Centrifugal micro-fluidic chip and nucleic acid detection method thereof

Abstract

The invention provides a centrifugal micro-fluidic chip and a nucleic acid detection method thereof, the chip comprises a chip body, a sample area and a plurality of sample processing units are arranged in the chip body, and the sample processing units are arranged around the sample area at intervals and located on the radial outer side of the sample area. Each sample processing unit comprises an air pressure waste liquid area, a quantitative area, a mixed amplification area, a detection area, a buffer area and a liquid channel area which are arranged from inside to outside in the radial direction of the chip body, the areas are communicated through corresponding connecting channels or siphon channels, and the radial inner sides of the buffer area and the mixed amplification area are communicated with the radial inner sides of the sample areas through exhaust pipes; and when the centrifugal micro-fluidic chip is in a centrifugal detection process, the sample injection hole of the sample area is sealed. According to the invention, 'sample input-result output 'is realized, the problems of operation error, sample pollution and the like are avoided, and the detection reliability and efficiency are improved.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics and biological detection technology, specifically relating to a centrifugal microfluidic chip and its nucleic acid detection method. Background Technology

[0002] In vitro diagnostics (IVD) is a technology that obtains clinical diagnostic information by testing human samples (such as blood and other bodily fluids) outside the human body. IVD plays a crucial role in assessing diseases and bodily functions. It includes sub-fields such as immunodiagnostics, biochemical diagnostics, and molecular diagnostics, with molecular diagnostics becoming the largest market segment within the IVD industry.

[0003] The core technology of molecular diagnostics is nucleic acid amplification technology. Current isothermal amplification technology basically involves many reactions in separate centrifuge tubes, which is cumbersome, highly dependent on manual pipetting, and prone to aerosol contamination, leading to false positive results. In addition, the traditional reaction mode still relies on manual operation to complete each step, making it difficult to integrate the steps of sample quantification, mixing, amplification and detection into one, and failing to achieve true "sample in - result out".

[0004] Currently, existing technologies still have many problems, such as large sample consumption, long detection time, high cost, and cumbersome operation.

[0005] The emergence of microfluidic chip technology has provided a revolutionary solution to the aforementioned problems. Microfluidics enables precise manipulation of tiny volumes of fluid within micrometer-scale channel structures. Combining isothermal amplification technology with microfluidic chips represents the forefront of current development in this field. This technology offers low reagent / sample consumption, short detection time, and high detection accuracy, achieving an integrated chip for "sample in—result out," exhibiting high integration and excellent portability. Therefore, providing a microfluidic chip capable of quantifying, mixing, isothermal amplifying, and detecting multiple indicators of sample solutions is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a centrifugal microfluidic chip and its nucleic acid detection method, which can overcome the shortcomings of the existing centrifugal microfluidic chip in terms of low functional integration and inability to achieve "sample in - result out".

[0007] To address the aforementioned problems, this invention provides a centrifugal microfluidic chip, comprising a chip body with a central spindle hole. A sample area for sample introduction is formed within the chip body, surrounding the spindle hole. Multiple sample processing units are also formed within the chip body, spaced apart around the sample area and located radially outward from it. Each sample processing unit includes, from the inside out, a pneumatic waste liquid area, a quantitative area, a mixed amplification area, a detection area, a buffer area, and a liquid channel area arranged radially from the inside out along the chip body. The sample area is connected to the liquid channel area via a first connecting channel, the liquid channel area is connected to the quantitative area via a second connecting channel, the quantitative area is connected to the pneumatic waste liquid area via a pneumatic channel, the detection area is connected to the buffer area via a third connecting channel, the buffer area is connected to the mixed amplification area via a first siphon channel, the quantitative area is connected to the mixed amplification area via a second siphon channel, and the radially inner sides of the buffer area and the mixed amplification area are connected to the radially inner side of the sample area via an exhaust pipe. During centrifugal detection, the sample inlet of the sample area is sealed.

[0008] In some embodiments, the first siphon channel has a portion located radially inside the hybrid amplification region.

[0009] In some embodiments, the second siphon channel has a portion located radially inside the gas pressure waste liquid zone.

[0010] In some embodiments, the pneumatic channel is located between the radially inner side of the metering zone and the radially outer side of the pneumatic waste liquid zone, the pneumatic channel and the pneumatic waste liquid zone are connected to a first interface, and the pneumatic waste liquid zone has a liquid storage portion located radially outer side of the first interface.

[0011] In some embodiments, spotting reagents or lyophilized bulbs are pre-embedded in the mixed amplification region; spotting reagents or lyophilized bulbs are pre-embedded in the detection region.

[0012] In some embodiments, each of the sample processing units is uniformly arranged around the sample area, and the liquid channel area is an annular ring concentrically arranged with the main shaft hole, and each quantitative area in each of the sample processing units is connected to the same liquid channel area via the second connecting channel.

[0013] In some embodiments, the chip body includes a chip body and a chip cover that is sealed and attached to a first end face of the chip body. The sample area, the gas pressure waste liquid area, the quantitative area, the mixed amplification area, the detection area, the buffer area, the liquid channel area, the first connection channel, the second connection channel, the first siphon channel, the second siphon channel, and the exhaust pipe are all formed on the first end face in the form of an open groove. The sample inlet is formed on the chip cover.

[0014] In some embodiments, the chip body is further provided with a fixing hole offset to one side of the spindle hole.

[0015] The present invention also provides a nucleic acid detection method using the above-mentioned centrifugal microfluidic chip, comprising the following steps: A preset amount of sample liquid is injected into the sample area through the injection port, and the injection port is sealed after the injection is completed. The centrifuge disc is controlled to rotate in the first direction of rotation so that the sample liquid in the sample area is evenly fed into each of the quantitative areas through the first connecting channel, the liquid channel area and the second connecting channel in sequence by using centrifugal force. After each quantitative area is filled with sample liquid, it enters the pneumatic waste liquid area through the pneumatic channel. After all the sample liquid in the sample area has been centrifuged and output, the centrifuge disc is controlled to decelerate and stop. The compressed gas in the gas pressure waste liquid area pushes the sample liquid in the quantitative area into the mixed amplification area through the second siphon channel. After a quantitative amount of sample solution has entered the mixing amplification zone, the centrifuge disc is controlled to rotate in both directions to mix the sample solution with the spotting reagent or lyophilized bulb pre-embedded in the mixing amplification zone. After mixing for a preset time, the centrifuge disc is controlled to decelerate and stop, and the heating module is controlled to heat the mixing amplification region to achieve nucleic acid amplification of the mixed liquid; After amplification is complete, the centrifuge disc is rotated again to allow the amplified mixture to enter the detection area sequentially through the first siphon channel, the buffer zone, and the third connecting channel. Optical nucleic acid detection is performed on the mixture that enters the detection area.

[0016] In some embodiments, a step of heating the detection zone is included after the mixture enters the detection zone and before optical nucleic acid detection is performed.

[0017] This invention provides a centrifugal microfluidic chip and its nucleic acid detection method. The centrifugal microfluidic chip integrates nucleic acid amplification, sample solution quantification, reagent mixing, and detection functions. It requires no pretreatment or reagent addition; only a single sample addition is needed. Driven by centrifugation, it can complete the joint detection of multiple nucleic acid indicators, achieving "sample in - result out," avoiding operational errors and sample contamination, and improving the reliability and efficiency of detection. More importantly, in this invention, the radial inner sides of the buffer zone and the mixing amplification zone of each sample processing unit are connected to the radial inner side of the sample area via exhaust pipes. This achieves gas-liquid displacement within a sealed (closed) space during centrifugation of the microfluidic chip. This ensures controllable transfer of liquid by centrifugal force while maintaining a completely sealed state throughout the nucleic acid processing, further preventing sample contamination. Attached Figure Description

[0018] Figure 1 This is an exploded view of the centrifugal microfluidic chip according to an embodiment of the present invention; Figure 2 Figure 1 Schematic diagram of the chip cover structure Figure 3 for Figure 1 A schematic diagram of the chip's main structure.

[0019] The reference numerals in the attached figures are as follows: 1. Top cover; 2. Chip body; 101. Sample inlet; 201. Spindle hole; 202. Sample area; 203. Fixing hole; 204. Exhaust pipe; 206. First siphon channel; 207. Liquid channel area; 208. Gas pressure channel; 209. Gas pressure waste liquid area; 210. Second connection channel; 211. Buffer zone; 212. Detection area; 213. Mixed amplification area; 214. Quantification area; 215. First connection channel; 216. Second siphon channel; 217. Third connection channel. Detailed Implementation

[0020] See also Figures 1 to 3As shown in the embodiment of the present invention, a centrifugal microfluidic chip is provided, including a chip body. A spindle hole 201 is provided at the center of the chip body. A sample area 202 for adding samples is formed within the chip body, surrounding the spindle hole 201. A plurality of sample processing units are also formed within the chip body. Each sample processing unit is spaced around the sample area 202 and located radially outward from the sample area 202. Each sample processing unit includes a pressurized waste liquid area 209, a quantification area 214, a mixed amplification area 213, a detection area 212, a buffer zone 211, and a liquid channel area 207 arranged radially from the inside to the outside of the chip body. The sample area 202 is connected to the liquid channel area via a first connecting channel 215. The liquid channel area 207 is connected to the quantitative area 214 via the second connecting channel 210. The quantitative area 214 is connected to the gas pressure waste liquid area 209 via the gas pressure channel 208. The detection area 212 is connected to the buffer zone 211 via the third connecting channel 217. The buffer zone 211 is connected to the mixed amplification area 213 via the first siphon channel 206. The quantitative area 214 is connected to the mixed amplification area 213 via the second siphon channel 216. The radially inner sides of the buffer zone 211 and the mixed amplification area 213 are connected to the radially inner side of the sample area 202 via the exhaust pipe 204. During the centrifugal detection process of the centrifugal microfluidic chip, the sample inlet 101 of the sample area 202 is sealed.

[0021] In this technical solution, the centrifugal microfluidic chip integrates nucleic acid amplification, sample solution quantification, reagent mixing, and detection functions. No pretreatment or reagent addition is required; only one sample addition is needed. Driven by centrifugation, it can complete the joint detection of multiple nucleic acid indicators, achieving "sample in - result out." This avoids operational errors and sample contamination, improving the reliability and efficiency of the detection. More importantly, in this invention, the radial inner sides of the buffer zone 211 and the mixing amplification zone 213 of each sample processing unit are connected to the radial inner side of the sample area 202 via an exhaust pipe 204. This achieves gas-liquid displacement within a sealed (closed) space during centrifugation of the microfluidic chip. This ensures controllable transfer of the liquid by centrifugal force while maintaining a completely sealed state throughout the nucleic acid processing, further preventing sample contamination.

[0022] In some embodiments, the first siphon channel 206 has a portion located radially inside the mixing amplification region 213, see details below. Figure 3 As shown, in a specific embodiment, the aforementioned first siphon channel 206 is S-shaped.

[0023] In this technical solution, the first siphon channel 206 has a portion located radially inside the mixing amplification region 213, which can effectively prevent the mixed liquid from entering the aforementioned buffer zone 211 through the first siphon channel 206 during the chip forward and reverse rotation process of the mixed sample in the mixing amplification region 213 and during the heating amplification process (increased pressure), thus ensuring that the sample liquid is fully mixed and amplified in the mixing amplification region 213.

[0024] In some embodiments, the second siphon channel 216 has a portion located radially inside the gas pressure waste liquid zone 209, see details below. Figure 3 As shown, in a specific embodiment, the aforementioned second siphon channel 216 is S-shaped.

[0025] In this technical solution, the second siphon channel 216 has a portion located radially inside the gas pressure waste liquid area 209. It can utilize the principle of communicating vessels to prevent the sample liquid from further entering the mixed amplification area 213 connected to it via the second siphon channel 216 after the quantitative area 214 is filled with sample liquid, thus preventing inaccurate quantification of the sample liquid.

[0026] In some embodiments, the pneumatic channel 208 is located between the radially inner side of the metering zone 214 and the radially outer side of the pneumatic waste liquid zone 209, the pneumatic channel 208 and the pneumatic waste liquid zone 209 are connected to a first interface (not shown in the figure), and the pneumatic waste liquid zone 209 has a liquid storage portion (not shown in the figure) located radially outer side of the first interface.

[0027] In this technical solution, by setting a liquid storage section on the radially outer side of the first interface in the gas pressure waste liquid area 209 to store the waste liquid, it can be ensured that when the microfluidic chip stops rotating (or the rotation speed is reduced to below a certain value), the compressed gas pressure drives the sample liquid in the quantitative area 214 into the mixed amplification area 213, while the waste liquid does not follow, thus ensuring the accuracy of quantification.

[0028] In some embodiments, spotting reagents or lyophilized bulbs are pre-embedded in the mixed amplification region 213; spotting reagents or lyophilized bulbs are pre-embedded in the detection region 212. The spotting reagents or lyophilized bulbs can be reasonably selected according to the actual detection requirements.

[0029] In some embodiments, each of the sample processing units is uniformly arranged around the sample area 202, and the liquid channel area 207 is an annular ring concentrically arranged with the spindle hole 201. Each quantitative area 214 in each of the sample processing units is connected to the same liquid channel area 207 via the second connecting channel 210.

[0030] In this technical solution, the liquid channel area 207 is an annular ring concentrically arranged with the spindle hole 201. This allows for uniform distribution of the sample liquid in the sample area 202 along the circumferential direction when the chip is driven to rotate, ensuring a more uniform amount of sample liquid in each sample processing unit and guaranteeing the quantitative accuracy of the sample liquid. It is understandable that the amount of sample liquid added to the aforementioned sample area 202 should be reasonably matched to the volume of the aforementioned liquid channel area 207, the first connecting channel 215 and the second connecting channel 210, the quantitative area 214, and the gas pressure waste liquid area 209. Excessive sample liquid addition will result in overfilling of the gas pressure waste liquid areas 209, causing waste liquid to further enter the quantitative area 214 when the gas pressure outputs the quantitative sample liquid.

[0031] In some embodiments, the chip body includes a chip body 2 and a chip cover 1 sealed and attached to a first end face of the chip body 2. The sample area 202, the gas pressure waste liquid area 209, the quantitative area 214, the mixed amplification area 213, the detection area 212, the buffer zone 211, the liquid channel area 207, the first connection channel 215, the second connection channel 210, the first siphon channel 206, the second siphon channel 216, and the exhaust pipe 204 are all formed on the first end face in the form of open slots. The sample inlet 101 is formed on the chip cover 1 to simplify the chip fabrication process.

[0032] In some embodiments, the chip body is further provided with a fixing hole 203 offset to one side of the spindle hole 201 to realize reliable rotational drive of the chip.

[0033] The diameters of the aforementioned first siphon channel 206, second siphon channel 216, first connecting channel 215, and second connecting channel 210, as well as the volumes of the aforementioned sample area 202, pressurized waste liquid area 209, quantitative area 214, mixed amplification area 213, detection area 212, buffer zone 211, and liquid channel area 207, can be reasonably selected according to actual needs. In principle, under the premise of meeting the requirements, the smaller the design value should be, so that the microfluidic chip forms microchambers and microchannels, reducing the consumption of samples / reagents, lowering the detection cost, and improving the accuracy, stability, and repeatability of the detection results, making it suitable for quantitative detection.

[0034] According to an embodiment of the present invention, a nucleic acid detection method using the above-described centrifugal microfluidic chip is also provided, comprising the following steps: A preset amount of sample liquid is injected into the sample area 202 through the injection port 101, and the injection port 101 is sealed after the injection is completed. The centrifuge disc is controlled to rotate along the first direction of rotation so that the sample liquid in the sample area 202 is evenly fed into each of the quantitative areas 214 through the first connecting channel 215, the liquid channel area 207, and the second connecting channel 210 in sequence using centrifugal force. After each quantitative area 214 is filled with sample liquid, it enters the pneumatic waste liquid area 209 through the pneumatic channel 208. After all the sample liquid in the sample area 202 has been centrifuged and output, the centrifuge plate is controlled to decelerate and stop. The compressed gas in the gas pressure waste liquid area 209 pushes the sample liquid in the quantitative area 214 into the mixed amplification area 213 through the second siphon channel 216. After a quantitative amount of sample solution has entered the mixing amplification zone 213, the centrifuge disc is controlled to rotate in both directions to mix the sample solution with the spotting reagent or lyophilized bulb pre-embedded in the mixing amplification zone 213. After mixing for a preset time, the centrifuge disc is controlled to decelerate and stop, and the heating module is controlled to heat (for example, isothermal amplification can be used) the mixing amplification region 213 to realize nucleic acid amplification of the mixed liquid; After amplification is complete, the centrifuge disc is rotated again to allow the amplified mixture to enter the detection area 212 sequentially through the first siphon channel 206, the buffer zone 211 and the third connecting channel 217. Optical nucleic acid detection is performed on the mixture that enters the detection area 212.

[0035] In some embodiments, after the mixture enters the detection zone 212 and before optical nucleic acid detection is performed, a step of heating the detection zone 212 is also included.

[0036] Specifically, in one particular embodiment, the optical detection of nucleic acids is achieved using the following steps: The sample (i.e., the sample solution) is injected into the sample area 202 through the injection port 101; The centrifuge disc rotates clockwise (the centrifuge disc drives the chip to rotate) at a speed of 3000-5000 r / min for 30-60 s. During the rotation of the centrifuge disc, the sample first enters the liquid channel area 207 through the first connecting channel 215. The liquid channel area 207 is filled with liquid. The liquid is evenly distributed into each quantitative area 214 through the second connecting channel 210. The quantitative area 214 is filled with liquid. Excess liquid enters the pneumatic waste liquid area 209 through the pneumatic channel 208. Then, the gas decelerates and stops. The compressed gas in the gas pressure waste liquid zone 209 pushes the liquid in the quantitative zone 214 into the mixed amplification zone 213 through the second siphon channel 216. Rotate in both directions at a speed of -1200 r / min to 1200 r / min. Select the appropriate running time based on the mixing effect. The sample liquid dissolves the pre-embedded reagent to achieve mixing. The centrifuge then slowed down and stopped, and began heating, starting nucleic acid amplification in the mixed liquid. Rotate clockwise at a speed of 2000-5000 r / min for 30-60 s. The amplified mixed liquid enters the detection area 212 through the first siphon channel 206, buffer zone 211 and third connecting channel 217. Then the amplified mixed liquid dissolves the reagents embedded in the detection area 212. The centrifuge is heated and the optical detection begins to produce the results.

[0037] The centrifugal microfluidic chip provided by this invention only requires a centrifuge to drive, which improves the portability of detection and broadens the application scenarios of this type of product.

[0038] It will be readily understood by those skilled in the art that the aforementioned advantageous methods can be freely combined and superimposed without conflict.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention. The above are merely preferred embodiments of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. A centrifugal microfluidic chip, characterized by, The chip comprises a chip body, a main shaft hole (201) is arranged in the center of the chip body, a sample area (202) for adding sample is formed in the chip body and surrounds the main shaft hole (201), a plurality of sample processing units are also formed in the chip body, each sample processing unit is arranged at intervals and is located in the radial outer side area of the sample area (202), each sample processing unit comprises a gas pressure waste liquid area (209), a quantitative area (214), a mixing and amplification area (213), a detection area (212), a buffer area (211) and a liquid channel area (207) which are arranged from inside to outside along the radial direction of the chip body, the sample area (202) is communicated with the liquid channel area (207) through a first connecting channel (215), the liquid channel area (207) is communicated with the quantitative area (214) through a second connecting channel (210), the quantitative area (214) is communicated with the gas pressure waste liquid area (209) through a gas pressure channel (208), the detection area (212) is communicated with the buffer area (211) through a third connecting channel (217), the buffer area (211) is communicated with the mixing and amplification area (213) through a first siphon channel (206), the quantitative area (214) is communicated with the mixing and amplification area (213) through a second siphon channel (216), the radial inner side of the buffer area (211) and the mixing and amplification area (213) is communicated with the radial inner side of the sample area (202) through an exhaust pipe (204), and the sample inlet hole (101) of the sample area (202) is sealed when the centrifugal microfluidic chip is in a centrifugal detection process.

2. The centrifugal microfluidic chip of claim 1, wherein, The first siphon channel (206) has a part which is located in the radial inner side of the mixing and amplification area (213).

3. The centrifugal microfluidic chip of claim 1, wherein, The second siphon channel (216) has a part which is located in the radial inner side of the gas pressure waste liquid area (209).

4. The centrifugal microfluidic chip of claim 1 wherein, The gas pressure channel (208) is located between the radial inner side of the quantitative area (214) and the radial outer side of the gas pressure waste liquid area (209), the gas pressure channel (208) is communicated with the gas pressure waste liquid area (209) at a first interface, and the gas pressure waste liquid area (209) has a liquid storage part which is located in the radial outer side of the first interface.

5. The centrifugal microfluidic chip of claim 1 wherein, A dot sample reagent or a freeze-dried ball is embedded in the mixing and amplification area (213), and a dot sample reagent or a freeze-dried ball is embedded in the detection area (212).

6. The centrifugal microfluidic chip of claim 1 wherein, Each sample processing unit is uniformly arranged around the sample area (202), and the liquid channel area (207) is a circular ring which is concentrically arranged with the main shaft hole (201), each quantitative area (214) in each sample processing unit is communicated with the same liquid channel area (207) through the second connecting channel (210).

7. The centrifugal microfluidic chip of claim 1 wherein, The chip body comprises a chip main body (2) and a chip cover (1) sealedly attached to a first end face of the chip main body (2), the sample area (202), the gas pressure waste liquid area (209), the quantitative area (214), the mixed amplification area (213), the detection area (212), the buffer area (211), the liquid channel area (207), the first connecting channel (215), the second connecting channel (210), the first siphon channel (206), the second siphon channel (216) and the exhaust pipe (204) are all formed on the first end face in the form of an open slot, and the sample inlet hole (101) is formed on the chip cover (1).

8. The centrifugal microfluidic chip of claim 1 wherein, The chip body is further provided with a fixing hole (203) offset to one side of the main shaft hole (201).

9. A method for detecting a nucleic acid using the centrifugal microfluidic chip according to any one of claims 1 to 8, characterized by, The method comprises the following steps: A preset amount of sample liquid is injected into the sample area (202) through the sample inlet hole (101), and after the injection is completed, the sample inlet hole (101) is closed; The centrifugal disc is controlled to rotate in a first rotation direction, so that the sample liquid in the sample area (202) is sequentially and uniformly introduced into each quantitative area (214) through the first connecting channel (215), the liquid channel area (207) and the second connecting channel (210) by using centrifugal force, and after the sample liquid fills each quantitative area (214), the sample liquid is introduced into the gas pressure waste liquid area (209) through the gas pressure channel (208); After the sample liquid in the sample area (202) is completely output by centrifugation, the centrifugal disc is controlled to decelerate and stop, and the compressed gas in the gas pressure waste liquid area (209) pushes the sample liquid in the quantitative area (214) to enter the mixed amplification area (213) through the second siphon channel (216); After the quantitative sample liquid completely enters the mixed amplification area (213), the centrifugal disc is controlled to rotate in a positive and negative direction, so that the sample liquid is mixed with the sample spotting reagent or lyophilized ball pre-embedded in the mixed amplification area (213); After mixing for a preset time, the centrifugal disc is controlled to decelerate and stop, and the heating module is controlled to heat the mixed amplification area (213) to realize nucleic acid amplification of the mixed liquid; After amplification is completed, the centrifugal disc is controlled to rotate again, so that the amplified mixed liquid sequentially enters the detection area (212) through the first siphon channel (206), the buffer area (211) and the third connecting channel (217); The mixed liquid in the detection area (212) is subjected to optical nucleic acid detection.

10. The nucleic acid detection method according to claim 9, wherein, After the mixed liquid enters the detection area (212), before the optical nucleic acid detection is performed, the detection area (212) is heated. After the mixed liquid enters the detection area (212), before the optical nucleic acid detection is performed, the detection area (212) is heated.

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