Centrifugal multi-digital pcr microdroplet generation chip

Abstract

The application provides a centrifugal type multiple digital PCR microdroplet generation chip, comprising: a disc body, the inside of the disc body is provided with a plurality of droplet generation units which are independent and sealed, the droplet generation unit comprises a reaction substrate pool, a microfluidic channel, a microdroplet collection pool, an oil pool connecting channel and an oil pool which are sequentially communicated, the center of the disc body is provided with a central axis mounting hole, the reaction substrate pool, the oil pool and the microdroplet collection pool are sequentially arranged in the direction away from the central axis mounting hole, and the disc body is provided with a sample adding hole communicated with the reaction substrate pool and an oil adding hole communicated with the oil pool. The application adopts the principle of multiple flux step emulsification driven by centrifugal force to generate multiple monodisperse volume droplets, realizes rapid segmentation and multiple automation of the amplification system solution, realizes multiple flux generation of nanoliter droplets, and the sample partitioning speed is more efficient and simple, and more uniform and stable microdroplets can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, in particular to a centrifugal multi-reaction digital PCR micro-droplet generation chip. BACKGROUND

[0002] A large reaction system is micro-dropletized before traditional PCR amplification, and the reaction system containing nucleic acid molecules is divided into thousands of water-in-oil micro-droplets, i.e. thousands of independent PCR reaction systems, by using a micro-droplet forming device. The micro-droplet digital PCR technology combining microfluidic chip technology and PCR technology has greatly improved sensitivity and precision compared to traditional digital PCR, and is simple to operate and has low sample consumption.

[0003] In addition, due to the reaction characteristics of micro-droplet digital PCR, the required sample amount is extremely small, and the possibility of a single DNA template appearing in a single micro-droplet system without being detected is very low, which is a great progress compared to the second generation of real-time fluorescent quantitative PCR. Micro-droplet digital PCR has many irreplaceable advantages in the detection field, especially for micro-DNA detection and quantification, and is flexible to use, can adjust the throughput and sensitivity as needed, and provides more reliable results for nucleic acid detection.

[0004] Droplet microfluidic technology is a technology that uses the shear force of the continuous phase to break the surface tension of the dispersed phase at the microscale, and divide the dispersed phase into nanoliter or even picoliter droplets. Droplet microfluidic chip has the advantages of small volume, high precision, complete isolation between droplets, etc., and is a very excellent micro-reactor. Common droplet generation methods include "T" channel method, flow focusing method and coaxial method. Under the driving of stable positive pressure or negative pressure, the three droplet generation methods have good consistency and generation rate.

[0005] Micro-droplet digital PCR technology combining microfluidic technology and PCR technology is a breakthrough technology for detecting and quantifying nucleic acids that has developed rapidly in recent years. To realize micro-droplet digital nucleic acid amplification analysis, the formation of micro-droplets is the key link of micro-droplet digital PCR technology, which must rely on a device that can efficiently generate highly monodisperse droplets, and the droplet generation process must be as simple as possible. The device is compatible with general standard equipment, which can reduce the complexity of the entire system, and even can be directly used by non-professional users. Centrifugal microfluidic technology is one of many microfluidic driving methods, and its advantage is that it eliminates the complex design of relying on external driving liquid flow, and the stress on the centrifugal turntable is uniform in all directions, which can support concurrent operation of multiple microfluidic chips / structures on the turntable.

[0006] At present, the centrifugal microfluidic technology has been well integrated with basic liquid flow operation. Through microfluidic control under centrifugal force, the oil-water two-phase step emulsion droplet generation technology is used to synchronously and efficiently produce multiple volume monodisperse droplets with different sizes. The PCR reaction substrate (polymerase, deoxyribonucleotide triphosphate, primer, probe, buffer, etc.) and detection target template are mixed and rapidly on-line segmented into a pL-nL volume multiple volume micro-reactor single layer array for subsequent PCR rapid amplification. The centrifugal microfluidic chip is also the most competitive form and carrier of microfluidic technology facing market application in the future.

[0007] The number, generation rate and consistency of droplets are key factors affecting the application of droplet microtechnology. To achieve higher throughput droplet generation, most researchers use the "T" channel method, flow focusing method and coaxial method for array and parallel droplet preparation. Although this array structure improves the generation rate, the size and flow rate error of each droplet generation structure is large, which affects the equal distribution of samples and the final precision and sensitivity. Another method is to use a multi-stage droplet segmentation method to gradually segment the generated droplets into small droplets to improve the generation rate. However, the processing difference of each micro-segmentation structure causes the droplet segmentation to be asymmetric, and the size of the prepared droplets is different. The above two multi-throughput droplet generation technologies have a great impact on the generation of droplets due to the large size difference.

[0008] In recent years, step emulsification has become an important method for producing stable size droplets. Its main feature is that it can use parallel large-scale nozzles, microfluidic channels and other arrays to achieve high-throughput droplet production. The traditional step emulsification method uses pressure or injection pump to drive droplet generation, and the whole device is bulky, complex and low in integration. Moreover, droplets are easily accumulated at the nozzle, which not only interferes with the formation of subsequent droplets, reduces the droplet generation rate, but also causes the droplet size distribution to be wide.

[0009] The PCR micro-droplet generation chip in the prior art does not use a sealing structure, which may be affected by other mixed factors. In addition, the current domestic research mainly uses flexible micro-pipes, but the production of flexible micro-pipes is very complex and requires manual production. SUMMARY

[0010] The present application provides a centrifugal multiple digital PCR micro-droplet generation chip to solve at least one of the above technical problems.

[0011] To address the aforementioned problems, as one aspect of the present invention, a centrifugal multiplex digital PCR microdroplet generation chip is provided, comprising: a disk body, wherein multiple independent and sealed droplet generation units are disposed inside the disk body, each droplet generation unit comprising a reaction substrate cell, a microfluidic channel, a microdroplet collection cell, an oil cell connection channel, and an oil cell connected in sequence, the disk body having a central axis mounting hole, the reaction substrate cell, the oil cell, and the microdroplet collection cell being arranged in sequence along a direction away from the central axis mounting hole, and the disk body having a sample loading hole communicating with the reaction substrate cell and an oil filling hole communicating with the oil cell.

[0012] Preferably, the microfluidic channel extends in an arc shape.

[0013] Preferably, the microfluidic channel is L-shaped.

[0014] Preferably, the cross-section of the reaction substrate cell is fan-shaped.

[0015] Preferably, the cross-section of the microdroplet collection pool is circular.

[0016] Preferably, the disc body has multiple positioning holes.

[0017] This invention enables the pumping of liquid along a microfluidic channel using centrifugal force, eliminating the need for complex fluid drive equipment. Microfluidic actuation can be achieved using only a common motor, achieving droplet formation without any pumps. Monodisperse droplets are simply and quickly obtained by utilizing density differences and centrifugal force, and the generated droplets can be removed from the nozzle during centrifugation. Compared to other droplet generation devices, this invention offers high throughput per nozzle, a simple device design, and no dead volume.

[0018] This invention utilizes a centrifugally driven multi-throughput stepped emulsification principle to generate multiple monodisperse volumetric droplets, achieving rapid segmentation and automation of the amplification system solution, and realizing multi-throughput generation of nano-level droplets. Compared with traditional droplet generation techniques such as T-shaped cross-linking or flow focusing, this method offers more efficient and convenient sample segmentation, while also yielding more uniform and stable microdroplets.

[0019] This invention utilizes centrifugal force and the principle of step emulsification to generate multiple monodisperse volume droplets. The magnitude of centrifugal force (rotation speed and acceleration) and the microstructure size of the centrifugal multiplex digital PCR microdroplet generation chip were repeatedly simulated and verified, realizing the possibility of simultaneous operation of the two methods.

[0020] This invention replaces the complex cleanroom processing procedure with a microfluidic channel, which greatly reduces the processing difficulty and processing time. It also has good sample injection capability. The sealed structure effectively avoids the influence of other contaminating factors and eliminates the complicated design that previously relied on externally driven liquid flow. At the same time, the centrifugal force is uniform in all aspects and can support the concurrent operation of multi-unit structures. Attached Figure Description

[0021] Figure 1 A perspective view of the invention is shown schematically;

[0022] Figure 2 A schematic diagram of a droplet generation unit is shown.

[0023] Figure 3 A cross-sectional view of the present invention is shown schematically. Figure 1 ;

[0024] Figure 4 A cross-sectional view of the present invention is shown schematically. Figure 2 ;

[0025] Figure 5 A perspective view of the present invention is shown schematically.

[0026] The attached diagram is labeled as follows: 1. Disc; 2. Reaction substrate cell; 3. Microfluidic channel; 4. Microdroplet collection cell; 5. Oil cell connection channel; 6. Oil cell; 7. Central shaft mounting hole; 8. Sample dispensing hole; 9. Oil filling hole; 10. Positioning hole. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0028] As one aspect of the present invention, a centrifugal multiplex digital PCR microdroplet generation chip is provided, comprising: a disk body 1, wherein the disk body 1 is provided with multiple independent and sealed droplet generation units, the disk body 1 is disc-shaped, and the disk body 1 has multiple independent microdroplet generation units with the same structure, each independent unit being in a sealed state.

[0029] Furthermore, the droplet generation unit includes a reaction substrate pool 2, a microfluidic channel 3, a microdroplet collection pool 4, an oil pool connection channel 5, and an oil pool 6 connected in sequence. A central axis mounting hole 7 is provided at the center of the disk body 1. The reaction substrate pool 2, the oil pool 6, and the microdroplet collection pool 4 are arranged in sequence in a direction away from the central axis mounting hole 7. The disk body 1 is provided with a sample addition hole 8 communicating with the reaction substrate pool 2 and an oil filling hole 9 communicating with the oil pool 6.

[0030] The microdroplet collection pool 4 and the oil pool 6 are connected by the oil pool connection channel 5. The sample is controlled by the microfluidic channel 3 to generate droplets, and centrifugal force is used to drive the droplets to be collected in the microdroplet collection pool 4.

[0031] Preferably, the microfluidic channel 3 extends in an arc shape. More preferably, the microfluidic channel 3 is L-shaped. Thus, the microfluidic channel 3 is an L-shaped arc. More preferably, the microfluidic channel is generally zigzag-shaped, with arc-shaped corners. This zigzag and arc-shaped design is primarily to ensure the sample smoothly enters the oil pool from the sample cell, achieving the ideal effect of rapid droplet formation without sample loss.

[0032] Preferably, the reaction substrate pool 2 has a fan-shaped cross-section. Preferably, the microdroplet collection pool 4 has a circular cross-section.

[0033] Preferably, the disc body 1 has multiple positioning holes 10, and can be connected and fixed to the centrifugal drive device through four positioning holes 10 and one positioning hole 10.

[0034] In use, the reaction substrate is injected into the reaction substrate pool 2 through the sample loading port 8. Under centrifugal force, the reaction substrate passes through the microfluidic channel 3 and generates highly uniform water-in-oil microdroplets in the microdroplet collection pool 4. The oil phase is injected into the oil pool 6 through the oil filling port 9. The oil phase in the oil pool 6 enters the microdroplet collection pool 4 through the oil pool connecting channel 5. Under centrifugal force, the oil phase will completely enter the microdroplet collection pool 4.

[0035] In the above technical solution, the present invention can be based on microfluidic control driven by centrifugal force, that is, the size of the microfluidic channel and the magnitude of the centrifugal force are determined. When the rotation speed reaches about 1500 rpm, a large number (>15000) of highly uniform water-in-oil reaction droplets with a diameter of about 120 μm can be generated in the microdroplet collection pool 4.

[0036] exist Figures 1 to 5 In the illustrated embodiment, the centrifugal droplet generation chip of the present invention includes eight independent droplet generation units distributed along its circumference to improve throughput and meet the need for simultaneous droplet generation from eight samples. During droplet generation, all microfluidic channels simultaneously prepare droplets, achieving high-throughput droplet preparation. The centrifugal force generated by the circular motion driven by the micromotor can be used as the driving force for the liquid flow, and it can be combined with a stepped emulsification device.

[0037] This invention enables the pumping of liquid along a microfluidic channel using centrifugal force, eliminating the need for complex fluid drive equipment. Microfluidic actuation can be achieved using only a common motor, achieving droplet formation without any pumps. Monodisperse droplets are simply and quickly obtained by utilizing density differences and centrifugal force, and the generated droplets can be removed from the nozzle during centrifugation. Compared to other droplet generation devices, this invention offers high throughput per nozzle, a simple device design, and no dead volume.

[0038] This invention utilizes a centrifugally driven multi-throughput stepped emulsification principle to generate multiple monodisperse volumetric droplets, achieving rapid segmentation and automation of the amplification system solution, and realizing multi-throughput generation of nano-level droplets. Compared with traditional droplet generation techniques such as T-shaped cross-linking or flow focusing, this method offers more efficient and convenient sample segmentation, while also yielding more uniform and stable microdroplets.

[0039] This invention utilizes centrifugal force and the principle of step emulsification to generate multiple monodisperse volume droplets. The magnitude of centrifugal force (rotation speed and acceleration) and the microstructure size of the centrifugal multiplex digital PCR microdroplet generation chip were repeatedly simulated and verified, realizing the possibility of simultaneous operation of the two methods.

[0040] This invention replaces the complex cleanroom processing procedure with a microfluidic channel, which greatly reduces the processing difficulty and processing time. It also has good sample injection capability. The sealed structure effectively avoids the influence of other contaminating factors and eliminates the complicated design that previously relied on externally driven liquid flow. At the same time, the centrifugal force is uniform in all aspects and can support the concurrent operation of multi-unit structures.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A centrifugal multiplex digital PCR microdroplet generation chip, characterized in that, include: The disk body (1) has multiple independent and sealed droplet generating units inside. Each droplet generating unit includes a reaction substrate pool (2), a microfluidic channel (3), a microdroplet collection pool (4), an oil pool connection channel (5), and an oil pool (6) connected in sequence. The center of the disk body (1) has a central axis mounting hole (7). The reaction substrate pool (2), the oil pool (6), and the microdroplet collection pool (4) are arranged in sequence away from the central axis mounting hole (7). The disk body (1) has a sample feeding hole (8) connected to the reaction substrate pool (2) and an oil filling hole (9) connected to the oil pool (6). The microfluidic channel (3) is zigzag-shaped with arc-shaped corners. In use, the reaction substrate is injected into the reaction substrate pool (2) through the sample loading hole (8). Under the action of centrifugal force, the reaction substrate passes through the microfluidic channel (3) and generates water-in-oil microdroplets with strong uniformity in the microdroplet collection pool (4). The oil phase is injected into the oil pool (6) through the oil filling hole (9). The oil phase in the oil pool (6) enters the microdroplet collection pool (4) through the oil pool connecting channel (5). Under the action of centrifugal force, the oil phase will completely enter the microdroplet collection pool (4).

2. The centrifugal multiplex digital PCR microdroplet generation chip according to claim 1, characterized in that, The cross-section of the reaction substrate pool (2) is fan-shaped.

3. The centrifugal multiplex digital PCR microdroplet generation chip according to claim 2, characterized in that, The cross-section of the microdroplet collection pool (4) is circular.

4. The centrifugal multiplex digital PCR microdroplet generation chip according to claim 1, characterized in that, The disc body (1) has multiple positioning holes (10).

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