Droplet pcr chip and droplet pcr detection system

Abstract

The application discloses a droplet PCR chip and a droplet PCR detection system. The droplet PCR chip comprises at least one unit, and the unit comprises: an oil phase reservoir and a PCR reagent reservoir, the PCR reagent reservoir is connected with the oil phase reservoir through a fluid flow path, and the two reservoirs are combined and flow to a droplet generation channel; a droplet spreading cavity, one end of the droplet spreading cavity is communicated with the droplet generation channel to receive the generated droplet, and the droplet spreading cavity provides a place for the droplet to perform a PCR amplification reaction; and an outlet reservoir connected to the other end of the droplet spreading cavity to discharge the air in the droplet spreading cavity which is pressed by the droplet; wherein each reservoir is also used to receive mineral oil to seal the droplet spreading cavity. Thus, the air bubbles generated in the oil phase by heating are prevented from leaking out and expelling the droplets out of the droplet spreading cavity, and since the sealing, the vapor pressure generated in the oil phase by heating is retained inside the droplet PCR chip, the vapor pressure inside the droplet spreading cavity is improved, and thus the air bubbles generated in the droplet PCR chip can be reduced or avoided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidic droplet digital polymerase chain reaction (PCR), and particularly relates to a droplet PCR chip and a droplet PCR detection system. BACKGROUND

[0002] The microdroplet chip is developed on the basis of the traditional single-phase microfluidic chip technology. Compared with the single-phase microfluidic system, the microdroplet chip has the advantages of less consumption of samples and reagents, faster mixing speed, less cross contamination, and easy operation, due to the water / oil two-phase separation feature.

[0003] The existing microdroplet chip stores the generated droplets in the PCR reaction area. The boiling point of the commonly used oil for droplet generation is about 130 DEG C. Therefore, in the conventional PCR reaction, bubbles are formed when the heating temperature reaches 95 DEG C. The gradually increasing bubbles will expel the droplets out of the original flat position, finally leading to the failure of PCR. SUMMARY

[0004] The application provides a droplet PCR chip and a droplet PCR detection system to solve the technical problem that the microdroplet chip is prone to form bubbles in the PCR reaction process.

[0005] To solve the above technical problem, one technical solution adopted by the application is a droplet PCR chip, comprising at least one unit, wherein the unit comprises: an oil phase storage pool for storing oil phase; a PCR reagent storage pool for storing PCR reagents, and the PCR reagent storage pool and the oil phase storage pool are connected through a fluid flow path and merge into a droplet generation channel; a droplet flat cavity, one end of the droplet flat cavity is communicated with the droplet generation channel to receive the generated droplets, and the droplet flat cavity is used to provide a place for PCR amplification reaction of the droplets; and an outlet storage pool connected to the other end of the droplet flat cavity to discharge the air in the droplet flat cavity pressed by the droplets; wherein the oil phase storage pool, the PCR reagent storage pool and the outlet storage pool are also used to receive mineral oil to seal the droplet flat cavity.

[0006] According to an embodiment of the application, the droplet flat cavity comprises at least two first cavity segments and at least one second cavity segment, wherein the first cavity segments are arranged in a first preset direction, and the second cavity segment sequentially connects two adjacent first cavity segments in head-to-tail mode, so that the droplet flat cavity is arranged in a meandering mode.

[0007] According to an embodiment of the present application, the fluid flow path comprises a first flow path in communication with the oil phase reservoir and a second flow path in communication with the PCR reagent reservoir, the first flow path is provided in two, the two first flow paths and the second flow path converge at the same position, and the two first flow paths are respectively located on both sides of the second flow path.

[0008] According to an embodiment of the present application, the droplet PCR chip is provided with a tin foil or aluminum foil for sealing the open end of the oil phase reservoir, the PCR reagent reservoir and the outlet reservoir.

[0009] According to an embodiment of the present application, the droplet PCR chip comprises an upper structure and a lower structure, wherein the upper structure comprises the oil phase reservoir, the PCR reagent reservoir and the outlet reservoir, and the lower structure comprises the fluid flow path and the droplet generation channel.

[0010] According to an embodiment of the present application, the droplet PCR chip is made of a material that is transparent and can withstand the temperature of PCR reaction.

[0011] According to an embodiment of the present application, the droplet PCR chip is made of a cyclic olefin polymer or cyclic olefin copolymer material.

[0012] According to an embodiment of the present application, the droplet PCR chip is injection molded by a cyclic olefin polymer or cyclic olefin copolymer.

[0013] To solve the above technical problems, another technical solution adopted by the present application is: a droplet PCR detection system, comprising: a thermal cycle heating device for controlling PCR amplification reaction of the droplet PCR chip according to any one of the above; an oil injection device for adding mineral oil to the oil phase reservoir, the PCR reagent reservoir and the outlet reservoir of the droplet PCR chip to seal the droplet flat cavity.

[0014] According to an embodiment of the present application, the droplet PCR detection system comprises: a heat sealing device for tin foil or aluminum foil heat sealing of the open end of the oil phase reservoir, the PCR reagent reservoir and the outlet reservoir.

[0015] According to an embodiment of the present application, the droplet PCR detection system comprises: a fluorescence imaging device for fluorescence detection of the droplet after completing the PCR amplification reaction.

[0016] The beneficial effects of the present application are: different from the prior art, by setting an outlet liquid pool at the end of the droplet paving cavity, before the PCR amplification reaction, the oil phase liquid pool, the PCR reagent liquid pool and the outlet liquid pool are all used to accommodate mineral oil to seal the droplet paving cavity, avoiding the bubbles generated by heating in the oil phase to be discharged to expel the droplets out of the droplet paving cavity; in addition, since the oil phase is sealed, the vapor pressure generated by heating in the oil phase is retained inside the droplet PCR chip, further improving the vapor pressure inside the droplet paving cavity, so that the bubbles generated inside the droplet PCR chip can be reduced or avoided. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 is a schematic diagram of the overall structure of an embodiment of the droplet PCR chip of the present application;

[0019] Figure 2 is a schematic diagram of the side view structure of an embodiment of the droplet PCR chip of the present application;

[0020] Figure 3 is a schematic diagram of the local structure of an embodiment of the droplet PCR detection system of the present application. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0022] Please refer to Figures 1 to 2 , Figure 1 is a schematic diagram of the overall structure of an embodiment of the droplet PCR chip of the present application; Figure 2 is a schematic diagram of the side view structure of an embodiment of the droplet PCR chip of the present application.

[0023] An embodiment of the present application provides a droplet PCR chip 10, as shown in Figure 1 and Figure 2As shown, the droplet PCR chip 10 includes at least one unit 100, each unit 100 including an oil phase reservoir 110, a PCR reagent reservoir 120, a droplet spreading chamber 130, and an outlet reservoir 140. The oil phase reservoir 110 is used to store the oil phase, the PCR reagent reservoir 120 is used to store the PCR reagent, and the PCR reagent reservoir 120 and the oil phase reservoir 110 are merged through a fluid flow path 150 and merged into a droplet generation channel 160. One end of the droplet spreading chamber 130 is in communication with the droplet generation channel 160 to receive the droplets formed through the droplet generation channel 160, and the droplet spreading chamber 130 is used to provide a site for the droplets to perform the PCR amplification reaction. The outlet reservoir 140 is connected to the other end of the droplet spreading chamber 130 to discharge the air in the droplet spreading chamber 130 squeezed by the droplets.

[0024] Through the above structure, the oil phase in the oil phase reservoir 110 and the PCR reagent in the PCR reagent reservoir 120 can be merged through the fluid flow path 150 and then merged into the droplet generation channel 160, the oil phase and the PCR reagent are mixed and then pass through the droplet generation channel 160 to generate droplets of the oil phase wrapping the water phase, and the droplets enter the droplet spreading chamber 130 to perform the PCR amplification reaction. Since the droplets are easily generated bubbles during the planar PCR process, and the generation of bubbles is because the oil phase is heated to generate a large amount of bubbles, so that the droplets are squeezed out of the droplet spreading chamber 130, resulting in loss of droplets. The outlet reservoir 140 is provided at the end of the droplet spreading chamber 130, and before performing the PCR amplification reaction, the oil phase reservoir 110, the PCR reagent reservoir 120, and the outlet reservoir 140 are all used to receive mineral oil to seal the droplet spreading chamber 130, so as to avoid the bubbles generated by heating in the oil phase from leaking out and squeezing the droplets out of the droplet spreading chamber 130; in addition, since the vapor pressure generated by heating in the oil phase is retained inside the droplet PCR chip 10 after sealing, the vapor pressure inside the droplet spreading chamber 130 is further improved, thereby reducing or avoiding the generation of bubbles inside the droplet PCR chip 10.

[0025] In an embodiment, as shown in FIG. 1, the droplet PCR chip 10 is provided with a tin foil or aluminum foil for sealing the open end of the oil phase reservoir 110, the PCR reagent reservoir 120, and the outlet reservoir 140. Figure 1 After the oil phase reservoir 110, the PCR reagent reservoir 120, and the outlet reservoir 140 all receive mineral oil, the open end of the oil phase reservoir 110, the PCR reagent reservoir 120, and the outlet reservoir 140 can be further sealed with a tin foil or aluminum foil, so as to fully ensure the absolute sealing state of the entire internal environment of the droplet PCR chip 10 during the PCR process, and prevent the vapor pressure from leaking to the outside to cause problems such as droplet instability and aerosol. The tin foil or aluminum foil can be fixed to the open end of each reservoir by heat sealing.

[0026] In one embodiment, such as Figure 1 As shown, each droplet PCR chip 10 has three units 100 arrayed on it. In other embodiments, there may be one unit 100, two units 100, four units 100 or more units 100, which is not limited here.

[0027] In one embodiment, such as Figure 1 As shown, the fluid flow path 150 includes a first flow path 151 connected to the oil phase reservoir 110 and a second flow path 152 connected to the PCR reagent reservoir 120. The two first flow paths 151 and the second flow path 152 converge at the same position, and the two first flow paths 151 are respectively on both sides of the second flow path 152. Thus, the oil phase flows from the two first flow paths 151 to both sides of the PCR reagent in the second flow path 152 and merges into the droplet generation channel 160. Through the two-phase flow, droplets of the oil phase encapsulating the water phase can be successfully formed, so that the droplet generation density is consistent with the density of droplet spreading, thereby achieving droplet generation and single-layer droplet spreading at the same time.

[0028] It should be noted that, as Figure 1 As shown, the fluid flow path 150 may also include a third flow path 153 that connects the droplet spreading cavity 130 and the outlet liquid storage pool 140. Under normal circumstances without large internal pressure, the third flow path 153 is only used to discharge gas, and the droplets in the droplet spreading cavity 130 usually do not flow out from the third flow path 153.

[0029] In one embodiment, such as Figure 1 As shown, the droplet spreading cavity 130 includes at least one first cavity segment 131 and at least one second cavity segment 132. The first cavity segments 131 are arranged in an array along a first preset direction, and the second cavity segments 132 connect adjacent first cavity segments 131 end to end, so that the droplet spreading cavity 130 is meandering, thereby effectively reducing its volume while ensuring the length of the droplet spreading cavity 130. The droplets are spread in a single layer within the droplet spreading cavity 130, and the droplets are arranged closely together. Specifically, in this embodiment, the droplet spreading cavity 130 includes five first cavity segments 131 and four second cavity segments 132.

[0030] In one embodiment, such as Figure 1 As shown, the droplet-type PCR chip 10 includes an upper structure and a lower structure. The upper structure includes an oil phase reservoir 110, a PCR reagent reservoir 120, and an outlet reservoir 140. The lower structure includes a fluid flow path 150 and a droplet generation channel 160. Both the PCR reagent reservoir 120 and the outlet reservoir 140 are cylindrical and protrude from the surface of the droplet-type PCR chip 10. During production, the fluid flow path 150 and the droplet generation channel 160 can be formed by engraving.

[0031] In one embodiment, as shown in Figure 1 The droplet PCR chip 10 is made of a material that is optically transparent, optically clear, and can withstand the temperature of PCR reaction. As an example, the material can be a cyclic olefin polymer (COP) plastic or a cyclic olefin copolymer (COC) material, and in other embodiments, other materials that meet the above requirements can also be used, which are not limited here. When the droplet PCR chip 10 of the present application is made of a cyclic olefin polymer (COP) or a cyclic olefin copolymer (COC) material, a thermal compression bonding can be used to seal the droplet PCR chip 10, so that the entire PCR process is in a completely closed environment, thereby effectively avoiding the problem of DNA aerosol generated during PCR.

[0032] Further, as shown in Figure 1 The droplet PCR chip 10 of the present application is injection molded from a cyclic olefin polymer (COP) or a cyclic olefin copolymer (COC) material. Since the droplet PCR chip 10 according to the present application is injection molded and generally used once in application, the droplet PCR chip 10 according to the present application is very advantageous in cost compared to the traditional chip made of PDMS material. In addition, since the high-temperature-resistant COP or COC material is used to make the droplet PCR chip 10, the generated droplets do not need to be removed, and the PCR thermal cycling reaction (heating and cooling) can be directly performed through the droplet spreading cavity 130.

[0033] Please refer to Figure 1 , Figure 3 is a partial structure diagram of an embodiment of the droplet PCR detection system of the present application.

[0034] Another embodiment of the present application provides a droplet PCR detection system 200, as shown in Figure 3 The droplet PCR detection system 200 further includes an oil injection device 230 for adding mineral oil to the oil phase reservoir 110, the PCR reagent reservoir 120, and the outlet reservoir 140 of the droplet PCR chip 10 to seal the droplet spreading cavity 130, avoid the bubbles generated in the oil phase by heating from leaking out and expelling the droplets out of the droplet spreading cavity 130, and further improve the vapor pressure inside the droplet spreading cavity 130 since the vapor pressure generated by heating in the oil phase is retained inside the droplet PCR chip 10 after sealing, thereby reducing or avoiding the generation of bubbles inside the droplet PCR chip 10. Since the droplet PCR detection system 200 of the present application is provided with the oil injection device 230, mineral oil can be automatically added to the oil phase reservoir 110, the PCR reagent reservoir 120, and the outlet reservoir 140 of the droplet PCR chip 10, improving the work efficiency and making the equipment more intelligent.

[0035] Further, as shown in Figure 3 the droplet PCR detection system 200 further comprises a heat sealing device 210 for tin foil or aluminum foil heat sealing of the open end of the oil phase reservoir 110, the PCR reagent reservoir 120 and the outlet reservoir 140. After the oil injection device 230 completes the oil injection, the heat sealing device 210 seals the tin foil or aluminum foil to the open end of each reservoir, thereby fully ensuring the absolute sealing state of the entire internal environment of the droplet PCR chip 10 during the PCR process.

[0036] Further, as shown in Figure 3 Figure 3 the droplet PCR detection system 200 further comprises a fluorescence imaging device 220 for fluorescence detection of the droplets after completing the PCR amplification reaction, which can realize direct fluorescence collection and analysis of the droplets after the PCR amplification reaction is completed.

[0037] Specifically, the fluorescence imaging device 220 comprises: a laser light source located directly above the droplet PCR chip 10 region and having a 45° incident direction; a variable focus lens and a CCD camera located directly above the droplet PCR chip 10 region; and a bandpass fluorescence filter located between the variable focus lens and the CCD camera. The excitation light source comprises a light-emitting diode (LED) and a 15° lens, as well as a bandpass excitation light filter, wherein the bandpass excitation light filter has a center wavelength of 473 nm and a bandwidth of 10 nm. In addition, the bandpass fluorescence filter has a center wavelength of 535 nm and a bandwidth of 40 nm.

[0038] After the PCR amplification reaction is completed, the light-emitting diode in the system uniformly shines 45° from above the droplet PCR chip 10 on the droplet paving cavity 130 region of the droplet PCR chip 10 via the 15° lens and the bandpass excitation light filter. The 15° lens and the bandpass excitation light filter are used for focusing and filtering, respectively. Here, the 45° oblique light path can effectively reduce the excitation light scattering background, thereby improving the sensitivity of fluorescence detection. After exciting the fluorescence inside the droplet, the variable focus lens above can collect it, and after filtering through the bandpass fluorescence filter, it enters the CCD camera. The fluorescence picture is collected by the CCD camera to obtain the PCR reaction result.

[0039] The thermal cycling device, the oil injection device 230, the heat sealing device 210 and the fluorescence imaging device 220 in the present application can alternately act on the region of the droplet PCR chip 10 through a conversion mechanism. Of course, the droplet PCR chip 10 can also be transferred to the working region of each device in turn through a transfer mechanism.

[0040] The present application also provides a use method of a droplet PCR chip 10 and its detection system, which is specifically as follows:

[0041] I. Droplet generation and spreading.

[0042] Now 50 μl of oil phase is added into the oil phase reservoir 110. After the oil phase fills the entire oil phase reservoir 110 and flows through the fluid flow path 150 into the PCR reagent reservoir 120, 20-30 μl of prepared PCR reagent to be detected is added into the PCR reagent reservoir 120. Since the oil phase has already flowed into the PCR reagent reservoir 120, the PCR reagent can be in full contact and mixed with the oil phase, and the first flow path 151 has been filled with the oil phase, which can avoid the waste of PCR reagent backflowing into the first flow path 151.

[0043] The air pressure of 50-300 mbar is applied to the PCR reagent reservoir 120 and the oil phase reservoir 110 respectively for 1-5 min, and the mixed oil phase and PCR reagent generate oil phase wrapped water phase droplets through the droplet generation channel 160, and the droplets enter the droplet spreading chamber 130.

[0044] II. Sealing of the droplet PCR chip 10.

[0045] After the droplet generation is completed, the droplet PCR chip 10 is placed into the detection system, and the oil injection device 230 automatically adds 20-30 μl of mineral oil into the oil phase reservoir 110, the PCR reagent reservoir 120 and the outlet reservoir 140.

[0046] Subsequently, the heat sealing device 210 automatically heat seals the tin foil or aluminum foil at the open end of each reservoir, thereby achieving the sealing of the droplet PCR chip 10, avoiding the leakage of the air bubbles generated by heating in the oil phase to expel the droplets out of the droplet spreading chamber 130; in addition, since the vapor pressure generated by heating in the oil phase is retained inside the droplet PCR chip 10 after sealing, the vapor pressure inside the droplet spreading chamber 130 is further improved, thereby reducing or avoiding the generation of air bubbles inside the droplet PCR chip 10.

[0047] III. PCR fluorescence detection of the droplets.

[0048] The fluorescence imaging device 220 automatically performs fluorescence detection on the droplets after the completion of the PCR amplification reaction, which can realize the direct fluorescence collection and analysis of the droplets after the completion of the PCR amplification reaction.

[0049] The above only describes the embodiments of the present application, and does not limit the patent scope of the present application. Any equivalent structure or equivalent flow transformation using the content of the specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A droplet PCR detection system, characterized by, The application relates to a droplet PCR chip, a thermal cycle heating device for controlling the droplet PCR chip to perform a PCR amplification reaction, and an oil injection device for dripping mineral oil into an oil phase storage pool, a PCR reagent storage pool and an outlet storage pool of the droplet PCR chip to seal a droplet array cavity. The droplet PCR chip comprises at least one unit, and the unit comprises: The oil phase storage pool is used for storing an oil phase. The PCR reagent storage pool is used for storing PCR reagents, and the PCR reagent storage pool and the oil phase storage pool are connected through a fluid flow path and are combined into a droplet generation channel. The droplet array cavity is communicated with the droplet generation channel at one end to receive generated droplets, and the droplet array cavity is used for providing a place for the droplets to perform a PCR amplification reaction. The outlet storage pool is connected to the other end of the droplet array cavity to discharge air pressed by the droplets in the droplet array cavity. The oil phase storage pool, the PCR reagent storage pool and the outlet storage pool are also used for receiving mineral oil to seal the droplet array cavity. The fluid flow path comprises a first flow path communicated with the oil phase storage pool and a second flow path communicated with the PCR reagent storage pool. The droplet PCR chip comprises an upper structure and a lower structure. The droplet PCR chip comprises an upper structure and a lower structure.

2. The droplet PCR detection system of claim 1, wherein, The droplet PCR chip is made of a material with light transmission and capable of resisting a PCR reaction temperature.

3. The droplet PCR detection system of claim 1, wherein, The droplet PCR chip is made of a cyclic olefin polymer or a cyclic olefin copolymer material.

4. The droplet PCR detection system of claim 1, wherein, The droplet PCR chip is injection molded by a cyclic olefin polymer or a cyclic olefin copolymer.

5. The droplet PCR detection system of claim 2, wherein, The application relates to a droplet PCR chip, a thermal cycle heating device for controlling the droplet PCR chip to perform a PCR amplification reaction, and an oil injection device for dripping mineral oil into an oil phase storage pool, a PCR reagent storage pool and an outlet storage pool of the droplet PCR chip to seal a droplet array cavity.

6. The droplet PCR detection system of claim 1, wherein, The application relates to a droplet PCR chip, a thermal cycle heating device for controlling the droplet PCR chip to perform a PCR amplification reaction, and an oil injection device for dripping mineral oil into an oil phase storage pool, a PCR reagent storage pool and an outlet storage pool of the droplet PCR chip to seal a droplet array cavity.

7. The droplet PCR detection system of claim 6, wherein, The application relates to a droplet PCR chip, a thermal cycle heating device for controlling the droplet PCR chip to perform a PCR amplification reaction, and an oil injection device for dripping mineral oil into an oil phase storage pool, a PCR reagent storage pool and an outlet storage pool of the droplet PCR chip to seal a droplet array cavity.

8. The droplet PCR detection system of claim 7, wherein, ​ 9. The droplet PCR detection system according to any one of claims 1-8, wherein, ​ ​ 10. The droplet PCR detection system of claim 1, wherein, ​ ​ 11. The droplet PCR detection system of claim 9, wherein, ​ ​

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