CN113817577B - Liquid drop micro-fluidic chip with automatic nucleic acid extraction and purification functions - Google Patents
Liquid drop micro-fluidic chip with automatic nucleic acid extraction and purification functions Download PDFInfo
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Abstract
The invention provides a liquid drop micro-fluidic chip with an automatic nucleic acid extraction and purification function, which comprises a magnetic bead method nucleic acid extraction and purification area, a reagent sample mixing area and a micro-liquid drop generation area, wherein each area is communicated by a micro-channel; the magnetic bead method nucleic acid extraction and purification area comprises a sample storage cavity, an eluent storage cavity, a magnet, a rectangular magnetic bead deflection flow channel, a first negative pressure cavity, a differential pressure elution flow channel and a magnetic separation flow channel; the reagent mixing area comprises a T-shaped three-way flow passage, a reagent cavity and an asymmetric mixer; the micro-droplet generation area comprises a cross-shaped four-way runner, an oil storage cavity and a second negative pressure cavity. The invention integrates nucleic acid extraction and purification, reagent mixing and micro-droplet generation on a chip, can realize automatic extraction and droplet generation of nucleic acid, provides a simple and feasible operation scheme for pretreatment and digital analysis of nucleic acid, and is particularly suitable for on-site rapid detection of infectious diseases.
Description
Technical Field
The invention relates to the technical field of microfluidic biochips, in particular to a droplet microfluidic chip with an automatic nucleic acid extraction and purification function.
Background
The existing nucleic acid extraction and purification modes mainly comprise phenol chloroform extraction, centrifugal column purification and magnetic bead nucleic acid separation technology. Wherein, phenol chloroform extraction method uses phenol to denature protein, sodium dodecyl sulfonate to crack cell membrane, and protein or polypeptide is digested under the action of protease K, EDTA to free DNA from nucleoprotein. And obtaining purified nucleic acid after precipitation, extraction, multiple times of washing and other processes. However, reagents such as phenol and chloroform have high toxicity, low nucleic acid recovery rate, high handling difficulty, and difficulty in performing a trace amount of operations. The centrifugal column and the magnetic bead method are used for extracting and purifying nucleic acid, and the principle that a silica gel carrier or nano magnetic beads only have strong affinity and adsorption force on nucleic acid but do not adsorb other biochemical components is utilized, so that a nucleic acid purification product with better quality can be obtained at low cost, and the two methods are main stream extraction methods in the current market. However, the above extraction method still requires multiple steps, and the extraction and purification process is very complicated.
In order to simplify the extraction and purification process, a plurality of mainstream molecular diagnostic companies at home and abroad sequentially develop a plurality of magnetic bead-based automated nucleic acid extraction devices, such as a GeneRotex-series nucleic acid extractor, an MGISP automated nucleic acid extraction system manufactured by Hua Dazhi, a KingFisher Presto nucleic acid extraction system developed by Thermol Fisher, a MagNA-series full-automatic nucleic acid purifier manufactured by Roche, and the like, which are proposed by Tianlong technology limited companies. The full-automatic nucleic acid extraction and purification equipment replaces heavy manual operation through system integration, and meanwhile, the detection accuracy and reliability are remarkably improved. However, these instruments and devices only have a single function of nucleic acid extraction and purification, and cannot integrate functions of sample addition, reagent mixing and the like into one device or one system, which is not beneficial to on-site rapid real-time detection and analysis such as instant detection, accompanying diagnosis and the like. Therefore, it is important to establish a detection method with high efficiency, high integration level, less sample space exposure, high detection sensitivity and accurate and reliable result.
Disclosure of Invention
In order to solve the above problems, it is necessary to provide a droplet microfluidic chip having an automatic nucleic acid extraction and purification function.
The invention provides a liquid drop micro-fluidic chip with an automatic nucleic acid extraction and purification function, which comprises a magnetic bead method nucleic acid extraction and purification area, a reagent sample mixing area and a micro-liquid drop generation area, wherein each area is communicated by a micro-channel;
the magnetic bead method nucleic acid extraction and purification area comprises a sample storage cavity, an eluent storage cavity, a magnet, a rectangular magnetic bead deflection flow channel, a first negative pressure cavity, a differential pressure elution flow channel and a magnetic separation flow channel;
the sample storage cavity and the eluent storage cavity are arranged on one side of the short side of the rectangular magnetic bead deflection flow channel and are communicated with the rectangular magnetic bead deflection flow channel through a micro flow channel; the first negative pressure cavity and the differential pressure elution flow channel are arranged on one side of the other short side of the rectangular magnetic bead deflection flow channel and are communicated with the rectangular magnetic bead deflection flow channel through a micro flow channel; the outlet of the pressure difference elution flow passage is communicated with the magnetic separation flow passage; the magnet is arranged between the rectangular magnetic bead deflection flow channel and the magnetic separation flow channel;
the reagent mixing area comprises a T-shaped three-way flow passage, a reagent cavity and an asymmetric mixer;
the first inlet of the T-shaped three-way runner is communicated with the reagent cavity, the second inlet of the T-shaped three-way runner is communicated with the magnetic separation runner, and the outlet of the T-shaped three-way runner is communicated with the inlet of the asymmetric mixer;
the micro-droplet generation area comprises a cross-shaped four-way runner, an oil storage cavity and a second negative pressure cavity;
the first inlet of the cross four-way runner is communicated with the outlet of the asymmetric mixer, the second inlet and the third inlet which are opposite are communicated with the oil storage cavity, and the outlet runner is communicated with the second negative pressure cavity.
Based on the above, the sample storage chamber and the first negative pressure chamber are located on the same side, and the eluent storage chamber and the differential pressure elution flow channel are located on the same side.
Based on the above, the micro-channel is processed by a milling machine or a soft lithography process.
Based on the above, the width ratio of the high-pressure narrow flow channel and the low-pressure wide flow channel of the pressure difference elution flow channel is between 1:1.5 and 1:15.
Based on the above, the geometric dimension of the cross-shaped four-way runner is selected to be 40-150 μm high and 30-200 μm wide.
Based on the above, the micro flow channel communicated with the oil storage cavity is symmetrically designed and surrounds the magnetic bead method nucleic acid extraction and purification area and the reagent sample mixing area, and the magnetic bead method nucleic acid extraction and purification area is designed below the reagent sample mixing area.
Based on the above, the first negative pressure cavity and the second negative pressure cavity are respectively provided with an injection pump.
The invention also provides application of the liquid drop micro-fluidic chip with the automatic nucleic acid extraction and purification function, and the liquid drop micro-fluidic chip with the automatic nucleic acid extraction and purification function is applied to digital RT-PCR, digital LAMP, digital RPA, digital CRISPR, single cell analysis or single molecule detection.
The beneficial effects of the invention are as follows: by integrating nucleic acid extraction and purification, reagent mixing and micro-droplet generation on one chip, the automatic extraction and droplet generation of nucleic acid can be realized, a simple and feasible operation scheme is provided for pretreatment and digital analysis of nucleic acid, and the method is particularly suitable for on-site rapid detection of infectious diseases.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
fig. 1 is a schematic structural diagram of a microfluidic chip according to embodiment 1 of the present invention.
FIG. 2 is a schematic diagram of a magnetic adsorption bead for a magnet according to example 1 of the present invention.
FIG. 3 is a graph showing the results of detection of a negative specimen by droplet digital RT-PCR using SARS-CoV-2 according to example 2 of the present invention.
FIG. 4 is a graph showing the results of the detection of a positive specimen by droplet digital RT-PCR in example 2 SARS-CoV-2 of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those described herein, and therefore the scope of the present invention is not limited to the specific embodiments disclosed below.
Example 1
As shown in fig. 1 and fig. 2, the invention provides a droplet microfluidic chip with an automatic nucleic acid extraction and purification function, which comprises a magnetic bead method nucleic acid extraction and purification area, a reagent sample mixing area and a micro droplet generation area, wherein each area is communicated by a micro flow channel;
the magnetic bead method nucleic acid extraction and purification area comprises a sample storage cavity 1, an eluent storage cavity 2, a first negative pressure cavity 3, a differential pressure elution flow channel 4, a rectangular magnetic bead deflection flow channel 5, a magnet 6 and a magnetic separation flow channel 7; the sample storage cavity 1 and the eluent storage cavity 2 are arranged on one side of the short side of the rectangular magnetic bead deflection flow channel 5 and are communicated with the rectangular magnetic bead deflection flow channel 5 through a micro flow channel; the first negative pressure cavity 3 and the differential pressure elution flow channel 4 are arranged on one side of the other short side of the rectangular magnetic bead deflection flow channel 5 and are communicated with the rectangular magnetic bead deflection flow channel 5 through a micro flow channel; the outlet of the pressure difference elution flow passage 4 is communicated with the magnetic separation flow passage 7; the magnet 6 is arranged between the rectangular magnetic bead deflection flow channel 5 and the magnetic separation flow channel 7 and respectively plays a role in deflecting magnetic beads and magnetic particle clusters 71;
the reagent mixing area comprises a reagent cavity 8, a T-shaped three-way runner 9 and an asymmetric mixer 10; the first inlet of the T-shaped three-way runner 9 is communicated with the reagent cavity 8, the second inlet is communicated with the magnetic separation runner 7, and the outlet is communicated with the inlet of the asymmetric mixer 10;
the micro-droplet generation area comprises a cross four-way runner 11, an oil storage cavity 12 and a second negative pressure cavity 13; the first inlet of the cross four-way runner 11 is communicated with the outlet of the asymmetric mixer 10, the second and third inlets are communicated with the oil storage cavity 12, and the outlet runner is communicated with the second negative pressure cavity 13. The mixed reagent flowing out of the asymmetric mixer 10 generates a large number of water-in-oil droplets under the flow focusing action of the droplet generation oil flowing in from the second inlet and the third inlet of the cross four-way channel 11. The second negative pressure cavity 13 can be used as a droplet storage pool for storing monodisperse droplets for subsequent testing.
Preferably, the sample storage chamber 1 and the first negative pressure chamber 3 are located on the same side, and the eluent storage chamber 2 and the differential pressure elution flow channel 4 are located on the same side. The micro flow channel communicated with the oil storage cavity 12 is symmetrically designed and surrounds the magnetic bead method nucleic acid extraction and purification area and the reagent sample mixing area, and the magnetic bead method nucleic acid extraction and purification area is designed below the reagent sample mixing area.
Preferably, the micro-channel is processed by a milling machine or a soft lithography process.
Preferably, the width ratio of the high-pressure narrow flow channel and the low-pressure wide flow channel of the differential pressure elution flow channel 4 is between 1:1.5 and 1:15.
Preferably, the geometric dimension of the cross-shaped four-way runner 11 is selected to be 40-150 μm in height and 30-200 μm in width.
Preferably, a syringe pump is disposed in each of the first negative pressure chamber 3 and the second negative pressure chamber 13.
The application method of the invention is as follows:
(1) The first negative pressure chamber 3 and the second negative pressure chamber 13 are respectively communicated with the syringe pump to provide stable negative pressure. Placing a magnet 6 at a position between the magnetic bead deflection flow channel 5 and the magnetic separation flow channel 7; adding the nucleic acid extract and magnetic beads into the throat swab storage liquid, and fully and uniformly mixing.
(2) The magnetic bead mixture, the eluent, the reaction reagent and the droplet generation oil are respectively added to the sample storage chamber 1, the eluent storage chamber 2, the reagent chamber 8 and the oil storage chamber 12.
(3) Both syringe pumps are running simultaneously and all solutions flow into the chip simultaneously. When the magnetic bead mixed solution and the eluent flow through the magnetic bead deflection flow channel 5 at the same time, the magnetic beads in the magnetic bead mixed solution deflect under the action of the magnet 6, and are transferred to the eluent from the sample solution and flow through the pressure difference elution flow channel 4. Subjecting the magnetic beads adsorbed with nucleic acid to rapid high-pressure and slow low-pressure elution to desorb the nucleic acid; when the magnetic beads flow through the magnetic separation flow channel 7, all the magnetic beads are adsorbed on the side wall of the flow channel of the magnetic separation flow channel 7 under the magnetic force of the magnet 6 to form a magnetic particle group 71; the desorbed nucleic acid solution is converged with the reaction reagent in a T-shaped three-way flow channel 9 and enters an asymmetric mixer 10 for full mixing; the mixed solution flows through the cross four-way flow channel 11 and generates water-in-oil droplets with droplet generation oil under the action of flow focusing.
(4) The droplets may be collected in the second negative pressure chamber 13 for subsequent digital analysis of the nucleic acids.
Example 2
The embodiment provides application of a liquid drop micro-fluidic chip with an automatic nucleic acid extraction and purification function, which is applied to digital RT-PCR, digital LAMP, digital RPA, digital CRISPR, single-cell analysis or single-molecule detection.
Application example of digital RT-PCR detection of SARS-CoV-2 (COVID-19)
SARS-CoV-2 (COVID-19) was pretreated and droplet generation was performed using the chip of example 1. Micro-droplets were subjected to droplet statistics and data analysis using a Bio-Rad QX200 digital RT-PCR system. The detailed operation process is as follows:
s1: SARS-CoV-2 (COVID-19) target sequence selection, sample preparation and PCR primer design.
Sample information
RT-PCR primers and probes
S2: chip preparation
The first negative pressure cavity 3 and the second negative pressure cavity 13 are respectively communicated with a syringe pump, the volume flow of the syringe pump of the first negative pressure cavity 3 is 50 mu L/min, and the volume flow of the syringe pump of the second negative pressure cavity 13 is 40 mu L/min. Placing a magnet 6 in a proper position between the magnetic bead deflection flow channel 5 and the magnetic separation flow channel 7; adding 10 mg magnetic beads into the throat swab storage liquid of a 1 mL normal person, and fully and uniformly mixing; meanwhile, 10 mg magnetic beads are added into a SARS-CoV-2 sequence solution synthesized by 10 fM to simulate the detection of a positive specimen.
S3: SARS-CoV-2 detection procedure
100. Mu.L of the magnetic bead-nucleic acid premix, 100. Mu.L of the eluent, 30. Mu.L of the digital RT-PCR reaction reagent and 100. Mu.L of the droplet-forming oil were added to the sample storage chamber 1, the eluent storage chamber 2, the reagent chamber 8 and the oil storage chamber 12, respectively.
Both syringe pumps are operated simultaneously and all solutions flow into the chip simultaneously. When the magnetic bead mixed solution and the eluent flow in a laminar flow mode in the magnetic bead deflection flow channel 5, the magnetic beads in the magnetic bead mixed solution deflect under the action of the magnet 6 and are transferred into the eluent, the magnetic beads adsorbed with nucleic acid are subjected to differential pressure elution process of high speed and low speed in sequence to desorb the nucleic acid, and the nucleic acid is adsorbed on the side wall of the flow channel under the magnetic force of the magnet 6 to form a magnetic particle group 71; the desorbed nucleic acid solution is converged with the digital RT-PCR reaction reagent in a T-shaped three-way flow channel 9 and enters an asymmetric mixer 10 for full mixing; the mixed solution flows through the cross-shaped four-way flow channel 11 and generates 2-3 ten thousand water-in-oil droplets with droplet generation oil under the flow focusing action, and the droplets are stored in the second negative pressure cavity.
1.5 After min, all syringe pumps were turned off. The droplets were collected in 96-well plates and fluorescent signals of the droplets were collected one by one using a Droplet Reader (QX 200 Droplet Reader) of a Bio-Rad QX200 digital RT-PCR system.
S4: data analysis
When the liquid drop contains the target nucleic acid sequence, a fluorescent signal is generated, and when the liquid drop does not contain the target sequence, the fluorescent signal is not generated, so that the number of the fluorescent liquid drops can represent the content of the target nucleic acid in the sample. As shown in FIG. 3, the throat swab of a normal person has a detection result that neither droplet generates fluorescence (negative signal), and the droplet is a negative result; as shown in FIG. 4, the detection result of the simulated positive sample shows that fluorescent droplets (positive signals) are obviously distinguished from background droplets (negative signals), and the detection concentration of the positive sample is 637 copies/. Mu.L.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (4)
1. A liquid drop micro-fluidic chip with a nucleic acid automatic extraction and purification function is characterized in that: the method comprises a magnetic bead method nucleic acid extraction and purification area, a reagent sample mixing area and a micro-droplet generation area, wherein each area is communicated by a micro-channel;
the magnetic bead method nucleic acid extraction and purification area comprises a sample storage cavity, an eluent storage cavity, a magnet, a rectangular magnetic bead deflection flow channel, a first negative pressure cavity, a differential pressure elution flow channel and a magnetic separation flow channel;
the sample storage cavity and the eluent storage cavity are arranged on one side of the short side of the rectangular magnetic bead deflection flow channel and are communicated with the rectangular magnetic bead deflection flow channel through a micro flow channel; the first negative pressure cavity and the differential pressure elution flow channel are arranged on one side of the other short side of the rectangular magnetic bead deflection flow channel and are communicated with the rectangular magnetic bead deflection flow channel through a micro flow channel; the outlet of the pressure difference elution flow passage is communicated with the magnetic separation flow passage; the magnet is arranged between the rectangular magnetic bead deflection flow channel and the magnetic separation flow channel; the sample storage cavity and the first negative pressure cavity are positioned at the same side, and the eluent storage cavity and the differential pressure elution flow channel are positioned at the same side; the width ratio of the high-pressure narrow flow channel to the low-pressure wide flow channel of the pressure difference elution flow channel is between 1:1.5 and 1:15;
the reagent mixing area comprises a T-shaped three-way flow passage, a reagent cavity and an asymmetric mixer;
the first inlet of the T-shaped three-way runner is communicated with the reagent cavity, the second inlet of the T-shaped three-way runner is communicated with the magnetic separation runner, and the outlet of the T-shaped three-way runner is communicated with the inlet of the asymmetric mixer;
the micro-droplet generation area comprises a cross-shaped four-way runner, an oil storage cavity and a second negative pressure cavity;
the first inlet of the cross four-way runner is communicated with the outlet of the asymmetric mixer, the second inlet and the third inlet which are opposite are communicated with the oil storage cavity, and the outlet runner is communicated with the second negative pressure cavity;
the first negative pressure cavity and the second negative pressure cavity are respectively provided with an injection pump;
the using method of the liquid drop micro-fluidic chip comprises the following steps:
(1) Communicating the first negative pressure cavity and the second negative pressure cavity with a syringe pump respectively to provide stable negative pressure; placing the magnet in a position between the rectangular magnetic bead deflection flow channel and the magnetic separation flow channel; adding the nucleic acid extract and the magnetic beads into the throat swab storage liquid, and fully and uniformly mixing;
(2) Adding a magnetic bead mixed solution, an eluent, a reaction reagent and liquid drop generated oil into the sample storage cavity, the eluent storage cavity, the reagent cavity and the oil storage cavity respectively;
(3) Two injection pumps are operated simultaneously, and all solutions flow into the chip simultaneously;
when the magnetic bead mixed solution and the eluent flow through the rectangular magnetic bead deflection flow channel at the same time, the magnetic beads in the magnetic bead mixed solution deflect under the action of the magnet, are transferred from the sample solution to the eluent and flow through the pressure difference elution flow channel; subjecting the magnetic beads adsorbed with nucleic acid to rapid high-pressure and slow low-pressure elution to desorb the nucleic acid;
when the magnetic beads flow through the magnetic separation flow channel, all the magnetic beads are adsorbed on the side wall of the flow channel of the magnetic separation flow channel under the magnetic force of the magnet to form magnetic particle clusters; the desorbed nucleic acid solution is converged with the reaction reagent in the T-shaped three-way flow channel and enters the asymmetric mixer for full mixing; the mixed solution flows through the cross four-way flow channel and generates water-in-oil droplets with droplet generation oil under the flow focusing action;
(4) Droplets may be collected in the second negative pressure chamber for subsequent digital RT-PCR, digital LAMP, digital RPA, digital CRISPR, single cell analysis or single molecule detection.
2. The droplet microfluidic chip with automatic nucleic acid extraction and purification function according to claim 1, wherein: and the micro-flow channel is processed by a milling machine or a soft lithography process.
3. The droplet microfluidic chip with automatic nucleic acid extraction and purification function according to claim 1, wherein: the geometric dimension of the cross-shaped four-way runner is selected to be 40-150 mu m in height and 30-200 mu m in width.
4. The droplet microfluidic chip with automatic nucleic acid extraction and purification function according to claim 1, wherein: the micro-flow channel communicated with the oil storage cavity is symmetrically designed and surrounds the magnetic bead method nucleic acid extraction and purification area and the reagent sample mixing area, and the magnetic bead method nucleic acid extraction and purification area is designed below the reagent sample mixing area.
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| CN114308155B (en) * | 2021-12-23 | 2023-05-12 | 广东工业大学 | Microfluidic chip and digital fluorescent quantitative PCR (polymerase chain reaction) amplification instrument |
| CN115487881A (en) * | 2022-09-19 | 2022-12-20 | 英诺维尔智能科技（苏州）有限公司 | Micro-fluidic protein purification chip |
| 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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