CN115353949A - Centrifugal multiple digital PCR micro-droplet generation chip - Google Patents
Centrifugal multiple digital PCR micro-droplet generation chip Download PDFInfo
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- CN115353949A CN115353949A CN202211043281.XA CN202211043281A CN115353949A CN 115353949 A CN115353949 A CN 115353949A CN 202211043281 A CN202211043281 A CN 202211043281A CN 115353949 A CN115353949 A CN 115353949A
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Abstract
The invention provides a centrifugal multiple digital PCR micro-droplet generation chip, which comprises: the dish body, the inside of dish body is provided with a plurality of droplet generation units that mutually independent and sealed, the droplet generation unit is including reaction substrate pond, micro-fluidic channel, micro-droplet collecting tank, oil bath linking channel and the oil bath that communicates in proper order, the center of dish body is provided with the axis mounting hole, reaction substrate pond, oil bath, micro-droplet collecting tank are along keeping away from the direction of axis mounting hole sets gradually, seted up on the dish body with the application of sample hole of reaction substrate pond intercommunication and with the tank filler sleeve of oil bath intercommunication. The invention adopts the multi-flux stepped emulsification principle driven by centrifugal force to generate multiple single dispersed volume droplets, realizes the rapid division, multiple and automation of the solution of an amplification system, realizes the multi-flux generation of nano-grade droplets, has more efficient and simple sample zoning speed, and can obtain more uniform and stable micro-droplets.
Description
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a centrifugal multiple digital PCR micro-droplet generation chip.
Background
Before the traditional PCR amplification, a large reaction system is subjected to micro-droplet treatment, and a micro-droplet forming device is utilized to divide the reaction system containing nucleic acid molecules into thousands of water-in-oil micro-droplets, so that thousands of independent PCR reaction systems are formed. Compared with the traditional digital PCR, the micro-fluidic chip technology and the PCR technology combined micro-droplet type digital PCR technology have the advantages that the sensitivity and the precision are greatly improved, the operation is simple, and the sample consumption is low.
In addition, due to the reaction characteristics of the droplet-type digital PCR, the required sample size is very small, the probability that a single DNA template appears in a single droplet system without being detected is very low, and the method is a great progress compared with the second generation real-time fluorescence quantitative PCR. The micro-drop digital PCR has various irreplaceable advantages in the detection field, is particularly suitable for micro-DNA detection and quantification, is flexible to use, can adjust flux and sensitivity as required, and provides more reliable results for nucleic acid detection.
The droplet microfluidics technology is a technology which utilizes the fluid shear force of a continuous phase to destroy the surface tension of a discrete phase at a microscale and divides the discrete phase into nanoliter-level or even picoliter-level droplets. The liquid drop micro-fluidic chip has the advantages of small volume, high precision, complete separation between liquid drops and the like, and is a very excellent micro-reactor. Commonly used droplet generation methods include a "T" channel method, a flow focusing method, and a coaxial method. Under the drive of stable positive pressure or negative pressure, the 3 liquid drop generating methods have good consistency and generating rate.
The micro-drop digital PCR technology combining the micro-fluidic technology and the PCR technology is a breakthrough technology for detecting and quantifying nucleic acid, which is rapidly developed in recent years. To realize the micro-drop digital nucleic acid amplification analysis, the formation of micro-drops is a key link for realizing the micro-drop digital PCR technology, a device capable of generating highly monodisperse liquid drops with high efficiency is required to be relied on, and the generation process of the liquid drops is required to be as simple as possible. The device is compatible with universal standard equipment, so that the complexity of the whole system can be reduced, and even the device can be provided for non-professional users to directly use. The centrifugal micro-fluidic technology is one of a plurality of micro-fluidic driving means, and has the advantages that the traditional complicated design of depending on external driving liquid flow is omitted, meanwhile, the centrifugal turntable has uniform stress in all directions, and the concurrent operation of a plurality of micro-fluidic chips/structures on the turntable can be supported.
At present, the centrifugal microfluidic technology is well integrated and perfected with basic liquid flow operation, multi-volume monodisperse droplets with different volumes are synchronously and efficiently produced by an oil-water two-phase step emulsification droplet generation technology through microfluidic control under the drive of centrifugal force, and a PCR reaction substrate (polymerase, deoxyribonucleotide triphosphate, a primer, a probe, a buffer solution and the like) and a detection target template which are mixed with the PCR reaction substrate are rapidly divided into a multi-volume microreactor single-layer array with a pL-nL volume on line to carry out subsequent PCR rapid amplification. The centrifugal microfluidic chip is also the most competitive form and carrier for the future market application of the microfluidic technology.
The number, the generation rate and the consistency of the liquid drops are key factors influencing the application of the liquid drop micro-technology, most researchers adopt a T-shaped channel method, a flow focusing method and a coaxial method to carry out array and parallel liquid drop preparation for realizing the generation of liquid drops with higher flux, although the generation rate is improved by the array structure, the size difference of the generated liquid drops is large due to the size and the flow rate error of each generated liquid drop structure, and the equal distribution of samples and the final precision and sensitivity are influenced. The other method is to adopt a droplet multistage segmentation method to divide the generated droplets into small droplets step by step so as to improve the generation rate, however, the processing difference exists in each micro-segmentation structure, so that the droplet segmentation is asymmetric, and the prepared droplets have large size difference. The two multi-flux droplet generation technologies have influence on the generation effect of the droplets due to overlarge size difference.
In recent years, step emulsification has become another important method for producing stable size droplets, and the main feature is that parallel large-scale nozzles, microfluidic channels and other arrays can be used simultaneously to achieve high-throughput droplet production. The conventional step emulsification method uses pressure or syringe pump driving to generate liquid droplets, and the whole device is heavy, complex and low in integration level. Also, droplets tend to accumulate at the nozzle, not only interfering with subsequent droplet formation, reducing the rate at which droplets are produced, but also leading to a broadening of the droplet size distribution.
The PCR micro-droplet generation chip in the prior art does not adopt a sealing structure, and can be influenced by other mixed factors. In addition, at present, domestic research mainly comprises flexible microtubes, but the flexible microtubes are very complicated to manufacture and need to be manufactured by manpower.
Disclosure of Invention
The invention provides a centrifugal multi-digital PCR micro-droplet generation chip, which is used for solving at least one technical problem.
To solve the above problems, as an aspect of the present invention, there is provided a centrifugal multiplex digital PCR micro-droplet generating chip comprising: the dish body, the inside of dish body is provided with a plurality of droplet generation units that mutually independent and sealed, the droplet generation unit is including reaction substrate pond, micro-fluidic channel, micro-droplet collecting tank, oil bath linking channel and the oil bath that communicates in proper order, the center of dish body is provided with the axis mounting hole, reaction substrate pond, oil bath, micro-droplet collecting tank are along keeping away from the direction of axis mounting hole sets gradually, seted up on the dish body with the application of sample hole of reaction substrate pond intercommunication and with the tank filler sleeve of oil bath intercommunication.
Preferably, the microfluidic channel extends in an arc.
Preferably, the microfluidic channel is L-shaped.
Preferably, the reaction substrate pool is fan-shaped in cross section.
Preferably, the micro-droplet collection pool is circular in cross section.
Preferably, the tray body is provided with a plurality of positioning holes.
The invention can pump liquid along the microfluidic channel by centrifugal force, does not need complex fluid driving equipment, can realize the driving of microfluid by only using a common motor, realizes the formation of liquid drops under the condition of not using any pump, simply and quickly obtains monodisperse liquid drops by using the action of density difference and centrifugal force, and can remove the generated liquid drops from the nozzle in the centrifugal process. Compared with other droplet generating devices, the device has the advantages of high flux of each nozzle, simple device and no dead volume.
The invention adopts the multi-flux stepped emulsification principle driven by centrifugal force to generate multiple single dispersed volume liquid drops, realizes the rapid division, multiple and automation of the solution of an amplification system, and realizes the multi-flux generation of nano-grade liquid drops. Compared with the traditional liquid drop generation technologies such as T-shaped crossing or flow focusing, and the like, which are used for generating homogeneous liquid drops, the sample zoning speed is more efficient and simpler, and more uniform and stable micro liquid drops can be obtained.
The invention utilizes the centrifugal force and the stepped emulsification principle to generate multiple single dispersed volume droplets, and repeatedly simulates and verifies the centrifugal force (rotating speed and rotating acceleration) and the microstructure size of the centrifugal multiple digital PCR micro-droplet generation chip, thereby realizing the possibility of synchronous working of the two modes.
The invention replaces the complex ultra-clean room processing flow with the micro-fluidic channel, greatly reduces the processing difficulty and the processing time, has good sample introduction capability, adopts a sealing structure to effectively avoid the influence caused by other mixed factors, saves the complex design of externally driving liquid flow in the past, has uniform stress of each centrifugal force, and can support the concurrent operation of a multi-unit structure.
Drawings
FIG. 1 schematically illustrates a perspective view of the present invention;
figure 2 schematically shows a schematic view of a droplet generation unit;
FIG. 3 schematically illustrates a cross-sectional view of the present invention 1;
FIG. 4 schematically illustrates a cross-sectional view of the present invention 2;
fig. 5 schematically shows a perspective view of the invention.
Reference numbers in the figures: 1. a tray body; 2. a reaction substrate pool; 3. a microfluidic channel; 4. a micro-droplet collection tank; 5. the oil pool is connected with the channel; 6. an oil sump; 7. a middle shaft mounting hole; 8. a sample application hole; 9. an oil filling hole; 10. And (7) positioning the holes.
Detailed Description
The following detailed description of embodiments of the invention, but the invention can be practiced in many different ways, as defined and covered by the claims.
As an aspect of the present invention, there is provided a centrifugal type multiple digital PCR micro-droplet generation chip, comprising: the disk body 1, the inside of disk body 1 is provided with a plurality of droplet generation units that mutually independent and sealed, and disk body 1 is discoid, has the independent droplet generation unit of a plurality of looks isostructures on the disk body 1, and each independent unit is encapsulated situation.
Further, the droplet generation unit comprises a reaction substrate pool 2, a micro-fluidic channel 3, a micro-droplet collection pool 4, an oil pool connecting channel 5 and an oil pool 6 which are sequentially communicated, a center shaft mounting hole 7 is formed in the center of the plate body 1, the reaction substrate pool 2, the oil pool 6 and the micro-droplet collection pool 4 are sequentially arranged along the direction far away from the center shaft mounting hole 7, and a sample adding hole 8 communicated with the reaction substrate pool 2 and an oil adding hole 9 communicated with the oil pool 6 are formed in the plate body 1.
The micro-droplet collecting pool 4 is communicated with the oil pool 6 through an oil pool connecting channel 5, a sample controls the size of droplets generated through the micro-fluidic channel 3, and the droplets are driven to be collected in the micro-droplet collecting pool 4 by using centrifugal force.
Preferably, the microfluidic channel 3 extends in an arc. Preferably, the microfluidic channel 3 is L-shaped. Thus, the microfluidic channel 3 has an arcuately curved L-shape. Preferably, the microfluidic channel is substantially dogleg shaped and has an arc-shaped corner. The design of the fold line shape and the arc shape is mainly used for enabling a sample to smoothly enter the oil pool from the sample pool, and the optimal effect that liquid drops are quickly generated without sample loss is achieved.
Preferably, the reaction substrate pool 2 has a fan-shaped cross section. Preferably, the section of the micro-droplet collecting tank 4 is circular.
Preferably, the tray body 1 is provided with a plurality of positioning holes 10, and four positioning holes 10 and one positioning hole 10 can be connected and fixed with the centrifugal driving device.
When the device is used, a reaction substrate is injected into the reaction substrate pool 2 from the sample adding hole 8, passes through the microfluidic channel 3 under the action of centrifugal force, and water-in-oil micro-droplets with strong uniformity are generated in the micro-droplet collecting pool 4. The oil phase is injected into the oil pool 6 from the oil filling hole 9, the oil phase in the oil pool 6 enters the micro-droplet collecting pool 4 through the oil pool connecting channel 5, and the oil phase completely enters the micro-droplet collecting pool 4 under the action of centrifugal force.
In the above technical solution, the present invention can determine the size of the microfluidic channel and the magnitude of the centrifugal force based on the microfluidic control driven by the centrifugal force, and when the rotation speed reaches about 1500rpm, a large number (> 15000) of water-in-oil liquid reaction droplets with a diameter of about 120 μm and strong uniformity can be generated in the micro-droplet collection pool 4.
In the embodiment shown in fig. 1 to 5, the centrifugal droplet generation chip of the present invention comprises eight independent droplet generation units distributed along its circumference to increase throughput, which can meet the requirement of generating droplets simultaneously with eight samples. When the liquid drops are generated, all the microfluidic channels simultaneously prepare the liquid drops, high-flux liquid drop preparation can be realized, and the centrifugal force generated when the micro-motor is driven to do circular motion can be used as the driving force of liquid flow and is combined with a stepped emulsifying device for use.
The invention can pump liquid along the microfluidic channel by centrifugal force, does not need complex fluid driving equipment, can realize the driving of microfluid by only using a common motor, realizes the formation of liquid drops under the condition of not using any pump, simply and quickly obtains monodisperse liquid drops by using the action of density difference and centrifugal force, and can remove the generated liquid drops from the nozzle in the centrifugal process. Compared with other liquid drop generating devices, the device has the advantages of high flux of each nozzle, simplicity and no dead volume.
The invention adopts the multi-flux stepped emulsification principle driven by centrifugal force to generate multiple single dispersed volume liquid drops, realizes the rapid division, multiple and automation of the solution of an amplification system, and realizes the multi-flux generation of nano-grade liquid drops. Compared with the traditional liquid drop generation technologies such as T-shaped crossing or flow focusing, the method for generating the homogeneous liquid drops has the advantages that the sample zoning speed is more efficient, the method is simpler and more convenient, and more uniform and stable micro liquid drops can be obtained.
The invention utilizes the centrifugal force and the stepped emulsification principle to generate multiple single dispersed volume droplets, and repeatedly simulates and verifies the centrifugal force (rotating speed and rotating acceleration) and the microstructure size of the centrifugal multiple digital PCR micro-droplet generation chip, thereby realizing the possibility of synchronous working of the two modes.
The invention replaces the complex ultra-clean room processing flow with the micro-fluidic channel, greatly reduces the processing difficulty and the processing time, has good sample introduction capability, adopts a sealing structure to effectively avoid the influence caused by other mixed factors, saves the complex design of externally driving liquid flow in the past, has uniform stress of each centrifugal force, and can support the concurrent operation of a multi-unit structure.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A centrifugal, multiple digital PCR micro-droplet generation chip, comprising: disk body (1), the inside of disk body (1) is provided with a plurality of liquid drop generating unit that mutually independent and sealed, the liquid drop generating unit is including reaction substrate pond (2), micro-fluidic channel (3), little liquid drop collecting pit (4), oil bath linking channel (5) and oil bath (6) that communicate in proper order, the center of disk body (1) is provided with axis mounting hole (7), keep away from along reaction substrate pond (2), oil bath (6), little liquid drop collecting pit (4) the direction of axis mounting hole (7) sets gradually, set up on disk body (1) with sample adding hole (8) of reaction substrate pond (2) intercommunication and with tank filler opening (9) of oil bath (6) intercommunication.
2. The centrifugal multiplex digital PCR micro-droplet generating chip according to claim 1, characterized in that the microfluidic channels (3) extend in an arc.
3. The centrifugal multi-digital PCR micro-droplet generation chip according to claim 2, wherein the microfluidic channel (3) is L-shaped.
4. The centrifugal multiplex digital PCR micro-droplet generation chip according to claim 1, characterized in that the reaction substrate pool (2) has a sector-shaped cross section.
5. The centrifugal multiplex digital PCR micro-droplet generation chip according to claim 4, wherein the micro-droplet collection pool (4) is circular in cross-section.
6. The centrifugal type multiple digital PCR micro-droplet generation chip according to claim 1, wherein the tray body (1) is provided with a plurality of positioning holes (10).
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| CN202211043281.XA CN115353949A (en) | 2022-08-29 | 2022-08-29 | Centrifugal multiple digital PCR micro-droplet generation chip |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116237103A (en) * | 2023-05-11 | 2023-06-09 | 杭州博日科技股份有限公司 | Microfluidic chip |
| CN117511721A (en) * | 2024-01-08 | 2024-02-06 | 英诺维尔智能科技（苏州）有限公司 | Real-time fluorescence quantitative PCR instrument based on high-flux microfluidic chip |
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2022
- 2022-08-29 CN CN202211043281.XA patent/CN115353949A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116237103A (en) * | 2023-05-11 | 2023-06-09 | 杭州博日科技股份有限公司 | Microfluidic chip |
| CN116237103B (en) * | 2023-05-11 | 2024-06-21 | 杭州博日科技股份有限公司 | Microfluidic Chip |
| CN117511721A (en) * | 2024-01-08 | 2024-02-06 | 英诺维尔智能科技（苏州）有限公司 | Real-time fluorescence quantitative PCR instrument based on high-flux microfluidic chip |
| CN117511721B (en) * | 2024-01-08 | 2024-03-19 | 英诺维尔智能科技（苏州）有限公司 | Real-time fluorescence quantitative PCR instrument based on high-flux microfluidic chip |
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