are currently available, but some problems still remain. Through search, the Chinese patent application number is found: 202111538358.6 "proposes a microfluidic chip and detection method for nucleic acid detection, which only integrates amplification detection on the chip, and does not integrate the nucleic acid purification step. Chinese patent application number: 202011131328.9 "proposes a finger-pressure type microfluidic chip for nucleic acid detection, which cannot realize high-throughput detection. In summary, the micro-fluidic chip based on the external source can realize automatic detection, but the defects that the sample pretreatment, such as purification, is not integrated on the chip, the instrument is complex, the application to an outdoor nucleic acid detection scene is difficult, the portability is poor and the like exist at the same time.
Disclosure of Invention
The invention aims at overcoming the background defect, and develops a centrifugal chip integrating nucleic acid purification, LAMP amplification and real-time fluorescence detection based on a microfluidic technology aiming at a high-flux nucleic acid detection scene.
The aim of the invention is achieved by the following technical scheme:
the centrifugal fully-integrated high-flux nucleic acid detection micro-fluidic chip is characterized in that the chip structure comprises a top cover layer and a runner layer, and is sealed by adopting a lamination technology; the chip comprises a nucleic acid extraction and purification unit, a reactant mixing unit and a nucleic acid isothermal amplification and detection unit; the nucleic acid detection method is based on a real-time fluorescent nucleic acid amplification detection technology.
The high-throughput chip is characterized in that the nucleic acid extraction and purification unit comprises a pre-stored sample liquid chamber, a nucleic acid cleaning liquid chamber, a nucleic acid eluent chamber, a silica gel film and a waste liquid chamber; the sample liquid, the cleaning liquid and the eluent are automatically and sequentially released through rotation speed control, wherein firstly the sample liquid flows through the silica gel film, nucleic acid in the sample liquid is adsorbed on the silica gel film, secondly the cleaning liquid flows through the silica gel film to clean impurities such as protein on the silica gel film, the impurities flow into a waste liquid chamber through steering control fluid, finally the nucleic acid extract flows through the silica gel film to elute the nucleic acid in the waste liquid chamber, and the nucleic acid extract flows into a chamber opposite to the waste liquid chamber through steering control fluid.
The nucleic acid purification concentration unit is characterized by comprising compact silica gel particles, wherein the compact silica gel particles have a larger adsorption area, and DNA flowing through the compact silica gel particles can be adsorbed on a silica gel film.
The reactant mixing unit is characterized by comprising an LAMP amplification reaction liquid chamber, an eluted nucleic acid solution chamber and a mixing chamber, wherein the LAMP amplification reaction liquid and the eluted nucleic acid solution flow out simultaneously through rotation speed control and flow into the mixing chamber after complete mixing through a snake-shaped flow channel.
The composite chip for nucleic acid amplification and detection is characterized in that the nucleic acid isothermal amplification and detection unit comprises a plurality of isothermal amplification chambers and a plurality of flow channels; the temperature of the isothermal amplification chamber is kept at 65-75 ℃ by contacting with an external isothermal control board, and the cyclic amplification condition is reflected by placing the chip in a fluorescence detection platform and detecting the change of fluorescence intensity in real time.
The high-throughput chip is characterized in that the nucleic acid detection steps can be summarized as follows:
(a) Respectively adding 20-30 mu L of cracked sample liquid into the sample liquid chamber by using a pipetting gun, adding 50-60 mu L of cleaning liquid into the cleaning liquid chamber, adding 20-30 mu L of eluent into the eluent chamber, and adding 20-30 mu L of LAMP amplification reaction liquid into the LAMP amplification reaction liquid chamber;
(b) Performing counter-clockwise centrifugation at 3000-5000RPM, slowly suspending, and allowing the sample liquid to flow into the waste liquid chamber;
(c) Performing counter-clockwise centrifugation at 3000-5000RPM, decelerating at 30000RPM/s, and filling the flow channel with cleaning liquid;
(d) Performing counter-clockwise 3000-5000RPM rotation, and allowing cleaning liquid to flow into the waste liquid chamber;
(e) Rotating at 3000-5000RPM clockwise, decelerating at 30000-50000RPM/s, and filling the pipeline with eluent;
(f) Clockwise rotating at 3000-5000RPM, and flowing the eluted solution into the right side chamber;
(g) Rotating at 3000-5000RPM clockwise, decelerating at 30000-50000RPM/s, and filling the pipeline with LAMP amplification reaction solution and eluted nucleic acid solution;
(h) Rotating clockwise at 3000-5000RPM, mixing LAMP amplification reaction solution and eluent in a serpentine flow channel, and flowing into a mixing chamber;
(i) Flowing the mixed solution into a liquid separation chamber, performing high-speed centrifugation twice, and uniformly separating the mixed solution into each detection chamber and mixing the mixed solution with the primer;
(j) Controlling the temperature of the nucleic acid isothermal amplification and detection unit to be 65-75 ℃, observing the fluorescence intensity of the reactant under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
The chip for detecting nucleic acid is characterized in that the materials of the top cover layer and the runner layer are not limited to common medical plastics such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) and ABS; the top cover layer and the runner layer are not limited to various molding modes such as compression molding thermoplastic molding.
Compared with the prior art, the invention has the following advantages:
firstly, the invention realizes the integration of nucleic acid purification and amplification, integrates two steps of operations on the same chip, and improves the nucleic acid detection efficiency; secondly, the chip can enable the fluid to be released sequentially through the design of the valve, so that the detection process can be automatically controlled through the rotating speed and the rotating direction of the centrifugal machine, automatic detection is realized, manual operation can be omitted, and the cost of nucleic acid detection is further reduced; meanwhile, in the process of realizing nucleic acid purification and amplification detection, only one external force is needed for controlling the centrifugal force, and the control system is simple and suitable for mass production; in addition, the design can realize detection of a plurality of items, different primers are embedded in the detection chamber in advance, and high-throughput detection of the plurality of items can be realized.
Drawings
FIG. 1 is a schematic structural diagram of a centrifugal fully integrated microfluidic chip for high-throughput nucleic acid detection;
FIG. 2 is a schematic view of the top cover layer (1) in FIG. 1;
fig. 3 is a schematic structural diagram of the runner layer (2) in fig. 1.
Detailed Description
The following describes embodiments of the present invention in detail. It should be emphasized that the following description is merely exemplary in nature and is in no way intended to limit the scope of the invention or its applications.
It will be understood that when an element is referred to as being "mounted" 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 both a fixing action and a coupling or communication action.
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 merely for convenience in describing embodiments of the invention and to simplify the description by referring to the figures, rather than to indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless explicitly defined otherwise.
As shown in FIG. 1, the nucleic acid detecting chip comprises a top cover layer (1) and a flow channel layer (2).
As shown in fig. 2, the top cover layer (1) comprises a sample liquid chamber vent hole (3), a cleaning liquid chamber vent hole (9), an eluent chamber vent hole (4), an LAMP amplification reaction liquid chamber vent hole (5), an eluted nucleic acid solution chamber vent hole (6), a mixing chamber vent hole (7), an amplification chamber vent hole (8), a waste liquid chamber vent hole (10) and a plurality of flow channels which are communicated with each other.
As shown in fig. 3, the flow channel layer comprises a sample liquid chamber (11), an eluent chamber (12), an LAMP amplification reaction liquid (13), an eluted nucleic acid solution chamber (14), a mixed liquid chamber (15), an amplification detection chamber (16), a cleaning liquid chamber (17), a silica gel membrane (18), a waste liquid chamber (19) and a plurality of flow channels which are mutually communicated.
The materials of the top cover layer and the runner layer are common medical plastics, such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polymethyl methacrylate (PMMA) and ABS, and various plastic forming modes, such as compression molding, thermoplastic forming, injection molding and the like are adopted; for example, a method of injection molding is adopted, a mold is processed in advance, then polypropylene (PP) material is melted in a constant-temperature charging barrel (220-280 ℃), then the melted PP material is injected into the mold under pressure (800-140 MPa), and then the mold is subjected to pressure maintaining and cooling molding. The nucleic acid extraction unit in the chip uses a machining mode to process a microarray with a certain shape in the cavity, and the effect of adsorbing nucleic acid can be achieved after surface treatment.
The nucleic acid detection step specifically comprises:
(a) 20 μl of the lysed sample solution is added to the sample solution chamber (11) through the sample solution chamber vent holes (3) by using a pipette, 50 μl of the washing solution is added to the washing solution chamber (17) through the washing solution chamber vent holes (9), 20 μl of the washing solution is added to the washing solution chamber (12) through the washing solution chamber vent holes (6), and 20 μl of the LAMP amplification reaction solution is added to the LAMP amplification reaction solution chamber (16) through the LAMP amplification reaction solution chamber vent holes (5);
(b) Performing a 3000RPM counter-clockwise centrifugation, slowly pausing the sample flow to the waste chamber (19);
(c) Performing 3000RPM anticlockwise centrifugation, decelerating at 30000RPM/s, and filling the flow channel with cleaning liquid;
(d) Performing a counter-clockwise 3000RPM rotation, the cleaning liquid flowing into the waste liquid chamber (19);
(e) Rotating at 3000RPM clockwise, decelerating at 30000RPM/s, and filling the pipeline with eluent;
(f) Clockwise rotation at 3000RPM was performed and the eluted solution flowed into the right chamber (14);
(g) Rotating at 3000RPM clockwise, decelerating at 30000RPM/s, and filling the pipeline with the LAMP amplification reaction solution and the eluted nucleic acid solution;
(h) The LAMP amplification reaction solution and the eluent are mixed in a serpentine flow channel and flow into a mixing chamber (15) by rotating clockwise at 3000 RPM;
(i) Flowing the mixed solution into a liquid separation chamber, performing high-speed centrifugation twice, and uniformly separating the mixed solution into each detection chamber and mixing the mixed solution with the primer;
(j) The temperature of the nucleic acid isothermal amplification and detection unit is controlled to be 65 ℃, and the fluorescence intensity of the detection chamber (16) is observed under a microscope, and the concentration of the nucleic acid is quantitatively analyzed.
The background section of the present invention may contain background information about the problems or environments of the present invention and is not necessarily descriptive of the prior art. Accordingly, inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a further detailed description of the invention in connection with specific/preferred embodiments, and it is not intended that the invention be limited to such description. It will be apparent to those skilled in the art that several alternatives or modifications can be made to the described embodiments without departing from the spirit of the invention, and these alternatives or modifications should be considered to be within the scope of the invention. In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples," etc., means 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, schematic representations of the above terms are not necessarily directed 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. Those skilled in the art may combine and combine the features of the different embodiments or examples described in this specification and of the different embodiments or examples 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 invention as defined by the appended claims.
Claims (8)
1. The centrifugal fully-integrated high-flux nucleic acid detection micro-fluidic chip is characterized in that the chip structure comprises a top cover layer and a flow channel layer, and the chip is sealed by adopting a lamination technology; the chip comprises a nucleic acid extraction and purification unit, a reactant mixing unit and a nucleic acid isothermal amplification and detection unit.
2. The centrifugal fully-integrated high-throughput nucleic acid detection microfluidic chip according to claim 1, wherein the nucleic acid extraction and purification unit comprises a pre-stored sample liquid chamber, a nucleic acid cleaning liquid chamber, a nucleic acid eluent chamber, a silica gel film, and a waste liquid chamber; the sample liquid, the cleaning liquid and the eluent are automatically and sequentially released through rotation speed control, wherein firstly the sample liquid flows through the silica gel film, nucleic acid in the sample liquid is adsorbed on the silica gel film, secondly the cleaning liquid flows through the silica gel film to clean impurities such as protein on the silica gel film, the impurities flow into a waste liquid chamber through steering control fluid, finally the nucleic acid extract flows through the silica gel film to elute the nucleic acid in the waste liquid chamber, and the nucleic acid extract flows into a chamber opposite to the waste liquid chamber through steering control fluid.
3. The nucleic acid extraction and purification unit according to claim 1 or 2, comprising dense silica gel particles having a large adsorption area, wherein the DNA flowing therethrough can be adsorbed on the silica gel membrane.
4. The centrifugal fully-integrated high-throughput nucleic acid detection microfluidic chip of claim 1, wherein the reactant mixing unit comprises a LAMP amplification reaction solution chamber, an eluted nucleic acid solution chamber and a mixing chamber, wherein the LAMP amplification reaction solution and the eluted nucleic acid solution flow out simultaneously through rotation speed control and flow into the mixing chamber after complete full mixing through a serpentine flow channel.
5. The centrifugal fully-integrated high-throughput nucleic acid detection microfluidic chip of claim 1, wherein the nucleic acid isothermal amplification and detection unit comprises a plurality of isothermal amplification chambers and a plurality of flow channels; the temperature of the isothermal amplification chamber is kept at 65-75 ℃ by contacting with an external isothermal control board, and the cyclic amplification condition is reflected by placing the chip in a fluorescence detection platform and detecting the change of fluorescence intensity in real time.
6. The centrifugal fully-integrated high-throughput nucleic acid detection microfluidic chip of claims 1-5, wherein the nucleic acid detection method is based on a real-time fluorescent nucleic acid amplification detection technique.
7. The centrifugal fully-integrated high-throughput nucleic acid detection microfluidic chip of claims 1-6, wherein the nucleic acid detection steps can be summarized as:
(a) Respectively adding 20-30 mu L of cracked sample liquid into the sample liquid chamber by using a pipetting gun, adding 50-60 mu L of cleaning liquid into the cleaning liquid chamber, adding 20-30 mu L of eluent into the eluent chamber, and adding 20-30 mu L of LAMP amplification reaction liquid into the LAMP amplification reaction liquid chamber;
(b) Performing counter-clockwise centrifugation at 3000-5000RPM, slowly suspending, and allowing the sample liquid to flow into the waste liquid chamber;
(c) Performing counter-clockwise centrifugation at 3000-5000RPM, decelerating at 30000RPM/s, and filling the flow channel with cleaning liquid;
(d) Performing counter-clockwise 3000-5000RPM rotation, and allowing cleaning liquid to flow into the waste liquid chamber;
(e) Rotating at 3000-5000RPM clockwise, decelerating at 30000-50000RPM/s, and filling the pipeline with eluent;
(f) Clockwise rotating at 3000-5000RPM, and flowing the eluted solution into the right side chamber;
(g) Rotating at 3000-5000RPM clockwise, decelerating at 30000-50000RPM/s, and filling the pipeline with LAMP amplification reaction solution and eluted nucleic acid solution;
(h) Rotating clockwise at 3000-5000RPM, mixing LAMP amplification reaction solution and eluent in a serpentine flow channel, and flowing into a mixing chamber;
(i) Flowing the mixed solution into a liquid separation chamber, performing high-speed centrifugation twice, and uniformly separating the mixed solution into each detection chamber and mixing the mixed solution with the primer;
(j) Controlling the temperature of the nucleic acid isothermal amplification and detection unit to be 65-75 ℃, observing the fluorescence intensity of the reactant under a microscope, and quantitatively analyzing the concentration of the nucleic acid.
8. The centrifugal fully-integrated high-flux nucleic acid detection microfluidic chip according to claim 1, wherein materials of the top cover layer and the runner layer are not limited to common medical plastics such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC), ABS and the like; the top cover layer and the runner layer are not limited to various molding modes such as compression molding thermoplastic molding.
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| CN117384750A (en) * | 2023-12-06 | 2024-01-12 | 博奥生物集团有限公司 | Fully integrated digital nucleic acid analysis cartridge |
| CN117384750B (en) * | 2023-12-06 | 2024-03-08 | 博奥生物集团有限公司 | Fully integrated digital nucleic acid analysis cartridge |
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