Centrifugal microfluidic platform and nucleic acid extraction and amplification method therefor

Abstract

A centrifugal microfluidic platform and a nucleic acid extraction and amplification method therefor. The centrifugal microfluidic platform comprises a disk body and at least one microfluidic structure. Each of the at least one microfluidic structure comprises a sample storage tank, a first reaction tank, a non-polar solution storage tank, a non-polar solution accommodation tank, an elution tank, a polar solution storage tank, and a back-end reaction module. The first reaction tank is connected to the sample storage tank, and the non-polar solution storage tank is connected to the first reaction tank. A first end of the non-polar solution accommodation tank is connected to the first reaction tank by means of a bridge, and a first end of the elution tank is connected to a second end of the non-polar solution accommodation tank by means of a bridge. The polar solution storage tank is connected to the elution tank, and the back-end reaction module is connected to the elution tank.

Description

Centrifugal microfluidic platform and nucleic acid extraction and amplification method thereof

[0001] This application claims priority to Chinese Patent Application No. 202410839977.6, filed on June 26, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure relates to the field of microfluidic chip device technology for automated nucleic acid extraction and amplification, and in particular to a nucleic acid extraction method, a nucleic acid amplification method and microfluidic technology. BACKGROUND

[0003] With the rapid development of diagnostic technology, the improvement of disease cognition and the change of disease treatment methods, the in vitro diagnostic industry has developed rapidly in recent years. From the appearance of early blood cell analyzers, to the gradual popularization of biochemical detectors, to the application of chemiluminescence instruments, and finally to the emergence of genetic testing in the past five to ten years, the detection means has gone through the development process from cell morphology diagnosis, biochemical diagnosis, immunodiagnosis to molecular diagnosis.

[0004] The sensitivity of detection means is increasing, the specificity is rapidly enhancing, the application range is rapidly expanding, the operation threshold is gradually reducing, and the commercial value is becoming increasingly important. Molecular diagnostics is a branch of laboratory medicine or clinical pathology. This technology uses molecular biology to diagnose diseases, predict disease processes, select treatment methods, and monitor the effectiveness of treatment. Here, techniques such as quantitative polymerase chain reaction (qPCR) and quantitative real-time polymerase chain reaction (qRT-PCR) are widely used molecular biology techniques that can amplify and detect deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sequences for subsequent experimental purposes, and are still dominant in the market due to their simplicity and cost-effectiveness.

[0005] Compared with traditional biochemical detection or immunoprotein detection, molecular detection methods based on PCR technology have higher requirements in terms of cost, operation complexity, and laboratory construction. Due to the high purity requirement of PCR reaction itself for the sample to be tested, the complicated and time-consuming nucleic acid extraction preparation process, and other characteristics, a series of expensive instruments and complex laboratory manual operations are usually required to complete the PCR reaction.

[0006] With the current nucleic acid extraction method and the corresponding amplification method as an example, there are problems such as complicated operation steps, the need for multiple manual processing. Such a complex operation process is easy to introduce human factors, and also increases the errors that may occur in the operation process. For example, in multiple manual steps, the inaccuracy of the data may be caused by accidentally adding the wrong reagent or improper sample handling. Moreover, these multi-step operations also limit the speed and efficiency of extraction and amplification, affecting the work effect.

[0007] Furthermore, in the process of nucleic acid extraction and amplification, sample contamination of the experimental environment is an important problem. Such contamination may come from external DNA or RNA, affecting the accuracy and reliability of the experiment. Such contamination problems are particularly prominent in nucleic acid amplification, because even a small amount of contamination can cause significant deviation in the results.

[0008] In addition, a major problem in existing nucleic acid extraction methods is the high cost of required automated instruments. Existing automated instruments usually require a large amount of investment, including the cost of the equipment itself and the fees required for operation and maintenance. This makes it difficult for many small and medium-sized laboratories or research institutions to afford the high cost of automated instruments, limiting their ability to use these efficient methods for nucleic acid extraction and amplification.

[0009] At the same time, there are few products that can integrate the whole process of nucleic acid extraction and nucleic acid amplification. Existing methods often treat nucleic acid extraction and amplification as independent steps, and lack a solution that can efficiently integrate these two key links. This results in the need for multiple operations and sample transfers in actual operation, increasing the possibility of errors and wasting valuable time. SUMMARY

[0010] To solve the problems mentioned in the related art, the present disclosure provides a method to solve the problems of nucleic acid extraction and amplification methods in the related art. These innovative measures are expected to simplify the operation process, reduce equipment costs, and reduce sample contamination, thereby improving the efficiency and accuracy of nucleic acid extraction and amplification. This will be an important technical progress for the vast number of biological researchers and clinical laboratories, helping to accelerate the progress of related research and applications.

[0011] In some embodiments, the centrifugal microfluidic platform mainly includes a disc body and at least one microfluidic structure. Each of the microfluidic structures includes a sample storage tank, a first reaction tank, a non-polar solution storage tank, a non-polar solution containing tank, an elution tank, a polar solution storage tank, and a back-end reaction module.

[0012] The first reaction tank is connected to the sample storage tank, and the non-polar solution storage tank is connected to the first reaction tank. The non-polar solution accommodation tank bridges the first reaction tank at one end, and the elution tank bridges the other end of the non-polar solution accommodation tank. The polar solution storage tank is connected to the elution tank, and the rear-end reaction module is connected to the elution tank.

[0013] Based on the centrifugal microfluidic platform, the present disclosure also provides a nucleic acid extraction and amplification method of the centrifugal microfluidic platform. The nucleic acid extraction and amplification method of the centrifugal microfluidic platform provides the aforementioned centrifugal microfluidic platform in step (A). Then, step (B) centrifuges to make a sample in the sample storage tank, a non-polar solution in the non-polar solution storage tank, and a polar eluent in the polar solution storage tank flow into the first reaction tank.

[0014] In step (C), the sample, the non-polar solution, and the polar eluent are continuously centrifuged to stratify according to density, and the non-polar solution is only retained in the non-polar solution accommodation tank. Then, step (D) stops centrifugation and injects a plurality of magnetic beads into the first reaction tank, so that the plurality of magnetic beads capture target nucleic acids in the sample by an external magnetic field.

[0015] In step (E), the plurality of magnetic beads carrying impurities and the target nucleic acids pass through the non-polar solution accommodation tank retaining the non-polar solution by an external magnetic field to filter out the impurities and reach the elution tank. Further, step (F) centrifuges to separate the target nucleic acids from the plurality of magnetic beads under the action of the polar eluent.

[0016] Finally, step (G) suddenly changes the centrifugal direction to make the polar eluent with the target nucleic acids flow into the rear-end reaction module to perform a nucleic acid amplification reaction.

[0017] The above brief description of the present disclosure is intended to provide a basic description of several aspects and technical features of the present disclosure. The summary is not a detailed description of the present disclosure, and therefore its purpose is not to specifically list the key or important elements of the present disclosure, nor to define the scope of the present disclosure, but only to present several concepts of the present disclosure in a simple manner. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in some embodiments of the present disclosure or related technologies, the drawings needed in the embodiment or related technology description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and those skilled in the art can also obtain other drawings from these drawings without labor.

[0019] FIG. 1 is a schematic diagram of a centrifugal structure and data reading according to some embodiments;

[0020] FIG. 2 is a structural diagram of a microfluidic according to some embodiments;

[0021] FIG. 3 is a flowchart of a nucleic acid extraction and amplification method according to some embodiments.

[0022] In the drawings: 10 - centrifugal microfluidic platform, 100 - reader, 200 - disc body, 300 - fixed point, 400 - microfluidic structure, 401 - sample injection hole, 402 - sample storage tank, 403 - first connecting flow channel, 404 - first reaction tank vent hole, 405 - first reaction tank, 406 - non-polar solution containing tank, 407 - non-polar solution storage tank, 408 - elution tank vent hole, 409 - polar eluent injection hole, 410 - polar solution storage tank, 411 - second connecting flow channel, 412 - elution tank, 413 - siphon flow channel, 414 - gas compression tank, 415 - dispensing flow channel, 416 - dispensing tank, 417 - dispensing valve, 418 - second reaction tank, 419 - third connecting flow channel, 420 - phase change material storage tank, 421 - waste tank. DETAILED DESCRIPTION

[0023] The technology in some embodiments of the present disclosure will be described clearly and completely below with reference to the drawings in some embodiments of the present disclosure. Obviously, the described embodiments are only some of the embodiments of the present disclosure, not all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without labor are within the scope of protection of the present disclosure.

[0024] To solve the above technical problems, some embodiments of the present disclosure provide a centrifugal microfluidic platform and a nucleic acid extraction and amplification method thereof.

[0025] FIG. 1 is a schematic diagram of a centrifugal structure and data reading according to some embodiments. As shown in FIG. 1, the centrifugal microfluidic platform 10 in the present embodiment is mainly composed of a disc body 200. The disc body 200 is fixed to any machine with a centrifugal structure through a fixed point 300, which is not limited by the present disclosure. Accordingly, the disc body 200 can control the liquid flow in the microfluidic structure 400 by centrifugal rotation, exhibiting different microfluidic characteristics. Finally, after the test in the microfluidic structure 400 is completed, the reader 100 reads the results by optical reading, achieving the effect of automated detection.

[0026] FIG. 2 is a structural diagram of a microfluidic device according to some embodiments. FIG. 2 further illustrates the detailed structure of the microfluidic device 400 in the present embodiment. Referring to FIGS. 1 and 2, the microfluidic device 400 in the present embodiment mainly comprises a sample injection hole 401, a sample storage tank 402, a first connecting flow channel 403, a first reaction tank vent hole 404, a first reaction tank 405, a non-polar solution accommodating tank 406, a non-polar solution storage tank 407, an elution tank vent hole 408, a polar eluent injection hole 409, a polar solution storage tank 410, a second connecting flow channel 411, an elution tank 412, a siphon flow channel 413, a gas compression tank 414, a dispensing flow channel 415, a dispensing tank 416, a dispensing valve 417, a second reaction tank 418, a third connecting flow channel 419, a phase change material storage tank 420, and a waste tank 421.

[0027] In the present embodiment, the sample injection hole 401 is connected to the sample storage tank 402, and the sample storage tank 402 is connected to the first reaction tank 405 through the first connecting flow channel 403. In some embodiments, the first reaction tank 405 is provided with the first reaction tank vent hole 404 to balance the atmospheric pressure inside the first reaction tank 405. Then, the non-polar solution storage tank 407 is also connected to the first reaction tank 405. In the present embodiment, the non-polar solution storage tank 407 can also be provided with a non-polar solution injection hole (not shown), which is not limited by the present disclosure.

[0028] The non-polar solution accommodating tank 406 of the present embodiment is arranged at the bottom right corner of the first reaction tank 405. The non-polar solution accommodating tank 406 is in a U-shaped structure, which can be regarded as a configuration of a communicating pipe under a centrifugal state.

[0029] The non-polar solution accommodating tank 406 connects the first reaction tank 405 and the elution tank 412. For example, the non-polar solution accommodating tank 406 bridges the first reaction tank 405 at a first end and bridges the first end of the elution tank 412 at a second end. In the present embodiment, the elution tank 412 is in an inverted U-shaped structure, and the elution tank 412 is provided with the elution tank vent hole 408 to balance the atmospheric pressure inside the elution tank 412.

[0030] In some embodiments, the polar solution storage tank 410 is connected to the elution tank 412 through the second connecting flow channel 411, and the polar solution storage tank 410 is provided with the polar eluent injection hole 409.

[0031] In the present embodiment, the siphon flow channel 413, the gas compression groove 414, the dispensing flow channel 415, the at least one dispensing groove 416, the at least one dispensing valve 417, the at least one second reaction groove 418, the third connecting flow channel 419, the at least one phase change material storage groove 420, and the waste liquid groove 421 are collectively referred to as a rear-end reaction module. The rear-end reaction module is connected to the elution groove 412, for example, the rear-end reaction module is arranged at the second end of the elution groove 412 which is not connected to the non-polar solution containing groove.

[0032] Each of the at least one dispensing valve is connected to each of the at least one dispensing groove; each of the at least one second reaction groove is connected to each of the at least one dispensing valve; and each of the at least one phase change material storage groove is connected to each of the at least one second reaction groove through the third connecting flow channel.

[0033] Here, the gas compression groove 414 is arranged on the siphon flow channel 413. In the present embodiment, the siphon flow channel 413 is a valve structure controlled by capillary force and Euler force. Moreover, due to the long length of the siphon flow channel 413, the gas compression groove 414 can be used as an auxiliary force to pressurize and force the liquid in the elution groove 412 to flow out of the siphon flow channel 413 and into the dispensing flow channel 415 connected to the siphon flow channel 413 when the centrifugal force reaches a certain degree.

[0034] In the present embodiment, a plurality of dispensing grooves 416 are arranged on the dispensing flow channel 415. The dispensing grooves 416 are arranged as a larger recess structure, which can guide the eluted sample to enter the second reaction groove 418 after breaking through the dispensing valve 417 due to the centrifugal force.

[0035] Moreover, according to the reaction (for example, nucleic acid amplification reaction) required in the second reaction groove 418, the phase change material storage groove 420 can be heated by an external machine in the form of electric heating or light heating, so that the phase change material stored in the phase change material storage groove 420 is melted from solid to liquid and enters the second reaction groove 418 through the third connecting flow channel 419 to participate in the reaction. Or the reagent lyophilized block originally filled in the second reaction groove 418 is dissolved by the eluted sample, and biochemical reactions such as nucleic acid amplification are carried out, so as to facilitate subsequent result reading and detection.

[0036] Finally, the excess liquid is concentrated in the waste liquid groove 421 at the end of the dispensing flow channel 415 to prevent backflow into the second reaction groove 418 from affecting the reaction result, which is not limited in the present disclosure.

[0037] FIG. 3 is a flowchart of a nucleic acid extraction and amplification method according to some embodiments. Referring to FIG. 3, the embodiment of FIG. 3 will directly take the nucleic acid extraction and amplification as a detailed explanation of how the present embodiment should be performed.

[0038] First, step (A) is to provide a centrifugal microfluidic platform 10 as shown in FIG. 1 and FIG. 2, which includes a disc 200 with a microfluidic structure 400. Next, before step (B), the sample storage slot 402 in this embodiment can be pre-stored or injected by the sample injection hole 401. The sample in this embodiment can be bacteria, viruses, specific cells after being broken, whole blood or peripheral blood samples, etc. containing nucleic acid samples, which are not limited by the present disclosure.

[0039] In some embodiments, the non-polar solution storage slot 407 can also be filled by injecting or pre-storing non-polar solution. It should be noted that the density of the non-polar solution selected in this embodiment needs to be greater than the density of the sample itself or the sample and its solution. And the polar solution storage slot 410 also needs to be filled by injecting or pre-storing polar eluent through the polar eluent injection hole 409.

[0040] Therefore, when the sample storage slot 402, the non-polar solution storage slot 407, and the polar solution storage slot 410 are all prepared, step (B) can be performed, and the centrifugal disc 200 is centrifuged to make the sample and the non-polar solution in the sample storage slot 402, the non-polar solution storage slot 407, and the polar solution storage slot 410 flow into the first reaction slot 405; At the same time, the polar eluent flows into the elution slot 412.

[0041] Next, as shown in step (C), the centrifugal disc 200 continues to centrifuge, at which time the sample, the non-polar solution, and the polar eluent will be stratified according to their densities, and the non-polar solution will be retained in the non-polar solution containing slot 406. After step (C) is completed, the non-polar solution containing slot 406 will be filled with non-polar solution (such as oil phase solvent). Therefore, the non-polar solution containing slot 406 will effectively separate the first reaction slot 405 and the elution slot 412.

[0042] Next, after the so-called polar / non-polar / polar (first reaction slot 405 / non-polar solution containing slot 406 / elution slot 412) structure is formed, step (D) is performed. In step (D) of this embodiment, the centrifugation is first stopped, and a plurality of magnetic beads are injected into the first reaction slot 405, so that the plurality of magnetic beads capture the target nucleic acid in the sample in the first reaction slot 405 by applying a magnetic field outside the disc 200. In this embodiment, the plurality of magnetic beads can be added through the sample injection hole 401, which is not limited by the present disclosure.

[0043] In some embodiments, after the plurality of magnetic beads have captured the target nucleic acid, the plurality of magnetic beads carrying the impurities and the target nucleic acid are passed through the non-polar solution holding groove 406 by an external magnetic field. In this way, the impurities can be filtered out by the non-polar solution holding groove 406 before reaching the elution groove 412. Thus, the method can effectively filter out impurities that are polar, and by the principle that polar and non-polar solutions are immiscible, impurities other than the target nucleic acid are effectively filtered out, ensuring the purity and quality of the extracted target nucleic acid.

[0044] Next, after the plurality of magnetic beads carrying the target nucleic acid enter the elution groove 412, the target nucleic acid is separated in the polar eluent by the action of the polar eluent in the elution groove 412. After the nucleic acid to be extracted is substantially carried by the polar eluent, the embodiment performs step (G), which suddenly changes the centrifugal direction of the disc 200, causing the polar eluent carrying the target nucleic acid to flow into the rear-end reaction module shown in FIGS. 1 and 2 for nucleic acid amplification reaction.

[0045] The subsequent reactions in the rear-end reaction module of the embodiment are mainly designed to meet the structure of the nucleic acid amplification reaction. First, the polar eluent carrying the target nucleic acid breaks through the siphon flow channel 413 and enters the dispensing flow channel 415 due to the Euler force generated by step (G) and the pressure from the gas compression groove 414.

[0046] Next, after the polar eluent carrying the target nucleic acid enters the dispensing flow channel 415, the embodiment further increases the rotation speed of the centrifugal disc 200, causing the polar eluent carrying the target nucleic acid to pass through the at least one dispensing groove 416, break through the at least one dispensing valve 417, and enter the at least one second reaction groove 418 for the nucleic acid amplification reaction. As before, the dispensing groove 416 of the embodiment has a large recessed structure that can guide the eluted sample to break through the dispensing valve 417 and enter the second reaction groove 418 due to the centrifugal force.

[0047] Furthermore, depending on the reaction required in the second reaction groove 418 (e.g., nucleic acid amplification reaction), an external machine can be used to heat the phase change material stored in the phase change material storage groove 420 from solid to liquid, and then break through the third connecting flow channel 419 into the second reaction groove 418 to participate in the reaction; or the reagent lyophilized block originally filled in the second reaction groove 418 is dissolved by the eluted sample, and biochemical reactions such as nucleic acid amplification are performed for subsequent result reading and detection.

[0048] Finally, the excess liquid will be collected in the waste liquid tank 421 at the end of the dispensing flow channel 415 to prevent backflow into the second reaction tank 418 from affecting the results of the reaction. After the reaction is completed, the reader 100 shown in FIG. 1 can read the results of the reaction after amplification of the extracted nucleic acid of the sample by various reading methods such as reading the absorbance value.

[0049] The above description is only the preferred embodiments of the present disclosure, and cannot limit the scope of the present disclosure. Simple equivalent changes and modifications made in accordance with the patent scope and description of the present application are still within the scope of the present disclosure. The above is only the preferred embodiment of the present disclosure, and does not limit the patent scope of the present disclosure. Any equivalent structural transformation made in accordance with the disclosure description and drawings, or direct or indirect application in other related technical fields is included in the patent protection scope of the present disclosure.

Claims

1. A centrifugal microfluidic platform, comprising: One dish; At least one microfluidic structure is disposed on the disk, including: A sample storage slot; A first reaction tank is connected to the sample storage tank; A nonpolar solution storage tank is connected to the first reaction tank; A nonpolar solution container is bridged to the first reaction tank at a first end; A washing tank, the first end of which is bridged to the second end of the non-polar solution container tank; A polar solution storage tank is connected to the elution tank; and A back-end reaction module is connected to the elution tank.

2. The centrifugal microfluidic platform according to claim 1, wherein, The sample storage tank is also connected to a sample injection port.

3. The centrifugal microfluidic platform according to claim 1 or 2, wherein, The first reaction tank is also connected to a first reaction tank vent.

4. The centrifugal microfluidic platform according to any one of claims 1-3, wherein, The sample storage tank is connected to the first reaction tank via a first connecting channel.

5. The centrifugal microfluidic platform according to any one of claims 1-4, wherein, The nonpolar solution storage tank is also equipped with a nonpolar solution injection port.

6. The centrifugal microfluidic platform according to any one of claims 1-5, wherein, The nonpolar solution container has a U-shaped structure.

7. The centrifugal microfluidic platform according to claim 6, wherein, The elution tank has an inverted U-shaped structure.

8. The centrifugal microfluidic platform according to any one of claims 1-7, wherein, The washing tank is also equipped with a vent.

9. The centrifugal microfluidic platform according to any one of claims 1-8, wherein, The polar solution storage tank is also connected to the elution tank via a second connecting channel.

10. The centrifugal microfluidic platform according to any one of claims 1-9, wherein, The polar solution storage tank is also equipped with a polar elution injection port.

11. The centrifugal microfluidic platform according to any one of claims 1-10, wherein, The second end of the elution tank, which is not connected to the non-polar solution container, is equipped with the rear reaction module.

12. The centrifugal microfluidic platform according to claim 11, wherein, The back-end reaction module includes: One siphon channel; A gas compression tank is installed on the siphon channel; A dispensing channel is connected to the siphon channel; At least one dispensing slot is provided on the dispensing flow channel; At least one dispensing valve, each of the at least one dispensing valves being connected to each of the at least one dispensing slots; At least one second reaction tank, each of the at least one second reaction tank being connected to each of the at least one dispensing valves; At least one phase change material storage tank, each phase change material storage tank being connected to each second reaction tank in the at least one second reaction tank via a third connecting flow channel; and A waste liquid tank is located at the bottom of the dispensing channel.

13. A method for nucleic acid extraction and amplification using a centrifugal microfluidic platform, comprising: (A) Provides the centrifugal microfluidic platform according to claim 12; (B) Centrifugation causes a sample and a nonpolar solution in the sample storage tank, the nonpolar solution storage tank, and the polar solution storage tank to flow into the first reaction tank, and a polar elution solution to flow into the elution tank. (C) Continuous centrifugation causes the sample, the nonpolar solution, and the polar eluent to separate into layers according to density, and the nonpolar solution remains only in the nonpolar solution container. (D) Stop centrifugation and inject multiple magnetic beads into the first reaction tank, so that the multiple magnetic beads capture the target nucleic acid in the sample by means of an external magnetic field; (E) The multiple magnetic beads carrying impurities and the target nucleic acid are passed through the non-polar solution container containing the non-polar solution by means of an external magnetic field to filter out the impurities and then reach the elution tank. (F) The target nucleic acid is separated by centrifugation under the action of the polar elution solution; (G) Suddenly change the centrifugation direction to allow the polar elution buffer containing the target nucleic acid to flow into the back-end reaction module for nucleic acid amplification reaction.

14. The method for nucleic acid extraction and amplification using a centrifugal microfluidic platform according to claim 13, wherein, When the polar elution containing the target nucleic acid breaks through the siphon channel, it is propelled by pressure from the gas compression tank.

15. The method for nucleic acid extraction and amplification using a centrifugal microfluidic platform according to claim 14, wherein, After the polar elution containing the target nucleic acid enters the dispensing channel, the centrifugation speed is increased so that the polar elution containing the target nucleic acid is distributed through the at least one dispensing tank and then passes through the at least one dispensing valve into the at least one second reaction tank to carry out the nucleic acid amplification reaction.

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