Micro-fluidic dynamic mixing amplification chip and use method thereof
By designing the electrode array and reaction port on the microfluidic dynamic hybrid amplification chip, and by alternately energizing the electrode group, the droplets move back and forth inside the chip, the problems of poor liquid mixing effect and bubble generation during PCR amplification are solved, and better liquid mixing and temperature uniformity are achieved.
Patent Information
- Application Number
- CN202311776875.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-21
- Publication Date
- 2025-06-24
AI Technical Summary
The existing microfluidic chips have poor liquid mixing effect during PCR amplification, and the liquid moves easily and bubbles will be generated after heating, affecting subsequent liquid movement and fluorescence data acquisition.
A microfluidic dynamic hybrid amplification chip is designed, including a chip substrate, an isolation layer and an upper cover plate, and an electrode array and a reaction port are provided. The two electrode groups alternately powered up in turn, causing the droplets to move back and forth between the reaction port and the chip, and the liquid is stirred by capillary action and surface tension to enhance the mixing effect and reduce the generation of bubbles.
It significantly improves the mixing effect of the liquid, ensures the uniformity of the liquid temperature during the amplification process, and reduces the impact of bubbles on subsequent liquid movement and fluorescence data acquisition during the heating process.
Smart Images
Figure CN120192837A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microfluidics, and particularly to a microfluidic dynamic mixing and amplification chip and a method for using the same. Background Art
[0002] A microfluidic chip integrates operation units such as sample preparation, reaction, and detection in the analysis processes of chemistry, biology, medicine, etc. onto a carrier at the micron scale, and automatically completes the entire analysis process.
[0003] Currently, traditional PCR uses a static amplification method. During the amplification process, the components inside the liquid are mixed solely by thermal convection, and the mixing effect is actually not ideal. Currently, there is a method of using a digital microfluidic chip to heat the liquid while driving the liquid by energizing the electrodes, achieving the effect of mixing and amplifying simultaneously.
[0004] However, an ordinary digital microfluidic chip only simply moves the liquid, and its mixing effect is still not good. Moreover, bubbles are easily generated when the liquid moves after heating, and these bubbles pose a great risk to the subsequent liquid movement and fluorescence data collection. Summary of the Invention
[0005] In order to solve the problems existing in the above-mentioned prior art, the first object of the present invention is to provide a microfluidic dynamic mixing and amplification chip with a better mixing effect, which can reduce the influence of bubbles generated during the heating process on the subsequent liquid movement and fluorescence data collection.
[0006] The second object of the present invention is to provide a first method for using the above-mentioned microfluidic dynamic mixing and amplification chip.
[0007] In order to achieve the above first object, the microfluidic dynamic mixing and amplification chip provided by the present invention includes a chip substrate, an isolation layer, and an upper cover plate. An electrode array is provided on the chip substrate; the chip substrate, the isolation layer, and the upper cover plate enclose a liquid activity cavity, and the liquid activity cavity is correspondingly arranged with the electrode array; the electrode array includes a liquid transfer electrode area and a reaction electrode area connected in sequence; the upper cover plate further includes a cover plate reaction area; a reaction port is further provided at the upper cover plate, and the reaction port is communicated with the liquid activity cavity. The microfluidic dynamic mixing and amplification chip further includes a sealing cover, and the sealing cover covers the reaction port; the reaction electrode area includes a first reaction electrode group and a second reaction electrode group arranged in sequence along a first direction; in the projection on the chip substrate, the reaction electrode area is located within the cover plate reaction area, the reaction port is located within the reaction electrode area, both the first reaction electrode group and the second reaction electrode group partially overlap with the reaction port, and the area of the reaction port is smaller than the area of the reaction electrode area; the distance between the chip substrate and the cover plate reaction area is 0.3 mm to 1 mm.
[0008] As can be seen from the above, the height at the reaction port of the present invention is inconsistent with the height inside the chip, there is a drop. By alternately energizing the two electrode groups in sequence, the droplets move back and forth between the reaction port and the inside of the chip, and under the action of capillary action and surface tension, it plays a role in stirring the liquid, thereby enhancing the convection of each component inside the liquid, greatly improving the mixing effect of the liquid. This convection also makes the temperature inside the liquid more uniform during the amplification process. And since the liquid moves centered on the reaction port, most of the bubbles generated during the heating process of the amplification reaction are pushed away from the reaction port during the repeated movement of the liquid and stay in the area near the reaction port inside the chip. The remaining bubbles will also be pushed to the edge of the reaction port during the movement of the liquid, and the fluorescence reading position is set in the middle of the reaction port, and the reaction cover is made of a transparent material, so the acquisition of fluorescence data will not be affected by bubbles.
[0009] A further solution is that the thickness of the side wall of the reaction port is 0.6 mm to 2 mm.
[0010] A further solution is that the first reaction electrode group includes at least one reaction electrode; and / or the second reaction electrode group includes at least one reaction electrode.
[0011] A further solution is that the first reaction electrode group includes more than two reaction electrodes, and the multiple reaction electrodes are arranged along the second direction, and the second direction intersects with the first direction. In the projection on the chip substrate, the reaction port partially overlaps with all the reaction electrodes of the first reaction electrode group.
[0012] As can be seen from the above, the mixing effect can be fully improved by the movement and mixing of the reaction liquid in different directions.
[0013] A further solution is that there are mutually cooperating protrusions between the first reaction electrode group and the second reaction electrode group. In the projection on the chip substrate, the protrusions are located inside the reaction port; the protrusions include a first flange and a second flange. The first flange is located on the first reaction electrode group, and a second groove cooperating with the first flange is provided on the second reaction electrode group; the second flange is located on the second reaction electrode group, and a first groove cooperating with the second flange is provided on the first reaction electrode group.
[0014] A further solution is that a first heating wire is provided below the reaction electrode area.
[0015] As can be seen from the above, the first heating wire provided below the reaction electrode area is closer to the reaction liquid, and the heating effect is good.
[0016] A further solution is that the sealing cover is made of a transparent material.
[0017] As can be seen from the above, making the sealing cover of a transparent material can facilitate directly observing the amplification result in the reaction area.
[0018] Further, the sealing cover and the upper cover plate are integrally formed; or the sealing cover and the upper cover plate are detachably and sealingly connected.
[0019] To achieve the above second object, the present invention provides a method of using any of the above microfluidic dynamic mixing amplification chips. The reaction electrode region includes a first reaction electrode group and a second reaction electrode group arranged in sequence along a first direction. The method includes the following steps:
[0020] S1: Add a sample droplet into the sample inlet.
[0021] S2: Energize the pipetting electrode region to move the sample droplet into the liquid path active cavity corresponding to the reaction electrode region and fuse it with the reaction reagent in the reaction port.
[0022] S3: The first reaction electrode group and the second reaction electrode group are sequentially and cyclically energized, and the region where the reaction electrode region is located is heated until the sample droplet and the reaction reagent in the reaction port are uniformly mixed and then observed.
[0023] As can be seen from the above, by using the first reaction electrode and the second reaction electrode, the sample droplet is reciprocally moved and heated in the reaction port and inside the chip for amplification reaction. In this way, the method of mixing and amplifying simultaneously can effectively improve the amplification effect.
[0024] Further, after step S3 is executed, the following steps are further included:
[0025] S4: The first reaction electrode group and the second reaction electrode group stop being energized, the reaction electrode region continues to be heated, and fluorescence data is read.
[0026] S5: Repeat steps S3 and S4 until all fluorescence data is obtained.
[0027] Further, the first reaction electrode group further includes a first reaction electrode and a second reaction electrode arranged along a second direction; step S3 further includes: the first reaction electrode group and the second reaction electrode group are sequentially and cyclically energized, the first reaction electrode and the second reaction electrode are sequentially and cyclically energized, and the region where the reaction electrode region is located is heated.
[0028] In summary, the microfluidic dynamic mixing amplification chip of the present invention can greatly improve the mixing effect of the sample droplet, reduce the influence of bubbles generated during the heating process on the subsequent liquid movement and fluorescence data collection, and is convenient for observation. Description of the Drawings
[0029] Figure 1 It is a structural diagram of the first embodiment of the microfluidic dynamic mixing amplification chip of the present invention.
[0030] Figure 2Exploded view of the first embodiment of the microfluidic dynamic mixing and amplification chip of the present invention
[0031] Figure 3 Partial cross-sectional view of the first perspective of the first embodiment of the microfluidic dynamic mixing and amplification chip of the present invention.
[0032] Figure 4 Partial cross-sectional view of the second perspective of the first embodiment of the microfluidic dynamic mixing and amplification chip of the present invention.
[0033] Figure 5 Structural diagram of the chip substrate of the first embodiment of the microfluidic dynamic mixing and amplification chip of the present invention.
[0034] Figure 6 Position relationship diagram of the reaction electrode region and the reaction port of the first embodiment of the microfluidic dynamic mixing and amplification chip of the present invention.
[0035] Figure 7 Position relationship diagram of the reaction electrode region and the reaction port of the second embodiment of the microfluidic dynamic mixing and amplification chip of the present invention. Detailed implementation manners
[0036] First embodiment of the microfluidic dynamic mixing and amplification chip:
[0037] Refer to Figures 1 to 4 , the microfluidic dynamic mixing and amplification chip provided in this embodiment includes a chip substrate 1, a sealing cover 2, an isolation layer 3 and an upper cover plate 4. An electrode array 5 is provided on the chip substrate 1. The electrode array 5 includes a pipetting electrode region 6 and a reaction electrode region 7 that are connected in sequence. A first heating wire (not shown in the figure) is provided below the reaction electrode region 7. Optionally, a second heating wire (not shown in the figure) is provided below the pipetting electrode region 6.
[0038] Optionally, the first heating wire and the second heating wire may not be provided in the chip substrate 1, and the heating device on the microfluidic detection device can be used to heat the microfluidic dynamic mixing and amplification chip.
[0039] An injection port 40 and a reaction port 41 are provided on the upper cover plate 4. The sealing cover 2 covers the reaction port 41. The sealing cover 2 is made of a transparent material. The sealing cover 2 is detachably and sealingly connected to the upper cover plate 4.
[0040] Optionally, the sealing cover 2 and the upper cover plate 4 are integrally formed, and the reaction reagent is pre-stored on the chip substrate 1 and is correspondingly arranged with the reaction port 41.
[0041] Combined with Figure 5 and Figure 6, a liquid activity chamber 9 is formed by the chip substrate 1, the isolation layer 3 and the upper cover plate 4. The liquid activity chamber 9 is correspondingly arranged with the electrode array 5. Both the reaction port 41 and the sample injection port 40 are communicated with the liquid activity chamber 9. The upper cover plate 4 includes a cover plate reaction area 42. The reaction electrode area 7 includes a first reaction electrode 70 and a second reaction electrode 71 arranged in sequence along the moving direction of the liquid droplet. A protruding part 8 which cooperates with each other is arranged between the first reaction electrode 70 and the second reaction electrode 71. The protruding part 8 includes a first flange 80 arranged on the first reaction electrode 70 and a second flange 81 arranged on the second reaction electrode 71. A first groove which cooperates with the second reaction electrode 71 is arranged on the first reaction electrode 70, and a second groove which cooperates with the first reaction electrode 70 is arranged on the second reaction electrode 71. In the projection on the chip substrate 1, the protruding part 8 is located in the reaction port 41, the reaction electrode area 7 is located in the cover plate reaction area 42, the reaction port 41 is located in the reaction electrode area 7, and the area of the reaction port 41 is smaller than the area of the reaction electrode area 7. The distance a between the chip substrate 1 and the cover plate reaction area 42 is 0.3 mm to 1 mm; the thickness b of the side wall of the reaction port 41 is 0.6 mm to 2 mm.
[0042] Optionally, the thickness of the side wall of the reaction port 41 is 2 to 7 times the distance between the chip substrate 1 and the cover plate reaction area 42.
[0043] This embodiment also provides a method for using the above microfluidic dynamic mixing and amplification chip, and the method includes the following steps:
[0044] S1: Add the sample liquid droplet into the sample injection port 40.
[0045] S2: The electrodes 60, 61, 62, 63, 64 of the liquid transfer electrode area 6, the first reaction electrode 70 and the second reaction electrode 71 are energized in sequence or together, move the sample liquid droplet into the liquid activity chamber 9 corresponding to the reaction electrode area 7, and fuse with the reaction reagent in the reaction port 41.
[0046] S3: The first reaction electrode 70 and the second reaction electrode 71 are energized in sequence and cyclically, that is, when the first reaction electrode 70 is energized, the second reaction electrode 71 is powered off, and when the second reaction electrode 71 is energized, the first reaction electrode 70 is powered off, and this is cycled many times, and at the same time, the reaction electrode area 7 is heated, and the liquid moves back and forth between the first reaction electrode 70 and the second reaction electrode 71.
[0047] S4: The first reaction electrode 70 and the second reaction electrode 71 stop being energized, the reaction electrode area 7 continues to be heated, and the fluorescence data is read.
[0048] S5: Repeat steps S3 and S4 until all the fluorescence data is obtained.
[0049] Since the height at the reaction port 41 is much higher than the height inside the chip, ordinary small bubbles 12 cannot cut the liquid at this height. Moreover, the sample droplets move around the reaction port 41 as the center. During the heating process of the amplification reaction, most of the bubbles 12 generated are pushed away from the reaction port 41 during the repeated movement of the liquid and stay in the area near the reaction port 41 inside the chip. The remaining bubbles 12 will also be pushed to the edge area of the reaction port 41 during the movement of the liquid.
[0050] The second embodiment of the microfluidic dynamic mixing amplification chip:
[0051] See Figure 7 , the difference between this embodiment and the first embodiment is that the reaction electrode region 7 includes a first reaction electrode group 10 and a second reaction electrode group 11 arranged along the first direction. The first reaction electrode group 10 includes a first reaction electrode 100 and a second reaction electrode 101 arranged along the second direction. The second reaction electrode group 11 includes a third reaction electrode 110. A first flange 102 is provided on the first reaction electrode 100, a second flange 103 is provided on the second reaction electrode 101, a third flange 111 is provided on the third reaction electrode 110, a third groove cooperating with the first reaction electrode 100 is provided on the third reaction electrode 110, a first groove cooperating with the second flange 103 is provided on the first reaction electrode 100, and a second groove cooperating with the third flange 111 is provided on the second reaction electrode 101. The second direction is perpendicular to the first direction. In the projection on the chip substrate 1, the first flange 102, the second flange 103, and the third flange 111 are all located within the reaction port, and the first reaction electrode 100, the second reaction electrode 101, and the third reaction electrode 110 all partially overlap with the reaction port 41.
[0052] The difference in the usage method of the microfluidic dynamic mixing amplification chip of this embodiment from that of the first embodiment is that step S3 further includes: the first reaction electrode 100, the third reaction electrode 111, and the second reaction electrode 103 are sequentially and cyclically energized, and the reaction electrode region 7 is heated. The liquid moves back and forth between the first reaction electrode 100, the third reaction electrode 110, and the second reaction electrode 101. After a period of time, after the sample droplets are uniformly mixed with the reaction reagent in the reaction port, observation is carried out.
[0053] Optionally, the number of reaction electrodes in the first reaction electrode group 10 is three or more, or the number of reaction electrodes in the second reaction electrode group 11 is two or more. The setting of the flanges on each reaction electrode can be set according to the droplet movement requirements, and the number of flanges on each reaction electrode can also be two or more.
[0054] In summary, the microfluidic dynamic mixing and amplification chip of the present invention can greatly improve the mixing effect of sample droplets, reduce the influence of bubbles generated during the heating process on subsequent liquid movement and fluorescence data collection, and facilitate observation.
[0055] Finally, it should be emphasized that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A microfluidic dynamic mixing and amplification chip, comprising a chip substrate, an isolation layer and an upper cover plate. An electrode array is provided on the chip substrate; The chip substrate, the isolation layer and the upper cover plate enclose a liquid activity cavity, and the liquid activity cavity is correspondingly arranged with the electrode array; The electrode array includes a pipetting electrode area and a reaction electrode area connected in sequence; The upper cover plate further includes a cover plate reaction area; It is characterized in that: A reaction port is further provided at the upper cover plate, and the reaction port is communicated with the liquid activity cavity. The microfluidic dynamic mixing and amplification chip further includes a sealing cover, and the sealing cover covers the reaction port; The reaction electrode area includes a first reaction electrode group and a second reaction electrode group arranged in sequence along a first direction; In the projection on the chip substrate, the reaction electrode area is located in the cover plate reaction area, the reaction port is located in the reaction electrode area, both the first reaction electrode group and the second reaction electrode group partially overlap with the reaction port, and the area of the reaction port is smaller than the area of the reaction electrode area; The distance between the chip substrate and the cover plate reaction area is 0.3 mm to 1 mm.
2. The microfluidic dynamic mixing and amplification chip according to claim 1, characterized in that: The thickness of the side wall of the reaction port is 0.6 mm to 2 mm.
3. The microfluidic dynamic mixing and amplification chip according to claim 2, characterized in that: The first reaction electrode group includes at least one reaction electrode; And / or the second reaction electrode group includes at least one reaction electrode.
4. The microfluidic dynamic mixing and amplification chip according to claim 3, characterized in that: The first reaction electrode group includes more than two of the reaction electrodes, and the multiple reaction electrodes are arranged along a second direction, and the second direction intersects with the first direction. In the projection on the chip substrate, the reaction port partially overlaps with all the reaction electrodes of the first reaction electrode group.
5. The microfluidic dynamic mixing and amplification chip according to any one of claims 1 to 4, characterized in that: There are mutually cooperating protrusions between the first reaction electrode group and the second reaction electrode group. In the projection on the chip substrate, the protrusions are located in the reaction port; The protrusions include a first flange and a second flange. The first flange is located on the first reaction electrode group, and a second groove cooperating with the first flange is provided on the second reaction electrode group; The second flange is located on the second reaction electrode group, and a first groove cooperating with the second flange is provided on the first reaction electrode group.
6. The microfluidic dynamic mixing and amplification chip according to any one of claims 1 to 4, characterized in that: A first heating wire is provided below the reaction electrode area.
7. The microfluidic dynamic mixing and amplification chip according to any one of claims 1 to 4, characterized in that: The sealing cover is made of a transparent material.
8. Method for using a microfluidic dynamic mixing and amplification chip, characterized in that Applied to the microfluidic dynamic mixing and amplification chip according to any one of claims 1 to 7, the reaction electrode area includes a first reaction electrode group and a second reaction electrode group arranged in sequence along the first direction; The method includes the following steps: S1: Add the sample droplet into the injection port; S2: Energize the pipetting electrode region to move the sample droplet into the liquid path activity cavity corresponding to the reaction electrode region and fuse it with the reaction reagent in the reaction port; S3: Energize the first reaction electrode group and the second reaction electrode group in sequence and cyclically, and heat the region where the reaction electrode region is located.
9. The method for using the microfluidic dynamic mixing and amplification chip according to claim 8, wherein: After step S3 is completed, there is also the following steps: S4: The first reaction electrode group and the second reaction electrode group stop being energized, the reaction electrode region continues to be heated, and fluorescence data is read; S5: Repeat steps S3 and S4 until all the fluorescence data is obtained.
10. The method for using the microfluidic dynamic mixing and amplification chip according to claim 8 or 9, wherein: The first reaction electrode group further includes a first reaction electrode and a second reaction electrode arranged along the second direction; Step S3 further includes: The first reaction electrode group and the second reaction electrode group are energized in sequence and cyclically, the first reaction electrode and the second reaction electrode are energized in sequence and cyclically, and the region where the reaction electrode region is located is heated.