Ultrasonic droplet generation-based PCR microfluidic chip and application thereof

Abstract

The application relates to a PCR micro-fluidic chip based on ultrasonic droplet generation and application thereof, and belongs to the technical field of biological detection. The application provides a PCR micro-fluidic chip which comprises an L-shaped main chip, a sealing piston and a piston driving device. A sample cavity, a splash-proof well, a piston cavity and a sample adding hole are arranged on the vertical side of the L-shaped main chip. A flat cavity is arranged on the horizontal side of the L-shaped main chip. The sample cavity is located below the splash-proof well, and one end of the sample cavity close to the splash-proof well is connected with the splash-proof well. The piston cavity is located above the splash-proof well, and one end of the piston cavity close to the splash-proof well is connected with the splash-proof well. One end of the sample adding hole close to the splash-proof well is connected with the splash-proof well and / or the sample adding hole. One end of the flat cavity close to the splash-proof well is connected with the splash-proof well. One end of the sealing piston close to the splash-proof well is inserted into the splash-proof well through the piston cavity. The piston driving device is used for driving the sealing piston, so that the sealing piston closes or opens the communication position of the splash-proof well and the flat cavity under the driving of the piston driving device.

Description

Technical Field

[0001] This invention relates to a PCR microfluidic chip based on ultrasonic droplet generation and its application, belonging to the field of biodetection technology. Background Technology

[0002] Polymerase chain reaction (PCR) is a technique that uses a DNA template to amplify the DNA to a sufficient quantity for structural and functional analysis, with the participation of DNA polymerase and nucleotide substrates.

[0003] Droplet digital PCR involves microdividing the sample before traditional PCR amplification. This involves breaking down the reaction system containing nucleic acid molecules into thousands of nanoliter-sized droplets. Each droplet may contain no target nucleic acid molecule or contain one to several target molecules. After PCR amplification, each droplet is individually detected. Droplets with fluorescent signals are interpreted as 1, and those without are interpreted as 0. Based on the Poisson distribution principle and the number and proportion of positive droplets, the initial copy number or concentration of the target molecule can be determined. Compared to traditional PCR, droplet digital PCR does not rely on Ct values ​​or internal reference genes to determine the absolute number of target molecules down to a single copy. It is lower in cost and more practical, showing great promise for applications in copy number variation studies, detection of extremely low concentrations of nucleic acid molecules in samples from complex sources, NGS data validation, identification of NGS sequencing libraries, and studies of gene expression with subtle differences, such as miRNAs.

[0004] PCR microfluidic chips are a type of microfluidic chip used to realize droplet-based digital PCR. Traditional PCR microfluidic chips mostly use pneumatic or hydraulic methods to drive nucleic acid samples, which involves exchange and contact between gas or liquid and nucleic acid samples. This poses a risk of aerosol transmission, especially in the detection of certain infectious disease viruses. Furthermore, in the droplet generation process of traditional PCR microfluidic chips, a large number of discrete phases are generally generated continuously over a certain period of time, making it difficult to control droplet consistency. Therefore, developing a contactless PCR microfluidic chip with high droplet consistency is crucial. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a PCR microfluidic chip, comprising an "L"-shaped main plate, a sealing piston, and a piston driving device. The "L"-shaped main plate has a sample chamber, a splash guard, a piston chamber, and a sample application port on its vertical side, and a tiling chamber on its horizontal side. The sample chamber is located below the splash guard, and its end near the splash guard is connected to the splash guard. The piston chamber is located above the splash guard, and its end near the splash guard is connected to the splash guard. The sample application port's end near the splash guard is connected to the splash guard and / or the sample application port. The tiling chamber's end near the splash guard is connected to the splash guard. The sealing piston's end near the splash guard is inserted into the splash guard via the piston chamber. The piston driving device drives the sealing piston, causing it to close or open the connection between the splash guard and the tiling chamber.

[0006] In one embodiment of the present invention, the tiling cavity is horizontally arranged within the "L"-shaped main sheet, or the tiling cavity is inclined within the "L"-shaped main sheet.

[0007] In one embodiment of the present invention, when the tiling cavity is inclinedly arranged in the "L"-shaped main piece, the vertical distance between the tiling cavity and the horizontal plane gradually increases along the direction close to the splash guard, and the angle formed by the tiling cavity and the horizontal plane is 5 to 30°.

[0008] In one embodiment of the present invention, a sealing ring is fitted onto the end of the sealing piston near the splash guard.

[0009] In one embodiment of the present invention, the sample chamber is frustum-shaped; the diameter of the sample chamber gradually increases along the direction approaching the splash guard.

[0010] In one embodiment of the present invention, the splash guard and the piston chamber are cylindrical.

[0011] The present invention also provides a PCR detection device, which includes the above-mentioned PCR microfluidic chip and an ultrasonic generator; the ultrasonic generator is used to emit sound waves to the sample in the sample chamber, so that the sample forms droplets under the action of the sound waves.

[0012] In one embodiment of the present invention, the PCR detection device further includes a rotation drive device; the rotation drive device is used to drive the PCR microfluidic chip to rotate, so that the droplets enter the tiling cavity under the action of centrifugal force and gradually move to the periphery of the tiling cavity.

[0013] In one embodiment of the present invention, the ultrasonic generating device is a focused ultrasonic transducer, a phased array ultrasonic transducer, or a surface acoustic wave generator.

[0014] In one embodiment of the present invention, the acoustic impedance of the PCR microfluidic chip is close to that of the ultrasonic coupling agent.

[0015] The present invention also provides a PCR detection method, the method using the above-mentioned PCR detection device, comprising the following steps:

[0016] Oil layer pre-positioning steps: Invert the PCR microfluidic chip and add driving oil into the splash well through the sample addition port, so that the driving oil enters the spreading chamber;

[0017] Droplet generation steps: After the oil layer pre-placement step, seal the sample well and invert the PCR microfluidic chip; after inversion, open the sample well and add the sample into the sample chamber through the sample well; after addition, seal the sample well again and immerse the vertical side of the PCR microfluidic chip in the ultrasonic coupling agent; after immersion, turn on the ultrasonic generator and emit sound waves to the sample in the sample chamber through the ultrasonic coupling agent, so that the sample forms droplets under the action of the sound waves, and the formed droplets enter the flattening chamber under the action of gravity or centrifugal force;

[0018] Amplification Steps: After the droplet generation step is completed, the sealing piston is driven by the piston drive device, which blocks the connection between the splash guard and the tiling chamber. After the blocking is completed, the PCR microfluidic chip is temperature-cycled using the amplification tool to achieve PCR amplification.

[0019] Detection steps: After the amplification step is completed, the PCR microfluidic chip is read using an optical detection fixture to achieve PCR detection.

[0020] In one embodiment of the present invention, the ultrasonic generating device is a focused ultrasonic transducer; the parameters of the focused ultrasonic transducer are: focal length 6-18mm, frequency 1-150MHz, but not limited to the above parameters, mainly considering the liquid depth and droplet size for parameter selection; excitation power 0-50W adjustable; excitation duration 50-600μs adjustable, the excitation power and time are not limited to the above parameters, and are adjusted based on parameters such as sample characteristics and the required droplet size; excitation signal: a sine wave is used.

[0021] In one embodiment of the present invention, the detection steps are as follows: fluorescence excitation within the tiled cavity is performed using an LED light source; planar imaging is performed using a CCD or CMOS detector; image stitching and grayscale calibration are performed through algorithm processing, and the total number of droplets and the number of positive droplets are counted; the original concentration of the target DNA molecule is calculated.

[0022] The present invention also provides the application of the above-mentioned PCR microfluidic chip or PCR detection device or the above-mentioned PCR detection method in PCR detection.

[0023] The technical solution of this invention has the following advantages:

[0024] This invention provides a PCR microfluidic chip based on ultrasonic droplet generation, comprising an "L"-shaped main plate, a sealing piston, and a piston driving device. The "L"-shaped main plate has a sample chamber, a splash guard, a piston chamber, and a sample dispensing port on its vertical side, and a tiling chamber on its horizontal side. The sample chamber is located below the splash guard, and its end near the splash guard is connected to the splash guard. The piston chamber is located above the splash guard, and its end near the splash guard is connected to the splash guard. The sample dispensing port's end near the splash guard is connected to the splash guard and / or the sample dispensing port. The tiling chamber's end near the splash guard is connected to the splash guard. The sealing piston's end near the splash guard is inserted into the splash guard through the piston chamber. The piston driving device drives the sealing piston, causing it to close or open the connection between the splash guard and the tiling chamber. The PCR microfluidic chip described herein is simple in design and fabrication, requiring no complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The chip utilizes an external ultrasonic generator to generate droplets, allowing for precise control over individual droplet size and uniformity by adjusting the distance, frequency, power, excitation duration, and the excitation signal. The acoustic impedance of the PCR microfluidic chip is close to that of the ultrasonic coupling agent, ensuring maximum energy transfer and minimal reflection when using the external ultrasonic generator to generate droplets. The splash guard and sample chamber of the PCR microfluidic chip are of a certain length, reducing the speed at which droplets reach the droplet receiving area at the top of the chip cavity, preventing damage to the droplet morphology due to excessive kinetic energy. Furthermore, the presence of the splash guard also helps to prevent chip damage during rotation. To prevent liquid sloshing and splashing onto the droplet receiving area at the top of the chip; and, when the sample volume is large, the presence of the splash guard also prevents uncontrollable droplet formation when the liquid surface, due to the mound formed by the sound waves, comes into contact with the droplet receiving area at the top of the chip cavity when the droplets are generated using an external ultrasonic generator; the sealing piston of the PCR microfluidic chip can block the connection between the splash guard and the tiling cavity under the drive of the piston drive device, so that the tiling cavity forms a closed space, avoiding liquid evaporation during amplification due to the space inside the sample cavity and the splash guard; the PCR microfluidic chip uses an external ultrasonic generator, which generates droplets through the coupling contact between the ultrasonic generator and the PCR microfluidic chip. Compared with on-chip transducers (on-chip transducers require processing lithium niobate and metal electrodes onto the microfluidic chip, which has high cost and process requirements, and each chip is for single use, so the ultrasonic generator is also for single use), the ultrasonic generator of the PCR microfluidic chip can be reused multiple times, greatly reducing costs.In summary, the PCR microfluidic chip described above enables the control and encoding of individual droplet units for PCR detection, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing. Attached Figure Description

[0025] Figure 1 : A schematic diagram of the overall structure of one embodiment of the PCR microfluidic chip of the present invention.

[0026] Figure 2 : A cross-sectional structural schematic diagram of one embodiment of the PCR microfluidic chip of the present invention.

[0027] Figure 3 : A partial structural schematic diagram of one embodiment of the PCR microfluidic chip of the present invention.

[0028] Figure 4 : Schematic diagram of the oil layer pre-setting steps.

[0029] Figure 5 : A schematic diagram of the droplet generation process.

[0030] Figure 6 : A schematic diagram of the amplification steps.

[0031] Figure 7 Detection results of droplets obtained in Examples 1-3.

[0032] Figure 8 Detection results of droplets obtained in Examples 2-3.

[0033] Figures 1-3 In the middle, there are: 1. L-shaped main plate, 2. Sealed piston, 3. Piston drive device, 4. Sample chamber, 5. Anti-splash well, 6. Piston chamber, 7. Sample feeding hole, 8. Flattening chamber, and 9. Sealing ring. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0035] Example 1-1: A PCR microfluidic chip

[0036] like Figures 1-3This embodiment provides a PCR microfluidic chip, which includes an "L"-shaped main plate 1, a sealed piston 2, and a piston driving device 3. The "L"-shaped main plate 1 has a sample chamber 4, a splash guard 5, a piston chamber 6, and a sample application port 7 on its vertical side, and a tiling chamber 8 on its horizontal side. The sample chamber 4 is located below the splash guard 5, and the end of the sample chamber 4 near the splash guard 5 is connected to the splash guard 5. The piston chamber 6 is located above the splash guard 5, and the end of the piston chamber 6 near the splash guard 5 is connected to the splash guard 5. The end of the sample application port 7 near the splash guard 5 is connected to the splash guard 5. The end of the tiling chamber 8 near the splash guard 5 is connected to the splash guard 5. The sealed piston... 2. The end of the piston 2 closest to the splash guard 5 is inserted into the splash guard 5 via the piston chamber 6; the piston driving device 3 is used to drive the sealing piston 2, so that the sealing piston 2, driven by the piston driving device 3, closes or opens the connection between the splash guard 5 and the tiling chamber 8; the vertical distance between the tiling chamber 8 and the horizontal plane gradually increases along the direction close to the splash guard 5, and the angle formed by the tiling chamber 5 and the horizontal plane is 5-30°; a sealing ring 9 is fitted on the end of the sealing piston 2 closest to the splash guard 5; the sample chamber 4 is frustoconical, and the diameter of the sample chamber 4 gradually increases along the direction close to the splash guard 5; the splash guard 5 and the piston chamber 6 are cylindrical; the acoustic impedance of the PCR microfluidic chip is close to the acoustic impedance of the ultrasonic coupling agent to reduce energy reflection. The ultrasonic coupling agent is usually purified water, which has an acoustic impedance of 1.48 MRayl. Considering the amplification (temperature) and detection (transparency) characteristics that PCR microfluidic chips also need to have, PCR microfluidic chips generally use cyclic olefin copolymer (COC) materials with an acoustic impedance of 2.4 to 2.6 MRayl.

[0037] The PCR microfluidic chip described herein is simple in design and fabrication, requiring no complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The chip utilizes an external ultrasonic generator to generate droplets, allowing for precise control over individual droplet size and uniformity by adjusting the distance, frequency, power, excitation duration, and the excitation signal. The acoustic impedance of the PCR microfluidic chip is close to that of the ultrasonic coupling agent, ensuring maximum energy transfer and minimal reflection when using the external ultrasonic generator to generate droplets. The splash guard and sample chamber of the PCR microfluidic chip are of sufficient length to reduce the velocity of droplets reaching the droplet receiving area at the top of the chip cavity, preventing excessive kinetic energy from altering the droplet morphology. Damage to the sample volume; furthermore, when the sample volume is large, the presence of the splash guard can also prevent the liquid surface from contacting the droplet receiving area at the top of the chip cavity due to the mound formed by the sound waves when generating microdroplets using an external ultrasonic generator, thus preventing uncontrollable droplets from forming; the sealing piston of the PCR microfluidic chip can block the connection between the splash guard and the tiling cavity under the drive of the piston driving device, so that the tiling cavity forms a closed space, avoiding liquid evaporation during amplification caused by the space inside the sample cavity and the splash guard; the PCR microfluidic chip uses an external ultrasonic generator, which generates microdroplets through the coupling contact between the ultrasonic generator and the PCR microfluidic chip. Compared with on-chip transducers (on-chip transducers require processing lithium niobate and metal electrodes onto the microfluidic chip, which has high cost and process requirements, and each chip is for single use, so the ultrasonic generator is also for single use), the ultrasonic generator of the PCR microfluidic chip can be reused multiple times, greatly reducing the cost. In summary, the PCR microfluidic chip described above enables the control and encoding of individual droplet units for PCR detection, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing.

[0038] Examples 1-2: A PCR detection device

[0039] This embodiment provides a PCR detection device, which includes the PCR microfluidic chip of Embodiment 1-1 and an ultrasonic generator; the ultrasonic generator is used to emit sound waves to the sample in the sample chamber, so that the sample forms droplets under the action of the sound waves; the ultrasonic generator is a focused ultrasonic transducer, a phased array ultrasonic transducer or a surface acoustic wave generator.

[0040] When the ultrasonic generator emits sound waves to the sample in the sample chamber, the sound waves are focused on the liquid surface and vibrate. Due to the Rayleigh imbalance principle, tiny mounds will form on the liquid surface. When the energy is sufficient to overcome the surface tension of the liquid, the droplets will detach from the liquid and be sprayed vertically onto the sealed piston. The droplets sprayed onto the sealed piston enter the inclined tiling chamber under the action of gravity and are evenly spread in the tiling chamber.

[0041] The PCR microfluidic chip used in the PCR detection device has a simple design and manufacturing process, eliminating the need for complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The device utilizes an external ultrasonic generator to generate droplets, allowing for precise control of individual droplet size and high consistency through adjustments to the distance, frequency, power, excitation duration, and specific excitation signal control. The acoustic impedance of the PCR microfluidic chip is close to that of the ultrasonic coupling agent, ensuring maximum energy transfer and minimal reflection when using the external ultrasonic generator to generate droplets. The PCR microfluidic chip incorporates a splash guard and a sample chamber of sufficient length to minimize the risk of droplets reaching the top of the chip's inner cavity. The speed at which the droplets reach the receiving area is controlled to avoid damage to the droplet morphology due to excessive kinetic energy. Furthermore, when the sample volume is large, the presence of the splash guard prevents uncontrollable droplet formation when the liquid surface, formed by the acoustic waves, contacts the droplet receiving area at the top of the chip cavity during droplet generation using an external ultrasonic generator. The PCR microfluidic chip used in the PCR detection device is equipped with a sealing piston. Driven by the piston drive device, the sealing piston blocks the connection between the splash guard and the tiling cavity, creating a closed space within the tiling cavity and preventing liquid evaporation during amplification from the sample cavity and the space within the splash guard. The PCR microfluidic chip used in the PCR detection device employs an external ultrasonic generator. Droplets are generated through coupling contact between the ultrasonic generator and the PCR microfluidic chip. Compared to on-chip transducers (which require processing lithium niobate and metal electrodes onto the microfluidic chip, resulting in higher costs and process requirements, and each chip is for single use, thus the acoustic generator is also for single use), the cost is significantly reduced. In summary, the PCR detection device described above enables the control and encoding of individual droplet units, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing.

[0042] Examples 1-3: A PCR detection method

[0043] This embodiment provides a PCR detection method using the PCR detection device of Examples 1-2, and includes the following steps:

[0044] Oil layer pre-placement step: Invert the PCR microfluidic chip (the material of the PCR microfluidic chip is COC), and add driving oil through the sample application port into the splash guard. The driving oil (choose a driving oil of suitable viscosity, such as fluorinated oil 7500, to avoid oil dripping when the PCR microfluidic chip is upright) enters the spreading cavity under the influence of gravity and spreads evenly throughout the spreading cavity. In this step, the driving oil is used to encapsulate the droplets generated in the following steps, preventing droplet fusion and breakage. It also serves as a buffer for droplet ejection, preventing contact with the microdroplet receiving area at the top of the chip cavity during ejection, which could lead to splashing or breakage (see details). Figure 4 );

[0045] Droplet generation steps: After the oil layer pre-setting step, seal the sample well and invert the PCR microfluidic chip; after inversion, open the sample well and add the sample into the sample chamber through the sample well; after adding the sample, seal the sample well again and immerse the vertical side of the PCR microfluidic chip in an ultrasonic coupling agent (the ultrasonic coupling agent can be water or gel); after immersion, turn on the ultrasonic generator and emit sound waves to the sample in the sample chamber through the ultrasonic coupling agent, causing the sample to form droplets under the action of the sound waves. The formed droplets enter the spreading cavity under the action of gravity; in this step, when the ultrasonic generator emits sound waves to the sample in the sample chamber, the sound waves are focused on the liquid surface and generate vibration. Due to the Rayleigh imbalance principle, tiny mounds will form on the liquid surface. When the energy is sufficient to overcome the surface tension of the liquid, the droplets will detach from the liquid and be vertically sprayed onto the sealed piston. The droplets sprayed onto the sealed piston enter the tilted spreading cavity under the action of gravity and are evenly spread in the spreading cavity (see details). Figure 5 );

[0046] Amplification Steps: After the droplet generation step, a piston-driven device is used to drive a sealing piston, which blocks the connection between the splash guard and the tiling chamber. After blocking, the PCR microfluidic chip is temperature-cycled using an amplification fixture to achieve PCR amplification (see details). Figure 6 );

[0047] Detection steps: After the amplification step is completed, the PCR microfluidic chip is read using an optical detection fixture to achieve PCR detection.

[0048] The droplets formed using the method described above were statistically analyzed using a microscope combined with a CCD imaging algorithm (a focused ultrasonic transducer was used, with the following parameters: focal length 9, 12, 18 mm; frequency 10–30 MHz; power adjustable from 0 to 50 W; excitation duration adjustable from 50 to 600 μs; excitation signal: sine wave). The detection results are shown in […]. Figure 7 . Figure 7 In the study, the dimensions of 12 microdroplets with a theoretical diameter of 110 μm were measured, and the contact angle between the sample and the receiving plate was found to be approximately 75°. The actual calculated average equivalent diameter was 113 μm; CV: 1.63%.

[0049] The PCR microfluidic chip used in the described PCR detection method has a simple design and manufacturing process, eliminating the need for complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The method utilizes an external ultrasonic generator to generate droplets, allowing for precise control of individual droplet size and high consistency by adjusting the distance, frequency, power, excitation duration, and the excitation signal. The acoustic impedance of the PCR microfluidic chip is close to that of the ultrasonic coupling agent, ensuring maximum energy transfer and minimal reflection when using the external ultrasonic generator to generate droplets. The PCR microfluidic chip incorporates a splash guard and a sample chamber of sufficient length to minimize the risk of droplets reaching the top of the chip's inner cavity. The velocity in the droplet receiving area is controlled to avoid damage to the droplet morphology due to excessive kinetic energy. Furthermore, when the sample volume is large, the presence of the splash guard prevents uncontrollable droplet formation when the liquid surface, formed by the acoustic waves, contacts the droplet receiving area at the top of the chip cavity during droplet generation using an external ultrasonic generator. The PCR microfluidic chip used in the PCR detection method is equipped with a sealing piston. Driven by a piston drive device, the sealing piston blocks the connection between the splash guard and the tiling cavity, creating a closed space within the tiling cavity and preventing liquid evaporation during amplification from the sample cavity and the space within the splash guard. The PCR microfluidic chip used in the PCR detection method employs an external ultrasonic generator. Droplets are generated through coupling contact between the ultrasonic generator and the PCR microfluidic chip. This, combined with an on-chip transducer (which requires processing lithium niobate and metal electrodes onto the microfluidic chip, resulting in high cost and process requirements, and each chip is for single use only, thus the acoustic generator is also for single use), significantly reduces costs. In summary, the PCR detection method described above enables the control and encoding of individual droplet units, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing.

[0050] Example 2-1: A PCR microfluidic chip

[0051] like Figures 1-3 This embodiment provides a PCR microfluidic chip, which includes an "L"-shaped main plate 1, a sealed piston 2, and a piston driving device 3. The "L"-shaped main plate 1 has a sample chamber 4, a splash guard 5, a piston chamber 6, and a sample application port 7 on its vertical side, and a tiling chamber 8 on its horizontal side. The sample chamber 4 is located below the splash guard 5, and the end of the sample chamber 4 near the splash guard 5 is connected to the splash guard 5. The piston chamber 6 is located above the splash guard 5, and the end of the piston chamber 6 near the splash guard 5 is connected to the splash guard 5. The end of the sample application port 7 near the splash guard 5 is connected to the splash guard 5. The tiling chamber 8 is located near the splash guard 5. One end of the sealing piston 2 is connected to the splash well 5; the end of the sealing piston 2 near the splash well 5 is inserted into the splash well 5 through the piston cavity 6; the piston driving device 3 is used to drive the sealing piston 2, so that the sealing piston 2, under the drive of the piston driving device 3, closes or opens the connection between the splash well 5 and the flat cavity 8; the flat cavity 8 is horizontally arranged in the "L"-shaped main plate 1; a sealing ring 9 is fitted on the end of the sealing piston 2 near the splash well 5; the sample cavity 4 is frustoconical, and the diameter of the sample cavity 4 gradually increases along the direction close to the splash well 5; the splash well 5 and the piston cavity 6 are cylindrical; the acoustic impedance of the PCR microfluidic chip is close to the acoustic impedance of the ultrasonic coupling agent to reduce energy reflection. The ultrasonic coupling agent is usually purified water, which has an acoustic impedance of 1.48 MRayl. Considering the amplification (temperature) and detection (transparency) characteristics that PCR microfluidic chips also need to have, PCR microfluidic chips generally use cyclic olefin copolymer (COC) materials with an acoustic impedance of 2.4 to 2.6 MRayl.

[0052] The PCR microfluidic chip described herein is simple in design and fabrication, requiring no complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The chip utilizes an external ultrasonic generator to generate droplets, allowing for precise control over individual droplet size and uniformity by adjusting the distance, frequency, power, excitation duration, and the excitation signal. The acoustic impedance of the PCR microfluidic chip is close to that of the ultrasonic coupling agent, ensuring maximum energy transfer and minimal reflection when using the external ultrasonic generator to generate droplets. The splash guard and sample chamber of the PCR microfluidic chip are of a certain length, reducing the velocity of droplets reaching the droplet receiving area at the top of the chip cavity and preventing damage to the droplet morphology due to excessive kinetic energy. Furthermore, the presence of the splash guard also helps to mitigate the impact of chip rotation. To prevent liquid sloshing and splashing onto the droplet receiving area at the top of the chip cavity, the presence of the splash guard also prevents uncontrollable droplet formation when the liquid surface, due to the mound formed by the sound waves, comes into contact with the droplet receiving area at the top of the chip cavity when the sample volume is large. The sealing piston of the PCR microfluidic chip can block the connection between the splash guard and the tiling cavity under the drive of the piston drive device, so that the tiling cavity forms a closed space, avoiding liquid evaporation during amplification due to the space inside the sample cavity and the splash guard. The PCR microfluidic chip uses an external ultrasonic generator to generate droplets through the coupling contact between the ultrasonic generator and the PCR microfluidic chip. Compared with on-chip transducers (on-chip transducers require processing lithium niobate and metal electrodes onto the microfluidic chip, which has high cost and process requirements, and each chip is for single use, so the ultrasonic generator is also for single use), the ultrasonic generator of the PCR microfluidic chip can be reused multiple times, greatly reducing costs. In summary, the PCR microfluidic chip described above enables the control and encoding of individual droplet units for PCR detection, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing.

[0053] Example 2-2: A PCR detection device

[0054] This embodiment provides a PCR detection device, which includes the PCR microfluidic chip of Embodiment 2-1, an ultrasonic generator, and a rotation drive device. The ultrasonic generator is used to emit sound waves to the sample in the sample chamber, so that the sample forms droplets under the action of the sound waves. The ultrasonic generator is a focused ultrasonic transducer, a phased array ultrasonic transducer, or a surface acoustic wave generator. The rotation drive device is used to drive the PCR microfluidic chip to rotate, so that the droplets enter the tiling chamber under the action of centrifugal force and gradually move towards the periphery of the tiling chamber.

[0055] When the ultrasonic generator emits sound waves to the sample in the sample chamber, the sound waves are focused on the liquid surface and vibrate. Due to the Rayleigh imbalance principle, tiny mounds will form on the liquid surface. When the energy is sufficient to overcome the surface tension of the liquid, the droplets will detach from the liquid and be sprayed vertically onto the sealed piston. The droplets sprayed onto the sealed piston enter the inclined spreading chamber under the action of centrifugal force and spread evenly in the spreading chamber.

[0056] The PCR microfluidic chip used in the PCR detection device has a simple design and manufacturing process, eliminating the need for complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The device utilizes an external ultrasonic generator to generate droplets, allowing for precise control of individual droplets by adjusting the distance, frequency, power, excitation duration, and the excitation signal. This ensures controllable droplet size and high consistency. The PCR microfluidic chip's acoustic impedance is close to that of the ultrasonic coupling agent, maximizing energy transfer and minimizing reflection when using the external ultrasonic generator to generate droplets. The PCR microfluidic chip incorporates a splash guard and a sample chamber of sufficient length to reduce the velocity of droplets reaching the droplet receiving area at the top of the chip, preventing damage to the droplet morphology due to excessive kinetic energy. In addition, the presence of the splash guard during chip rotation prevents liquid sloshing from splashing onto the droplet receiving area at the top of the chip cavity. Furthermore, when the sample volume is large, the splash guard prevents uncontrollable droplet formation when the liquid surface, due to the mounds formed by the sound waves, comes into contact with the droplet receiving area at the top of the chip cavity during droplet generation using an external ultrasonic generator. The PCR microfluidic chip used in the PCR detection device is equipped with a sealing piston. Driven by the piston drive device, the sealing piston blocks the connection between the splash guard and the tiling cavity, creating a closed space within the tiling cavity and preventing liquid evaporation during amplification from the sample cavity and the space within the splash guard. The PCR detection device employs an external ultrasonic generator on the PCR microfluidic chip. Droplets are generated through the coupling contact between the ultrasonic generator and the PCR microfluidic chip. Compared to on-chip transducers (which require processing lithium niobate and metal electrodes onto the microfluidic chip, resulting in higher costs and process requirements, and each chip is for single use, thus the ultrasonic generator is also for single use), the ultrasonic generator can be reused multiple times, significantly reducing costs. In summary, the PCR detection device described above enables the control and encoding of individual droplet units, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing.

[0057] Examples 2-3: A PCR detection method

[0058] This embodiment provides a PCR detection method using the PCR detection device of Embodiment 2-2, comprising the following steps:

[0059] Oil layer pre-positioning step: Invert the PCR microfluidic chip (the material of the PCR microfluidic chip is COC) and add driving oil into the splash well through the sample application port; after adding, turn on the rotary drive device to control the rotation of the PCR microfluidic chip (speed 1-10 rpm), so that the driving oil (choose a driving oil with appropriate viscosity, such as fluorinated oil 7500, to avoid oil layer dripping when the PCR microfluidic chip is upright) enters the spreading chamber under the action of centrifugal force and is evenly spread in the spreading chamber; in this step, the driving oil is used to coat the droplets generated in the following steps to prevent droplet fusion and breakage, and also serves as a buffer for droplet spraying to prevent contact with the microdroplet receiving area at the top of the chip cavity during spraying, which could lead to splashing or breakage (see details). Figure 4 );

[0060] Droplet generation steps: After the oil layer pre-setting step, seal the sample well and invert the PCR microfluidic chip; after inversion, open the sample well and add the sample into the sample chamber through the sample well; after adding the sample, seal the sample well again and immerse the vertical side of the PCR microfluidic chip in an ultrasonic coupling agent (the ultrasonic coupling agent can be water or gel); after immersion, turn on the ultrasonic generator and emit sound waves to the sample in the sample chamber through the ultrasonic coupling agent, causing the sample to form droplets under the action of the sound waves. At the same time, turn on the rotation drive device to control the rotation of the PCR microfluidic chip (speed 1-10 rpm), so that the formed droplets enter the spreading chamber under the action of centrifugal force; in this step, when the ultrasonic generator emits sound waves to the sample in the sample chamber, the sound waves are focused on the liquid surface and generate vibration. Due to the Rayleigh imbalance principle, tiny mounds will form on the liquid surface. When the energy is sufficient to overcome the surface tension of the liquid, the droplets will detach from the liquid and be sprayed vertically onto the sealed piston. The droplets sprayed onto the sealed piston enter the horizontally set spreading chamber under the action of centrifugal force and are evenly spread in the spreading chamber (see details). Figure 5 );

[0061] Amplification Steps: After the droplet generation step, a piston-driven device is used to drive a sealing piston, which blocks the connection between the splash guard and the tiling chamber. After blocking, the PCR microfluidic chip is temperature-cycled using an amplification fixture to achieve PCR amplification (see details). Figure 6 );

[0062] Detection steps: After the amplification step is completed, the PCR microfluidic chip is read using an optical detection fixture to achieve PCR detection.

[0063] The droplets formed using the method described above were statistically analyzed using a microscope combined with a CCD imaging algorithm (a focused ultrasonic transducer was used, with the following parameters: focal length 9, 12, 18 mm; frequency 10–30 MHz; power adjustable from 0 to 50 W; excitation duration adjustable from 50 to 600 μs; excitation signal: sine wave). The detection results are shown in […]. Figure 8 . Figure 8 In the study, the size of 12 microdroplets with a theoretical diameter of 120 μm was measured, and the contact angle between the sample and the receiving plate was found to be approximately 75°. The actual calculated average diameter was 122 μm; CV: 1.31%.

[0064] The PCR microfluidic chip used in the described PCR detection method has a simple design and manufacturing process, eliminating the need for complex flow channels and resulting in low cost. The PCR microfluidic chip is completely sealed, with droplet generation, PCR amplification, and reading all occurring within the sealed chip, preventing aerosol contamination. The method utilizes an external ultrasonic generator to generate droplets, allowing for precise control of individual droplets by adjusting the distance, frequency, power, excitation duration, and the excitation signal. This ensures controllable droplet size and high consistency. The PCR microfluidic chip's acoustic impedance is close to that of the ultrasonic coupling agent, maximizing energy transfer and minimizing reflection when using the external ultrasonic generator to generate droplets. The PCR microfluidic chip incorporates a splash guard and a sample chamber of sufficient length to reduce the velocity of droplets reaching the droplet receiving area at the top of the chip cavity, preventing damage to the droplet morphology due to excessive kinetic energy. In addition, the presence of the splash guard during chip rotation prevents liquid sloshing from splashing onto the droplet receiving area at the top of the chip cavity. Furthermore, when the sample volume is large, the splash guard prevents uncontrollable droplet formation when the liquid surface, due to the mounds formed by the sound waves, contacts the droplet receiving area at the top of the chip cavity when using an external ultrasonic generator to generate droplets. The PCR microfluidic chip used in the PCR detection method is equipped with a sealing piston. Driven by a piston drive device, the sealing piston blocks the connection between the splash guard and the tiling cavity, creating a closed space within the tiling cavity and preventing liquid evaporation during amplification from the sample cavity and the space within the splash guard. The PCR detection method employs an external ultrasonic generator on the PCR microfluidic chip. Droplets are generated through coupling contact between the ultrasonic generator and the PCR microfluidic chip. Compared to on-chip transducers (which require processing lithium niobate and metal electrodes onto the microfluidic chip, resulting in higher costs and process requirements, and each chip is for single use, thus the ultrasonic generator is also for single use), the ultrasonic generator can be reused multiple times, significantly reducing costs. In summary, the PCR detection method described above enables the control and encoding of individual droplet units, making it suitable for molecular diagnostic fields such as single-molecule diagnostics and single-molecule sequencing.

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A droplet-based digital PCR detection device, characterized in that, The droplet-type digital PCR detection device includes a PCR microfluidic chip, an ultrasonic generator, and a rotation drive device; The PCR microfluidic chip includes an "L"-shaped main plate, a sealed piston, and a piston drive device. The "L"-shaped main plate has a sample chamber, a splash guard, a piston chamber, and a sample application port on its vertical side, and a tiling chamber on its horizontal side. The sample chamber is located below the splash guard, and its end near the splash guard is connected to the splash guard. The piston chamber is located above the splash guard, and its end near the splash guard is connected to the splash guard. The sample application port's end near the splash guard is connected to the splash guard. The tiling chamber's end near the splash guard is connected to the splash guard. The sealing piston is inserted into the splash well via a piston chamber at one end near the splash well. A piston drive device drives the sealing piston to close or open the connection between the splash well and the paving chamber. The vertical distance between the paving chamber and the horizontal plane gradually increases towards the splash well, and the angle between the paving chamber and the horizontal plane is 5-30°. A sealing ring is fitted onto the end of the sealing piston near the splash well. The sample chamber is frustum-shaped. The diameter of the sample chamber gradually increases towards the splash well. Both the splash well and the piston chamber are cylindrical. The ultrasonic generator is used to emit sound waves to the sample in the sample chamber, so that the sample forms droplets under the action of the sound waves. The rotary drive device is used to drive the PCR microfluidic chip to rotate, so that the droplets enter the tiling chamber under the action of centrifugal force and gradually move to the periphery of the tiling chamber.

2. A digital PCR detection method, characterized in that, The method uses the droplet digital PCR detection device according to claim 1, and includes the following steps: Oil layer pre-positioning steps: Invert the PCR microfluidic chip and add driving oil into the splash well through the sample addition port, so that the driving oil enters the spreading chamber; Droplet generation steps: After the oil layer pre-placement step, seal the sample well and invert the PCR microfluidic chip; after inversion, open the sample well and add the sample into the sample chamber through the sample well; after addition, seal the sample well again and immerse the vertical side of the PCR microfluidic chip in the ultrasonic coupling agent; after immersion, turn on the ultrasonic generator and emit sound waves to the sample in the sample chamber through the ultrasonic coupling agent, so that the sample forms droplets under the action of the sound waves, and the formed droplets enter the flattening chamber under the action of gravity or centrifugal force; Amplification Steps: After the droplet generation step is completed, the sealing piston is driven by the piston drive device, which blocks the connection between the splash guard and the tiling chamber. After the blocking is completed, the PCR microfluidic chip is temperature-cycled using the amplification tool to achieve PCR amplification. Detection steps: After the amplification step is completed, the PCR microfluidic chip is read using an optical detection fixture to achieve PCR detection.

3. The application of the droplet digital PCR detection device according to claim 1 or the digital PCR detection method according to claim 2 in PCR detection.

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