DIGITAL PCR SYSTEM AND DIGITAL PCR DROPLET FORMATION METHOD

Abstract

A digital PCR system has at least one droplet forming assembly and a droplet spraying hole assembly. The droplet forming assembly has at least one droplet collecting tank; the droplet spraying hole assembly is connected below the droplet forming assembly. The droplet spraying hole assembly has a plurality of droplet spraying holes in communication with the droplet collecting tank. Vaporization parts are provided in the droplet spraying holes and used for vaporizing digital PCR solution liquid layers in the droplet spraying holes and quickly pushing the vaporized digital PCR solution liquid layers into droplet forming oil in the droplet collecting tank to form digital PCR droplets.

Claims

1. A digital PCR system, wherein, comprising: at least one droplet generation component comprising at least one droplet collection groove; a droplet nozzle member, which is connected below the droplet generation component and comprises a plurality of droplet nozzles, said droplet nozzles have openings on the upper surface of said droplet nozzle member and extend toward, but not through, the lower surface of said droplet nozzle member, said droplet nozzles are in communication with said droplet collection groove, and said droplet nozzles have vaporizing parts for vaporizing liquid layers of digital PCR solution in said droplet nozzles, and rapidly pushing the vapor into droplet generating oil in said droplet collection groove to generate digital PCR droplets.

2. The digital PCR system of claim 1, wherein, said droplet nozzle member comprises thermal bubble print chips.

3. The digital PCR system of claim 1, wherein, said vaporizing part is arranged on a lower surface or sidewall of said droplet nozzle.

4. The digital PCR system of claim 1, wherein, the shape of the opening of said droplet nozzle comprises any one selected from the group consisting of round, ellipse and polygon.

5. The digital PCR system of claim 1, wherein, said vaporizing parts comprise heating elements for vaporizing liquid layers of said digital PCR solution by heating.

6. The digital PCR system of claim 5, wherein, said heating elements comprise at least one metal layer.

7. The digital PCR system of claim 1, wherein, a through groove is provided at the bottom of said droplet collection groove, and said through groove exposes a plurality of said droplet nozzles.

8. The digital PCR system of claim 1, wherein, said PCR system further comprises at least one PCR reagent chamber for storing digital PCR solution, said droplet nozzle member has a flow channel, and said droplet nozzles are in communication with said PCR reagent chamber through said flow channel.

9. The digital PCR system of claim 8, wherein, said flow channel comprise at least one main flow channel and a plurality of branch flow channels connecting to said main flow channel, and each of said droplet nozzles is connected to one of said branch flow channels, respectively.

10. The digital PCR system of claim 8, wherein, said digital PCR system further comprises a substrate, said PCR reagent chamber is arranged in said substrate, and said droplet nozzle member is connected above said substrate.

11. The digital PCR system of claim 10, wherein, said substrate comprises a first substrate component and a second substrate component, and said PCR reagent chamber comprises a PCR reagent upper chamber and a PCR reagent lower chamber, said PCR reagent upper chamber has an opening on a upper surface of said first substrate component and through the lower surface of said first substrate component, said PCR reagent lower chamber has an opening on a upper surface of said second substrate component and extends toward, but not through, a lower surface of said second substrate component, and said PCR reagent upper chamber is in communication with and partially overlapped with said PCR reagent lower chamber.

12. The digital PCR system of claim 11, wherein, at least one digital PCR solution injection hole is provided on the lower surface of said second substrate component, said digital PCR solution injection hole being connected to said PCR reagent lower chamber.

13. The digital PCR system of claim 11, wherein, said PCR reagent lower chamber comprises a first end and a second end, said digital PCR solution injection hole is in communication with said PCR reagent lower chamber at said first end, said PCR reagent lower chamber is in communication with said PCR reagent upper chamber at said second end, said PCR reagent lower chamber being firstly progressively increased and then gradually decreased in size from said first end to said second end.

14. The digital PCR system of claim 11, wherein, at least one exhaust port is provided on the lower surface of said second substrate component, said exhaust port is in communication with said PCR reagent upper chamber via a gas passage, said gas passage has an opening on the upper surface of said second substrate component and extends toward, but not through, the lower surface of said second substrate component.

15. The digital PCR system of claim 11, wherein, said first substrate component is fixed above said second substrate component by gluing.

16. The digital PCR system of claim 10, wherein, said digital PCR system further comprises a flexible circuit board, said flexible circuit board being connected above the upper surface of the substrate, said flexible circuit board has a through hole for accommodating said droplet nozzle member, a plurality of first connection pads and second connection pads are arranged on the surface of said flexible circuit board, and said droplet nozzle member is connected to said first connection pads by conducting wires.

17. The digital PCR system of claim 16, wherein, said flexible circuit board is connected to said substrate by gluing, a channel being provided on the surface of said substrate for preventing glue from flowing to said droplet nozzle member.

18. The digital PCR system of claim 16, wherein, at least two positioning through holes are arranged in said flexible circuit board, and positioning bumps at positions corresponding to the positioning through holes are provided on the surface of said substrate.

19. The digital PCR system of claim 16, wherein, said digital PCR system further comprises a controller, said controller comprises a controller housing and a controller circuit board arranged in said controller housing, said controller housing has a support for placing said substrate, a plurality of conductive pins for circuit connection connected to said circuit connection board of the controller are arranged on a surface of said support, said conductive pins for circuit connection are at positions corresponding to said second connection pads.

20. The digital PCR system of claim 19, wherein, at least one position-limiting slot is provided at one end of said substrate, and said controller housing has at least one position-limiting part corresponding to said position-limiting slot.

21-28. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1a is a perspective view of the digital PCR system of the present invention.

[0047] FIG. 1b is a top view of the digital PCR system of the present invention.

[0048] FIG. 1c is a bottom view of the digital PCR system of the present invention.

[0049] FIG. 1d to FIG. 1g are side views of the digital PCR system.

[0050] FIG. 2 is an exploded view of the digital PCR system of the present invention.

[0051] FIG. 3 is a perspective view of the droplet nozzle member in the digital PCR system of the present invention.

[0052] FIG. 4 is a partial cross-sectional view of the droplet nozzle member in the digital PCR system of the present invention.

[0053] FIG. 5a is a front perspective view of the droplet generation component in the digital PCR system of the present invention.

[0054] FIG. 5b is a back perspective view of the droplet generation component in the digital PCR system of the present invention.

[0055] FIG. 5c is a top view of the droplet generation component in the digital PCR system of the present invention.

[0056] FIG. 5d is a bottom view of the droplet generation component in the digital PCR system of the present invention.

[0057] FIG. 5e to FIG. 5h are side views of the droplet generation component in the digital PCR system of the present invention.

[0058] FIG. 6a is a perspective view of the substrate in the digital PCR system of the present invention.

[0059] FIG. 6b is a top view of the substrate in the digital PCR system of the present invention.

[0060] FIG. 6c is a bottom view of the substrate in the digital PCR system of the present invention.

[0061] FIG. 6d to FIG. 6g are side views of the substrate in the digital PCR system of the present invention.

[0062] FIG. 7 is a partial top view of the substrate in the digital PCR system of the present invention.

[0063] FIG. 8 is a front perspective view of the second substrate component in the digital PCR system of the present invention.

[0064] FIG. 9 is a back perspective view of the second substrate component in the digital PCR system of the present invention.

[0065] FIG. 10a is a front perspective view of the droplet nozzle member connected to the flexible circuit board in the digital PCR system of the present invention.

[0066] FIG. 10b is a back perspective view of the droplet nozzle member connected to the flexible circuit board in the digital PCR system of the present invention.

[0067] FIG. 10c is a top view of the droplet nozzle member connected to the flexible circuit board in the digital PCR system of the present invention.

[0068] FIG. 10d is a bottom view of the droplet nozzle member connected to the flexible circuit board in the digital PCR system of the present invention.

[0069] FIG. 10e to FIG. 10h are side views of the droplet nozzle member connected to the flexible circuit board in the digital PCR system of the present invention.

[0070] FIG. 11 is a perspective view of the controller in the digital PCR system of the present invention.

[0071] FIG. 12 is an optical microscope image of the digital PCR droplets generated by using the digital PCR system of the present invention.

LIST OF REFERRINGENCE NUMERALS

[0072] 1 droplet generation component [0073] 2 droplet collection groove [0074] 3 droplet nozzle member [0075] 4 droplet nozzle [0076] 5 vaporizing part [0077] 6 through groove [0078] 7 position-limiting hole [0079] 8 gas passage [0080] 9 main flow channel [0081] 10 branch flow channel [0082] 11 substrate [0083] 12 first substrate component [0084] 13 second substrate component [0085] 14 PCR reagent upper chamber [0086] 15 PCR reagent lower chamber [0087] 16 digital PCR solution injection hole [0088] 17 exhaust port [0089] 18 flexible circuit board [0090] 19 through hole [0091] 20 second connection pad [0092] 21 channel [0093] 22 positioning through hole [0094] 23 positioning bump [0095] 24 controller [0096] 25 controller housing [0097] 26 support [0098] 27 conductive pin for circuit connection [0099] 28 position-limiting slot [0100] 29, 30 position-limiting part [0101] 31 protuberance

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0102] Hereinafter, the embodiments of the present invention will be illustrated with specific examples, and other advantages and benefits of the present invention can be readily understood by those skilled in the art as disclosed in this specification. The present invention may also be implemented or applied with various other specific embodiments, and the details in this specification may be modified or altered in various ways based on different points of view and applications without departing from the spirit of the present invention.

[0103] Referring to FIG. 1a to FIG. 12. It should be noted that the illustrations provided in the examples only illustrate the basic concept of the present invention in a schematic manner, and that the drawings only show the components related to the present invention rather than the numbers, shapes and sizes of the components in actual implementation, and that the shape, number and ratio of each component in actual implementation may be changed arbitrarily, and the types of layout pattern of the components may be also more complicated.

Embodiment 1

[0104] The present invention provides a digital PCR system, as shown in FIG. 1a to FIG. 1g, among them, FIG. 1a is a perspective view of the digital PCR system, FIG. 1b displays a top view of the digital PCR system, FIG. 1c shows a bottom view of the digital PCR system, and FIG. 1d, FIG. 1e, FIG. 1f, and FIG. 1g are side views of the digital PCR system in four directions, respectively. FIG. 2 is an exploded view of the digital PCR system, in this embodiment, the digital PCR system comprises at least one droplet generation component 1 and a droplet nozzle member 3, the droplet nozzle member 3 is connected below the droplet generation component 1.

[0105] Specifically, the droplet generation component 1 comprises at least one droplet collection groove 2. FIG. 1a shows that the number of the droplet generation component 1 is one, and the droplet generation component 1 comprises four droplet collection grooves 2, wherein each droplet collection groove 2 is arranged in a row and two adjacent droplet collection grooves 2 share a common sidewall. Of course, in other embodiments, a plurality of droplet collection grooves 2 may also be arranged in other ways and may be arranged separately, the number of droplet generation components 1 may also be plural, and the scope of protection of the present invention should not be unduly limited herein.

[0106] Referring to FIG. 3, it shows a perspective view of droplet nozzle member 3, the droplet nozzle member 3 comprises a plurality of droplet nozzles 4. In the embodiment, droplet nozzles 4 are arranged in two rows and the droplet nozzles is evenly distributed in each row. In other embodiments, the droplet nozzles 4 may also be arranged in other ways, and the scope of protection of the present invention should not be unduly limited herein.

[0107] Referring to FIG. 4, it shows a partial cross-sectional view of the droplet nozzle member 3. In the embodiment, the droplet nozzles 4 have openings on the upper surface of droplet nozzle member 3, and extend toward, but not through, the lower surface of droplet nozzle member 3. The shape of the opening of each of droplet nozzles 4 comprises, but is not limited to, any one selected from the group consisting of round, ellipse and polygon. The volume of the digital PCR droplet to be generated is determined by the volume of the droplet nozzle 4.

[0108] As an example, the droplet nozzle member 3 may comprise thermal bubble print chips. Thermal bubble print technique is a major technique in the field of printers, the basic principle of the thermal bubble print technique is to eject ink droplets by heating ink. In the present invention, droplet nozzle member 3 may use existing thermal bubble print chips.

[0109] Referring to FIG. 5a to FIG. 5h, wherein, FIG. 5a is a front perspective view of the droplet generation component 1, FIG. 5b is a back perspective view of the droplet generation component 1, FIG. 5c is a top view of the droplet generation component 1, FIG. 5d is a bottom view of the droplet generation component 1, and FIG. 5e, FIG. 5f, FIG. 5g and FIG. 5h are side views of the droplet generation component 1 in four directions, respectively. In the embodiment, a through groove 6 is provided at the bottom of the droplet collection groove to expose a plurality of droplet nozzles 4, so that the droplet nozzles 4 are in communication with the droplet collection groove 2.

[0110] Specifically, as shown in FIG. 4, the droplet nozzle 4 is provided with a vaporizing part 5 for vaporizing liquid layers of the digital PCR solution inside the droplet nozzle and the vaporized solution is rapidly pushed into the droplet generating oil in the droplet collection groove 2 to generate digital PCR droplets.

[0111] As an example, the vaporizing part 5 is arranged on the bottom of droplet nozzle 4, and the vaporizing part 5 comprises a heating element for vaporizing the liquid layers of the digital PCR solution by heating. In the embodiment, the heating element comprises a heating plate, which may either a single metal layer or a composite multilayer metal layer. The shape of vaporizing part 5 comprises, but is not limited to, a round or square shape, and the area thereof may be 0.5 to 2 times the area of the bottom of the droplet nozzle 4. In other embodiments, the vaporizing part 5 may also be arranged on the sidewall of the droplet nozzle 4, and the scope of protection of the present invention should not be unduly limited herein.

[0112] Specifically, the PCR system further comprises at least one PCR reagent chamber for storing a digital PCR solution. As shown in FIG. 3 and FIG. 4, flow channels are provided in the droplet nozzle member 3, and the droplet nozzles 4 are in communication with the PCR reagent chamber through the flow channels.

[0113] As an example, the flow channels comprise at least one main flow channel 9 and a plurality of branch flow channels 10 in communication with the main flow channel 9, and each of the droplet nozzles 4 is in communication with one of the branch flow channels 10, respectively. FIG. 3 displays that the droplet nozzle member 3 comprises one main flow channel 9. In other embodiments, the number of the main flow channels 9 may also match the number of the droplet collection grooves 2. For example, as shown in FIG. 10b, the droplet nozzle member 3 comprises four main flow channels 9.

[0114] As an example, materials for constructing the flow channels and the droplet nozzles comprise, but are not limited to, silicon, polymers, photoresists, etc.

[0115] Specifically, as shown in FIG. 1a, the digital PCR system further comprises a substrate 11, the PCR reagent chamber is arranged in the substrate 11, and the droplet nozzle member 3 is connected above the upper surface of substrate 11.

[0116] As an example, a material for substrate 11 comprises, but is not limited to, any one of a transparent or opaque plastic, glass.

[0117] As an example, as shown in FIG. 2, the substrate 11 comprises a first substrate component 12 and a second substrate component 13, and the PCR reagent chamber comprises a PCR reagent upper chamber 14 and a PCR reagent lower chamber 15; the PCR reagent upper chamber 14 has an opening on the upper surface of the first substrate component 12 and extends through the lower surface of the first substrate component 12, the PCR reagent lower chamber 15 has an opening on the upper surface of the second substrate component 13 and extends toward, but not through, the lower surface of the second substrate component 13, and the PCR reagent upper chamber 14 is in communication with and partially overlapped with the PCR reagent lower chamber 15.

[0118] Referring to FIG. 6a to FIG. 6g, among them, FIG. 6a is a perspective view of substrate 11, FIG. 6b is a top view of substrate 11, FIG. 6c is a bottom view of substrate 11, and FIG. 6d, FIG. 6e, FIG. 6f and FIG. 6g are side views of substrate 11 in four directions, respectively.

[0119] As an example, the first substrate component 12 is fixed above the second substrate component 13 by gluing, e.g. with double-sided tapes or glue. In the embodiment, a sunken platform is provided on the surface of the second substrate component 13 for accommodating second substrate component 13, and arc-shaped extended spaces are provided at the four corners of the sunken platform, and the protrusions around the sunken platform function as a means for positioning when first substrate component 12 is glued to the sunken platform surface.

[0120] Referring to FIG. 7, it is a partial top view of the substrate, wherein it shows the relative position relationship between the PCR reagent upper chambers 14 and the PCR reagent lower chambers 15. In the embodiment, the PCR reagent is divided into two parts in order to hold enough digital PCR solution.

[0121] Referring to FIG. 8 and FIG. 9, they are front and back perspective views of the second substrate component 13, respectively. As shown in FIG. 7 and FIG. 9, in the embodiment, at least one digital PCR solution injection hole 16 is provided on the lower surface of the second substrate component 13, and digital PCR solution injection hole 16 is in communication with the PCR reagent lower chamber 15, for injecting the digital PCR solution into the PCR reagent chamber through the digital PCR solution injection hole 16.

[0122] Specifically, as shown in FIG. 7 and FIG. 8, the PCR reagent lower chamber 15 comprises a first end and a second end, the digital PCR solution injection hole 16 is in communication with the PCR reagent lower chamber 15 at the first end, the PCR reagent lower chamber 15 is in communication with the PCR reagent upper chamber 14 at the second end. In the embodiment, the PCR reagent lower chamber 15 is firstly progressively increased and then gradually decreased in size from the first end to the second end. With this design, it can prevent the formation of bubbles when adding liquid.

[0123] Specifically, as shown in FIG. 7 and FIG. 9, at least one exhaust port 17 is provided on the lower surface of the second substrate component 13, and the exhaust port 17 is in communication with the PCR reagent upper chamber 14 via a gas passage 8. As shown in FIG. 8, the gas passage 8 has an opening on the upper surface of the second substrate component 13 and extends toward, but not through, the lower surface of the second substrate component 13.

[0124] Specifically, in the embodiment, the opening area of the digital PCR solution injection hole 16 is greater than the opening area of the exhaust port 17, and the opening area of the digital PCR solution injection hole 16 is prepared slightly larger to support the tips of the pipetting gun. Due to capillarity, the liquid injected into the PCR reagent chamber does not flow out from the digital PCR solution injection hole 16 or the exhaust port 17.

[0125] Specifically, as shown in FIG. 1a and FIG. 2, the digital PCR system further comprises a flexible circuit board 18, the flexible circuit board 18 is connected to the upper surface of the substrate 11, and the droplet nozzle member 3 is connected in flexible circuit board 18.

[0126] Referring to FIG. 10a to FIG. 10h, wherein, FIG. 10a is a front perspective view of the droplet nozzle member 3 connected to the flexible circuit board 18; FIG. 10b is a back perspective view of the droplet nozzle member 3 connected to the flexible circuit board 18; FIG. 10c is a top view of the droplet nozzle member 3 connected to the flexible circuit board 18; FIG. 10d is a bottom view of droplet nozzle member 3 connected to the flexible circuit board 18; and FIG. 10e, FIG. 10f, FIG. 10g, and FIG. 10h are side views of the droplet nozzle member 3 connected to the flexible circuit board 18 in four directions, respectively.

[0127] Specifically, a through hole 19 is provided in the flexible circuit board 18 for accommodating the droplet nozzle member 3, a plurality of the first connection pads (not shown) and the second connection pads 20 are arranged on the surface of the flexible circuit board 18, the droplet nozzle member 3 is connected to the first connection pads by conducting wires, and the vaporizing parts 5 are connected to an external controller via flexible circuit board 18. The droplet nozzle member 3 is connected to the first connection pads by using a standard Wire Bond process.

[0128] As an example, the flexible circuit board 18 is connected to the substrate 11, as shown in FIG. 6a, the channels 21 are provided on the surface of the substrate 11 for preventing glue from flowing to the droplet nozzle member 3. The number of channels 21 is plural, and the channel distributes around the droplet nozzle member 3. The arrangement of channels 21 may be adjusted as required, and is not limited to the arrangement shown in FIG. 6a.

[0129] Specifically, the droplet generation component 1 is fixed on the flexible circuit board 18 by gluing. As shown in FIG. 5b, a protuberance 31 is arranged on the back of the droplet generation component 1, which ensures a strong bonding between the droplet generation component 1 and the flexible circuit board 18.

[0130] Specifically, as shown in FIG. 10a and FIG. 10b, at least two positioning through holes 22 are arranged in the flexible circuit board 18. As shown in FIG. 6a, the positioning bumps 23 at positions corresponding to the positioning through holes 22 are provided on the surface of the substrate 11. The positioning through holes 22 engage with the positioning bumps 23 to facilitate the accurate positioning of the flexible circuit board 18 on the substrate 11.

[0131] Specifically, the digital PCR system further comprises a controller. As shown in FIG. 11, it shows a perspective view of controller 24, which comprises a controller housing 25 and a controller circuit board arranged in the controller housing 25. The controller housing 25 has a support 26 for placing the substrate 11, a plurality of conductive pins for circuit connection 27 (also known as Pin) connected to the circuit connection board of the controller are arranged on the surface of support 26, and the conductive pins for circuit connection 27 are at positions corresponding to the second connection pads 30 on the flexible circuit board 18.

[0132] Specifically, the controller 24 is connected to the droplet nozzle member 3 via the flexible circuit board 18. The generation rate of the digital PCR droplets is controlled by controlling the heating time, the number of heatings and the time intervals of heating of the heating elements. Wherein, the control circuit of the controller 24 may employ an existing circuit structure.

[0133] Specifically, as shown in FIG. 9, at least one position-limiting slot 13 is provided at one end of the substrate, and as shown in FIG. 11, at least one position-limiting part 29 corresponding to the position-limiting slot 13 is provided in the controller housing 25.

[0134] Further, as shown in FIG. 8 and FIG. 9, a position-limiting hole 7 is provided in the substrate, the position-limiting hole 7 penetrates the front surface and the back surface of the substrate, and as shown in FIG. 11, a position-limiting part 30 corresponding to the position-limiting slot 7 is provided in the controller housing 25.

[0135] The digital PCR system of the present invention can be used for generating digital PCR droplets. The rapid droplet generation relies on the instantaneous vaporization of liquid layers with a thickness in nanometer-scale by using vaporizing parts in the droplet nozzles, so that the digital PCR solution inside the droplet nozzles is rapidly pushed into droplet generating oil to generate digital PCR droplets. Compared with the generation rate of 100 droplets per second on the market, a droplet generation rate of more than 1000 drops per second can be achieved by the droplet generation technique of the present invention. Compared with the method by which the oil and water phases move together to generate droplets, the oil phase in the technical solution of the present invention is static, so the consumption of oil is greatly reduced, reducing the amount of oil by about 50%. The technical solution of the present invention has an efficient utilization rate of digital PCR oil.

Embodiment 2

[0136] The present invention also provides a method for generating digital PCR droplets, comprising the following steps of: the digital PCR solution is vaporized by using vaporizing parts and rapidly pushed into droplet generating oil to generate digital PCR droplets.

[0137] As an example, the thermal bubble technique is used for high-speed digital PCR droplet generation. The vaporizing parts comprise heating elements for vaporizing the liquid layers of the digital PCR solution by heating.

[0138] Specifically, the generation rate of the digital PCR droplets is controlled by controlling the heating time, the number of heatings and the time intervals of heating of the heating element. The digital PCR droplet generation at a rate of more than 1000 droplets per second can be achieved by the method for generating digital PCR droplets of the present invention.

[0139] As an example, the method for generating digital PCR droplets comprises the following steps of:

[0140] S1: injecting a digital PCR solution into a PCR reagent chamber, so that the digital PCR solution enters the droplet nozzles in communication with the PCR reagent chamber to form liquid layers of the digital PCR solution;

[0141] S2: adding droplet generating oil into a droplet collection groove;

[0142] S3: the liquid layers are vaporized by using the vaporizing parts and rapidly pushed into the droplet generating oil in the droplet collection groove to generate the digital PCR droplets.

[0143] Specifically, the thicknesses of the liquid layers are in nanometer scale, and larger than 0.2 nm. In the embodiment, the thicknesses of the liquid layers are in the range of 0.2 nm to 30,000 nm.

[0144] Referring to FIG. 12, it is an optical microscope image of the digital PCR droplets generated by using the digital PCR system of the present invention, showing that the generated digital PCR droplets are symmetric and uniform in profile.

[0145] All of digital PCR biochemical reagents can be utilized when using the digital PCR system and the method for generating digital PCR droplets of the present invention. The concentration of many biomarker molecules in the blood is very low (e.g., circulating tumor DNA has only 3 DNA molecules per 2 ml of blood), but the digital PCR system and the method for generating digital PCR droplets of the present invention have the characteristics that the number of generated droplets is not limited by the amount of oil used and it is high speed, thus making it possible for the application of such assays in digital PCR.

[0146] In conclusion, thermal bubble technique is used in the digital PCR system and the method for generating digital PCR droplets of the present invention for high-speed digital PCR droplet generation. The rapid droplet generation relies on the instantaneous heating and vaporization of the liquid layers with a thickness in nanometer-scale by using the vaporizing parts in the droplet nozzles, so a digital PCR solution inside the droplet nozzles is quickly pushed into droplet generating oil to generate digital PCR droplets. Compared with the generation rate of 100 droplets per second on the market, a droplet generation rate of more than 1000 drops per second can be achieved by the droplet generation technique of the present invention. Compared with the method by which the oil and water phases move together to generate droplets, the oil phase in the technical solution of the present invention is static, so the consumption of oil is greatly reduced, reducing the amount of oil by about 50%. The technical solution of the present invention has an efficient utilization rate of digital PCR oil. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has a high utility value in industry.

[0147] The above-mentioned embodiments only illustrate the principle and efficacy of the present invention, and are not intended to limit the present invention. The above embodiments may be modified or altered by any person skilled in the art without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those with ordinary knowledge in the technical field, without departing from the spirit and technical ideas disclosed in the present invention, should still be covered by the claims of the present invention.