Digital PCR (Polymerase Chain Reaction) microdroplet generation device

Abstract

The invention provides a digital PCR (Polymerase Chain Reaction) microdroplet generation device, and relates to the field of microdroplet generation. The digital PCR microdroplet generation device comprises a liquid storage bin and a microflow plate which are communicated, the microflow plate is in an annular shape, a microflow channel is arranged in the microflow plate and used for circulating PCR reaction liquid, a cavity is further formed in the microflow plate, a microgroove is communicated between the cavity and the microflow channel, inert gas is injected into the cavity, and the inert gas is intermittently discharged from the microgroove; according to the digital PCR microdroplet generation device, the shearing rhythm of PCR is controlled through intermittent exhaust of inert gas, and the microgroove and the microfluidic channel are intersected at an acute angle, so that the size uniformity of the microdroplet is improved; a gas separation layer can be formed to prevent droplet fusion; the method is low in cost, easy to obtain and high in adaptability; after the inert gas cuts the PCR reaction liquid into the micro-droplets, the inert gas can separate the micro-droplets and continuously restrain the spatial arrangement of the micro-droplets, so that the micro-droplets are prevented from being extruded mutually during flowing, the retention and fracture of the micro-droplets are reduced, the integrity of the micro-droplets is improved, and the effective recovery rate of the micro-droplets is increased.

Description

Technical Field

[0001] This invention relates to the field of droplet generation, specifically to a digital PCR droplet generation device. Background Technology

[0002] Digital polymerase chain reaction (PCR), a cutting-edge technology for achieving absolute quantification of nucleic acids, divides the PCR reaction solution containing target nucleic acids into thousands or even millions of independent nanoliter or picoliter droplets (each droplet containing 0-1 target molecules). After PCR amplification, the number of positive droplets is counted based on a Poisson distribution, achieving absolute quantification of the initial template. Droplet generation is the core step of digital PCR; its generation efficiency, size uniformity, stability, and anti-contamination ability directly determine the quantitative accuracy, detection sensitivity, and experimental repeatability, becoming a key bottleneck restricting the development of digital PCR technology. Currently, digital PCR droplet generation devices are mainly based on microfluidic technology, and can be classified according to their generation principles as "T-channel shearing type," "flow focusing type," "membrane emulsification type," and "sonic wave driven type." In the droplet generation device, a droplet is a tiny, independent reaction unit with a "water-in-oil" core structure, constructed to achieve absolute quantification of nucleic acids. It is the core carrier for digital PCR technology to shift from "batch amplification" to "precise quantification at the single molecule level". Essentially, it disperses the PCR reaction system (aqueous phase) containing target nucleic acids into an inert oil phase (continuous phase) through microfluidics and other technologies, forming tiny droplets with uniform volume and stable structure.

[0003] Traditional single-stage shearing droplet generation devices use a T-shaped channel combined with an oscillating chamber to shear the PCR reaction solution into droplets. The shearing force only acts at the junction point and cannot continuously exert a stable constraint on the subsequent flow of droplets. This results in a large difference in the size of the generated droplets. Larger droplets have less fluid resistance and a faster flow rate, while smaller droplets have greater resistance and a slower flow rate. The velocity difference between the two can reach 20%-30%. This velocity difference makes it easy for subsequent smaller droplets to be "caught up" by the larger droplets in front, leading to unexpected passive collisions between droplets. This makes it easy for droplets to fuse or break apart. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a digital PCR droplet generation device that solves the problem of droplets easily undergoing unexpected passive collisions, leading to easy fusion or breakage between droplets.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a digital PCR droplet generation device, comprising a connected liquid storage tank and a microfluidic plate, wherein the microfluidic plate is ring-shaped and has microfluidic channels inside for the flow of PCR reaction solution.

[0006] The microfluidic plate also has a cavity, and a microgroove connects the cavity to the microfluidic channel. The cavity is used to inject inert gas, which is intermittently discharged from the microgroove and cuts the PCR reaction solution to form microdroplets. The gas and the PCR reaction solution in the microfluidic channel flow at an acute angle to each other.

[0007] Preferably, the microfluidic channels and cavities are each configured as a plurality of units, and are arranged circumferentially along the radial direction of the microfluidic plate.

[0008] Preferably, the inert gas is nitrogen.

[0009] Preferably, the inner wall of the microfluidic channel is provided with a hydrophobic coating.

[0010] Preferably, the width of the microgroove decreases sequentially from the cavity to the microfluidic channel.

[0011] Preferably, there is an angle between the microgroove and the microfluidic channel.

[0012] Preferably, the liquid storage chamber is used to inject PCR reaction solution, and a rotating cover is slidably installed at one end of the liquid storage chamber. When the rotating cover rotates, it drives the liquid storage chamber and the microfluidic plate to rotate.

[0013] Preferably, a fixing tube is fixedly installed at the bottom of the microfluidic plate, and each cavity is connected to the fixing tube. One end of the fixing tube is used to inject inert gas, and the other end is provided with several slots.

[0014] Preferably, a connector is rotatably installed at the bottom end of the fixed pipe, and a gas supply pipe connected to the fixed pipe is fixedly installed inside the connector.

[0015] Preferably, the bottom of the microfluidic plate is provided with an annular floating airbag.

[0016] Compared with existing technologies, this invention has the following advantages: By intermittently venting inert gas, the shearing rhythm of PCR is controlled; the microgroove and microfluidic channel intersect at an acute angle; after the inert gas is discharged from the microgroove, it forms a directional shearing reaction with the PCR reaction solution in the microfluidic channel; the inert gas has stable chemical properties and does not interfere with PCR. The reaction components are easily and precisely controlled, improving droplet size uniformity. Inert gas forms a gas-phase barrier to prevent droplet fusion. It is low-cost, readily available, and highly adaptable. After the inert gas cuts the PCR reaction solution into droplets, it also separates the droplets, continuously constraining their spatial arrangement and preventing them from being squeezed together during flow. The droplets flow along the annular microfluidic channel to the outlet, where they detach from the channel under the combined action of centrifugal force and airflow propulsion. The inert gas pushes the droplets and separates adjacent droplets, preventing fusion, ensuring independent reaction of each droplet, stabilizing droplet flow rate, improving flow uniformity, maintaining droplet size uniformity, and reducing quantitative errors. It also reduces droplet retention and breakage, improving droplet integrity and effective recovery rate, providing support for high-precision digital PCR operations. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of the present invention; Figure 2 This is an exploded view of the overall structure of the present invention; Figure 3 This is a cross-sectional view of the front view of the liquid storage tank of the present invention; Figure 4 This is a schematic diagram of the floating airbag of the present invention floating on an oily liquid in a container; Figure 5 This is a bottom view of the microfluidic plate of the present invention; Figure 6 This is a cross-sectional view of the top view of the microfluidic plate of the present invention; Figure 7 This is a cross-sectional view of the microfluidic plate of the present invention; Figure 8 For the present invention Figure 7 Enlarged schematic diagram of the structure at point A in the middle; Figure 9 This is a partial schematic diagram of the microfluidic plate of the present invention; Figure 10 This is a schematic diagram of the fixing tube of the present invention.

[0018] The components are: 1. Liquid storage tank; 2. Microfluidic plate; 3. Microfluidic channel; 4. Cavity; 5. Microgroove; 6. Rotating cap; 7. Fixing tube; 8. Groove opening; 9. Connector; 10. Gas delivery tube; 11. Floating airbag; 12. PCR reaction solution; 13. Container. Detailed Implementation

[0019] like Figures 1-10As shown, a digital PCR droplet generation device includes a connected reservoir 1 and a microfluidic plate 2. The reservoir 1 is used to inject PCR reaction solution 12. A rotating cover 6 is slidably installed at one end of the reservoir 1. When the rotating cover 6 rotates, it drives the reservoir 1 and the microfluidic plate 2 to rotate. The centrifugal force generated by the rotation can assist the PCR reaction solution 12 to flow in the microfluidic channels 3, reducing the stagnation of liquid flow caused by surface tension or channel resistance. This is especially suitable for high-viscosity PCR reaction solutions 12 (such as samples containing tissue lysates). The centrifugal force makes the liquid flow pressure in each radial channel more uniform, avoiding droplet size differences caused by uneven liquid supply, and improving the consistency of parallel generation in multiple channels. The sliding installation design of the rotating cover 6 facilitates operation, and the liquid flow rate can be flexibly controlled by adjusting the rotation speed to adapt to PCR reactions of different viscosities. To meet the droplet generation requirements of liquid 12, the microfluidic plate 2 is ring-shaped and has microfluidic channels 3 inside. A fixed tube 7 is fixedly installed at the bottom of the microfluidic plate 2. Each cavity 4 is connected to the fixed tube 7. One end of the fixed tube 7 is used to inject inert gas, and the other end is provided with several slots 8. A connector 9 is rotatably installed at the bottom of the fixed tube 7. A gas delivery tube 10 connected to the fixed tube 7 is fixedly installed in the connector 9. When the microfluidic plate 2 rotates, the connector 9 can rotate relative to the fixed tube 7 to ensure that the gas delivery tube 10 always remains stationary and to ensure a continuous and stable output of inert gas. The bottom of the microfluidic plate 2 is provided with a ring-shaped floating airbag 11. The floating airbag 11 can float on the oily liquid in the external container 13. When the droplets after the PCR reaction liquid 12 is cut are discharged, they can fall onto the oily liquid that is insoluble with it.

[0020] The microfluidic channels 3 and cavities 4 are each configured as multiple units, arranged circumferentially along the radial direction of the microfluidic plate 2. Multiple microfluidic channels 3 allow for the simultaneous processing of multiple PCR reaction solutions 12, significantly improving droplet generation efficiency and meeting the requirements for batch sample processing (such as clinical 96). The microfluidic channel 3 meets the detection requirements of well plate samples. The circumferential arrangement ensures that the distance from each channel to the center is consistent, making it more uniform under the influence of the liquid supply pressure of the reservoir 1 and the distribution of inert gas. This reduces the droplet size difference between different channels and improves the parallelism of multi-sample detection. The radial layout is compact, saving device space and adapting to the integration requirements of miniaturized digital PCR systems. The inner wall of the microfluidic channel 3 is provided with a hydrophobic coating. The hydrophobic coating (such as PTFE, PFOTS) can reduce the adhesion between the PCR reaction solution 12 (aqueous phase) and the channel wall, preventing droplets from lingering, fusing, or breaking in the channel, ensuring the integrity of the droplet morphology, reducing the frictional resistance between the liquid flow and the channel wall, making the flow of the PCR reaction solution 12 more stable, and indirectly improving the uniformity of droplet size when the inert gas is cut. The hydrophobic coating is chemically inert and does not adsorb nucleic acid and enzyme reaction components, avoiding the decrease in PCR amplification efficiency due to component loss and ensuring detection sensitivity. The microfluidic channel 3 is used to circulate the PCR reaction solution 12.

[0021] The microfluidic plate 2 also has a cavity 4, which is connected to the microfluidic channel 3 by a microgroove 5. The cavity 4 is used to inject an inert gas, namely nitrogen. Nitrogen is chemically inert and stable, and does not react with the enzymes, nucleic acids, and buffer components in the PCR reaction solution 12 at room temperature, thus avoiding interference with PCR amplification efficiency. Nitrogen has extremely low solubility in the PCR reaction solution 12 (far lower than argon), and will not change the osmotic pressure or pH of the reaction system due to gas dissolution. The microfluidic plate 2 is designed to ensure droplet stability. Nitrogen is low-cost and readily available (it can be prepared on-site in the laboratory using a nitrogen generator), and is compatible with the gas pressure drive module of mainstream digital PCR equipment, reducing experimental costs and improving operational convenience. Inert gas is intermittently discharged from the microchannel 5, cutting the PCR reaction solution 12 to form droplets. The gas and the flow direction of the PCR reaction solution 12 in the microfluidic channel 3 intersect at an acute angle. The annular microfluidic plate 2 provides a stable annular flow path for the PCR reaction solution 12. Combined with the directional exhaust of the microchannel 5, the shear force of the inert gas is more concentrated, improving the controllability of droplet cutting. The acute-angled gas-liquid flow direction can decompose into a propulsive component parallel to the liquid flow, reducing droplet retention while cutting droplets and improving generation efficiency. Intermittent exhaust can precisely control the cutting rhythm and avoid airflow turbulence caused by continuous gas supply, which is conducive to generating uniformly sized droplets, laying the foundation for subsequent PCR quantitative analysis. The width of the microchannel 5 decreases sequentially from the cavity 4 to the microfluidic channel 3. The microchannel 5 with decreasing width utilizes the "Venturi effect" to accelerate the inert gas flow. The wide inlet and narrow outlet of the gas flow concentrates gas kinetic energy, enhancing the shear force on the PCR reaction solution 12 and improving droplet cutting efficiency. The gradient width design reduces turbulence interference in gas flow, making the airflow more stable and further ensuring the controllability of the cutting rhythm. The angle between the microgroove 5 and the microfluidic channel 3 causes the gas and liquid flows to converge at an acute angle. The constraint of the wall of the microfluidic channel 3 concentrates the shear force, making the cutting more controllable and avoiding the chaotic droplet shape caused by the random diffusion of gas. The physical constraint of the wall of the microfluidic channel 3 concentrates the shear force of the inert gas in a preset direction, avoiding the dispersion of shear force caused by the random diffusion of gas, ensuring that the force and angle of each cut are consistent, and significantly improving the uniformity of droplet size (reducing the coefficient of variation CV value). The synergy between the acute angle direction and the liquid flow direction can reduce the reverse impact of gas on the liquid flow, avoid liquid flow turbulence, and ensure the continuity and stability of droplet generation. The directional shear force reduces the probability of droplet collision in the channel, reduces the risk of droplet fusion, and provides a guarantee for the "single droplet independent reaction" of subsequent PCR amplification.

[0022] In use, first open the rotating cover 6 of the storage chamber 1, and inject the PCR reaction solution 12 containing nucleic acid template, Taq enzyme, primers, and fluorescent probe into the storage chamber 1. Close the rotating cover 6 and ensure it is sealed to prevent contamination and evaporation. Then connect the rotating cover 6 to an external power source, such as a motor, to make the rotating cover 6 rotate. It drives the storage chamber 1 and the annular microfluidic plate 2 to rotate synchronously through sliding contact (the speed can be adjusted according to the viscosity of the reaction solution, such as 500-1000 rpm for high-viscosity samples (including tissue lysates). The centrifugal force generated by the rotation diffuses radially outward along the microfluidic plate 2, evenly pushing the PCR reaction solution 12 in the storage chamber 1 into several circumferentially arranged microfluidic channels 3, avoiding the stagnation of liquid flow caused by surface tension or resistance in traditional straight channels, while ensuring that the supply pressure difference of each microfluidic channel 3 is <5% (far lower than the 15% of traditional devices).

[0023] Nitrogen gas (inert gas, 99.999% purity) is injected into connector 9 through gas delivery pipe 10. Connector 9 is rotatably engaged with fixed pipe 7 (connector 9 remains stationary when microfluidic plate 2 rotates to avoid entanglement of the gas pipe). Nitrogen gas is dispersed through fixed pipe 7 to all cavities 4 of microfluidic plate 2 (the slot 8 of fixed pipe 7 can adjust the gas flow rate to ensure that the gas pressure deviation of each cavity 4 is <2%), achieving uniform gas distribution. Nitrogen gas in cavity 4 is discharged to microfluidic channel 3 through microgrooves 5. The width of microgrooves 5 decreases sequentially from cavity 4 to microfluidic channel 3 (e.g., inlet width 200μm, outlet width 50μm), utilizing the "Venturi effect" to increase the nitrogen gas flow rate from 10m / s. The flow rate is increased to 30-50 m / s, concentrating the gas kinetic energy at the outlet of microchannel 5, avoiding the airflow diffusion problem of traditional devices. Simultaneously, nitrogen is discharged in an "intermittent exhaust" mode (exhaust frequency linked to rotation speed, e.g., exhaust once per 1° rotation), precisely controlling the shearing rhythm. Microchannel 5 and microfluidic channel 3 intersect at an acute angle (30°-60°). After nitrogen is discharged from microchannel 5, it forms directional shear with the PCR reaction solution 12 (flow rate 0.1-0.5 μL / min) within microfluidic channel 3. The shearing force of nitrogen is concentrated at the gas-liquid junction (outlet of microchannel 5). The physical constraint of the microfluidic channel 3 wall prevents random gas diffusion, ensuring consistent shearing force and angle for each shearing operation. The acute-angle flow direction decomposes the kinetic energy of nitrogen into a "shearing component perpendicular to the liquid flow" (used to tear the reaction solution) and a "propulsion component parallel to the liquid flow." The microfluidic plate 2 uses a force (to propel the generated droplets along the annular channel, reducing stagnation) to solve the collision problem caused by droplet stagnation in traditional devices. The hydrophobic coating (e.g., PTFE, contact angle 120°-130°) on the inner wall of the microfluidic channel 3 reduces the adhesion between the PCR reaction solution 12 (aqueous phase) and the channel wall, preventing morphological distortion caused by droplet adhesion to the wall surface. It also reduces flow friction resistance (resistance reduced by 30%-50%), ensuring stable flow and indirectly improving droplet size uniformity. The cleaved droplets (50-100 μm in diameter, 100-500 pL in volume) flow along the annular microfluidic channel 3 to the outlet, where they detach from the channel under the combined action of centrifugal force and airflow propulsion. The annular floating airbag 11 at the bottom of the microfluidic plate 2 floats in the oily liquid (e.g., fluorinated oil, containing 0.5%) in the external container 13. The surface of the microfluidic plate 2 is made of perfluoropolyether surfactant to ensure that it is always horizontal (tilt angle < 0.5°) and the droplets fall vertically into the oily liquid (fall deviation < 100 μm), avoiding the problem of droplets hitting the wall of container 13 and breaking due to tilting in traditional devices; the oily liquid and the aqueous droplets are immiscible, further preventing droplet fusion (fusion rate < 1%) and water evaporation (evaporation rate < 0.5%), laying the foundation for subsequent PCR amplification.

[0024] It should be noted that, as Figure 9As shown, the inert gas forms a gas-phase barrier between adjacent droplets, utilizing the immiscibility of gas and liquid to completely block the direct contact path between droplets. Traditionally, droplets easily fuse due to surface tension and flow collisions after generation, leading to "multi-molecule encapsulation" that undermines the statistical premise of digital PCR. In this device, the gas barrier keeps the distance between droplets consistently at ≥1 / 2 of the droplet diameter, reducing the fusion rate to <1%, ensuring that each droplet independently carries 0 or 1 target molecule, providing a reliable basis for subsequent absolute quantification based on Poisson distribution. Simultaneously, the inert gas cuts the droplets, driving them forward along microfluidic channel 3 through airflow thrust, replacing or assisting traditional "simple liquid flow pressure drive." The gas flow propulsion direction and magnitude can be more precisely controlled through gas parameters (pressure, flow rate), ensuring that the velocity difference between droplets of different sizes is <5% (compared to 20%-30% under traditional liquid flow drive). This avoids the problem of "small droplets being caught up and collided with by large droplets" caused by uneven flow rates, further reducing the risk of merging, while ensuring the continuity and consistency of droplet movement within the channel. After the gas separates the droplets, it can continuously constrain their spatial arrangement, preventing morphological distortion caused by mutual compression and adsorption to the channel walls during flow. Traditional droplets are prone to aggregation, leading to a size variation coefficient (CV value) exceeding 8%. However, in this design, the gas separation combined with the hydrophobic coating of the microfluidic channel 3 ensures that the droplet size CV value is <3%, meeting the requirements of digital PCR for "monodispersed droplet swarms". The requirements are significantly reduced, thus minimizing the systematic error in Poisson distribution calculations. Inert gases (such as nitrogen) are chemically inert and have stable interfacial tension with PCR reaction solution 12 (aqueous phase). When separating droplets, the "low adhesion" of the gas-liquid interface can be utilized to reduce the interaction between droplets and the walls of microfluidic channels 3. Combined with the hydrophobic coating on the inner side of microfluidic channels 3, the retention rate of droplets on the channel walls can be reduced to <0.5% (compared to >8% in traditional devices). At the same time, droplet breakage due to "wall pulling" is avoided, improving the integrity of droplet generation and effective recovery rate.

[0025] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A digital PCR droplet generation device, characterized in that: It includes a connected liquid storage tank (1) and a microfluidic plate (2). The microfluidic plate (2) is ring-shaped and has a microfluidic channel (3) inside. The microfluidic channel (3) is used to flow PCR reaction solution (12). The microfluidic plate (2) is also provided with a cavity (4), and a microgroove (5) is connected between the cavity (4) and the microfluidic channel (3). The cavity (4) is used to inject inert gas. The inert gas is intermittently discharged from the microgroove (5) and cuts the PCR reaction solution (12) to form microdroplets. The gas and the PCR reaction solution (12) in the microfluidic channel (3) flow at an acute angle.

2. The digital PCR droplet generation device according to claim 1, characterized in that: The microfluidic channels (3) and cavities (4) are each configured as a plurality of such channels and are arranged in a circular pattern along the radial direction of the microfluidic plate (2).

3. The digital PCR droplet generation device according to claim 1, characterized in that: The inert gas is nitrogen.

4. The digital PCR droplet generation device according to claim 1, characterized in that: The inner wall of the microfluidic channel (3) is provided with a hydrophobic coating.

5. The digital PCR droplet generation device according to claim 1, characterized in that: The width of the microgroove (5) decreases sequentially from the cavity (4) to the microfluidic channel (3).

6. The digital PCR droplet generation device according to claim 1, characterized in that: There is an angle between the microgroove (5) and the microfluidic channel (3).

7. The digital PCR droplet generation device according to claim 1, characterized in that: The liquid storage chamber (1) is used to inject PCR reaction solution (12). A rotating cover (6) is slidably installed at one end of the liquid storage chamber (1). When the rotating cover (6) rotates, it drives the liquid storage chamber (1) and the microfluidic plate (2) to rotate.

8. The digital PCR droplet generation device according to claim 2, characterized in that: The bottom of the microfluidic plate (2) is fixedly installed with a fixed tube (7), and each cavity (4) is connected to the fixed tube (7). One end of the fixed tube (7) is used to inject inert gas, and the other end is provided with several slots (8).

9. The digital PCR droplet generation device according to claim 8, characterized in that: A connector (9) is rotatably installed at the bottom end of the fixed tube (7), and a gas supply pipe (10) connected to the fixed tube (7) is fixedly installed inside the connector (9).

10. The digital PCR droplet generation device according to claim 1, characterized in that: The bottom of the microfluidic plate (2) is provided with an annular floating airbag (11).

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