Nucleic acid extraction and detection integrated centrifugal microfluidic chip, detection method and application thereof

Abstract

The application discloses a nucleic acid extraction and detection integrated centrifugal micro-fluidic chip and a detection method and application thereof. The nucleic acid extraction and detection integrated centrifugal micro-fluidic chip comprises a micro-fluidic chip base body, the micro-fluidic chip base body comprises a nucleic acid extraction unit and a nucleic acid detection unit; the nucleic acid extraction unit is used for extracting nucleic acid in a sample, the nucleic acid extraction unit stores dry nucleic acid amplification reagents; the nucleic acid detection unit is used for detecting the nucleic acid extracted by the nucleic acid extraction unit, and the nucleic acid detection unit stores dry primers. The nucleic acid extraction and detection integrated centrifugal micro-fluidic chip of the application can quickly extract nucleic acid from a clinical sample and simultaneously perform detection by storing dry nucleic acid amplification reagents and primers prepared in advance, and the efficiency of nucleic acid extraction and detection is significantly improved. The detection result can be obtained within one hour.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to an integrated centrifugal microfluidic chip for nucleic acid extraction and detection, its detection method, and its application. Background Technology

[0002] Respiratory viruses are highly contagious and can cause widespread infection. Early detection is crucial for preventing economic losses and reducing mortality. Microfluidic chips, known for their small size, high integration, rapid analysis, and low reagent consumption, are widely used in pathogen detection. Centrifugal microfluidic chips, in particular, require only a small motor to precisely control liquids. By adjusting the rotation speed and direction, they can perform fluid pumping, valve control, metering, and mixing operations without manual pipetting and complex equipment. However, their small size and design limitations make integrating nucleic acid extraction, purification, and amplification challenging, significantly limiting their application in on-site pathogen detection.

[0003] Therefore, it is crucial to design an integrated centrifugal microfluidic chip for nucleic acid extraction and detection. Summary of the Invention

[0004] This invention provides an integrated centrifugal microfluidic chip for nucleic acid extraction and detection, along with its detection method and applications.

[0005] The first aspect of this invention provides an integrated centrifugal microfluidic chip for nucleic acid extraction and detection, comprising a microfluidic chip substrate, wherein the microfluidic chip substrate includes a nucleic acid extraction unit and a nucleic acid detection unit; wherein the nucleic acid extraction unit is used to extract nucleic acids from a sample, and the nucleic acid extraction unit stores dried nucleic acid amplification reagents; the nucleic acid detection unit is used to detect the nucleic acids extracted by the nucleic acid extraction unit, and the nucleic acid detection unit stores a dried primer set.

[0006] According to one embodiment of the present invention, the nucleic acid extraction unit includes: a sample chamber for adding a sample and a washing solution; an elution chamber for adding a nucleic acid elution solution; a nucleic acid extraction chamber connected to the sample chamber and the elution chamber for extracting nucleic acids from the sample; a nucleic acid collection chamber connected to the nucleic acid extraction chamber and storing the dried nucleic acid amplification reagent for amplification reaction with the nucleic acid entering from the nucleic acid extraction chamber; and a first waste liquid chamber connected to the nucleic acid extraction chamber for storing waste liquid flowing through the nucleic acid extraction chamber.

[0007] According to another embodiment of the present invention, the nucleic acid extraction chamber is equipped with glass fiber filter paper with a pore size of 0.7-2.0 μm.

[0008] According to another embodiment of the present invention, the nucleic acid detection unit includes: a paraffin chamber storing solid paraffin; a dispensing structure including multiple dispensing chambers and a second waste liquid chamber; and a nucleic acid amplification reaction structure including multiple nucleic acid amplification reaction chambers, each nucleic acid amplification reaction chamber communicating with each dispensing chamber, wherein the nucleic acid amplification reaction chambers store the dried primer set for nucleic acid detection; wherein each dispensing chamber is communicating with the paraffin chamber, and the solid paraffin, after melting, can enter each dispensing chamber to cut off the communication channel between the multiple nucleic acid amplification reaction chambers.

[0009] According to another embodiment of the present invention, the packaging structure includes 3-12 packaging chambers.

[0010] According to another embodiment of the present invention, the nucleic acid collection chamber and the dispensing structure are connected by a siphon channel.

[0011] According to another embodiment of the present invention, the nucleic acid extraction unit further includes: a transfer chamber, one side of which is connected to the nucleic acid extraction chamber, the nucleic acid collection chamber and the first waste liquid chamber, wherein the nucleic acid extracted by the nucleic acid extraction chamber through the transfer chamber and the waste liquid enter the nucleic acid collection chamber and the first waste liquid chamber respectively, and the other side of which is connected to the second waste liquid chamber; preferably, an exhaust valve is provided in the channel connecting the transfer chamber and the second waste liquid chamber.

[0012] According to another embodiment of the present invention, the first waste liquid chamber and the second waste liquid chamber are filled with a water-absorbing polymer for adsorbing the stored waste liquid.

[0013] According to another embodiment of the present invention, the microfluidic chip substrate is a rounded rectangle with a through hole at its center; the microfluidic chip substrate includes two functional modules with identical structures, which are symmetrically distributed radially on the substrate with the through hole as the center, and each functional module includes the nucleic acid extraction unit and the nucleic acid detection unit.

[0014] According to another embodiment of the present invention, it further includes: an encapsulation film for sealing the upper surface of the microfluidic chip substrate. Preferably, the encapsulation film is a pressure-sensitive adhesive film.

[0015] A second aspect of the present invention also provides a detection method based on the above-mentioned integrated centrifugal microfluidic chip for nucleic acid extraction and detection, comprising: extracting nucleic acid from the sample by the nucleic acid extraction unit under centrifugal force; and entering the nucleic acid detection unit for detection under centrifugal force.

[0016] According to one embodiment of the present invention, the detection method includes: adding a sample and lysis buffer into a sample chamber; rotating the microfluidic chip in a first rotation direction; under centrifugal force, the sample in the sample chamber enters a nucleic acid extraction chamber, where the nucleic acid in the sample is adsorbed onto the filter paper in the nucleic acid extraction chamber; and waste liquid entering a waste liquid chamber via a transfer chamber; adding a washing solution into the sample chamber; rotating the microfluidic chip in the first rotation direction; under centrifugal force, the washing solution sequentially passes through the nucleic acid extraction chamber and the transfer chamber, and enters the waste liquid chamber; adding an elution solution into an elution chamber; rotating the microfluidic chip in a second rotation direction; under centrifugal force, the elution solution passes through the nucleic acid extraction chamber, eluting the nucleic acid adhering to the filter paper; and entering a nucleic acid collection chamber via the transfer chamber, where it is mixed with the dried nucleic acid amplification reagent stored in the nucleic acid collection chamber. The microfluidic chip is rotated in a second direction at a first rotational speed. Under centrifugal force, the mixed solution in the nucleic acid collection chamber enters each dispensing chamber of the dispensing structure through a siphon channel, while excess liquid enters the second waste liquid chamber. The microfluidic chip is then rotated in a second direction at a second rotational speed. Under centrifugal force, the mixed solution in each dispensing chamber enters its corresponding nucleic acid amplification reaction chamber. The paraffin chamber is heated at a fixed point until the solid paraffin stored in the paraffin chamber melts. The microfluidic chip is then rotated in a second direction. Under centrifugal force, the melted paraffin enters each dispensing chamber and seals its corresponding nucleic acid amplification reaction chamber. The microfluidic chip is placed in a corresponding heating device to perform a nucleic acid amplification reaction, and then a judgment is made. The first rotational direction is opposite to the second rotational direction, and the first rotational speed is less than the second rotational speed.

[0017] A third aspect of the present invention provides an application of the above-mentioned integrated centrifugal microfluidic chip for nucleic acid extraction and detection in the fields of colorimetric, fluorescence or immunochromatographic analysis.

[0018] This invention relates to an integrated centrifugal microfluidic chip for nucleic acid extraction and detection. By storing pre-prepared dried nucleic acid amplification reagents and primer sets, it enables rapid extraction of nucleic acids from clinical samples and simultaneous detection, significantly improving the efficiency of nucleic acid extraction and detection. Results can be obtained within one hour. It meets the "sample in, result out" requirements for on-site testing, providing high nucleic acid extraction efficiency, accuracy, and specificity. It can be widely applied in healthcare, agricultural, and environmental systems, greatly improving the accuracy and efficiency of on-site testing. Attached Figure Description

[0019] Figure 1A This is a schematic diagram of the microfluidic chip substrate of the integrated centrifugal microfluidic chip for nucleic acid extraction and detection of the present invention.

[0020] Figure 1B This is a partial physical image of the integrated centrifugal microfluidic chip for nucleic acid extraction and detection of the present invention.

[0021] Figure 2 This is a schematic diagram of the integrated centrifugal microfluidic chip for nucleic acid extraction and detection of the present invention.

[0022] Figure 3 This is a physical image of the integrated centrifugal microfluidic chip for nucleic acid extraction and detection based on the present invention, and a schematic diagram of the liquid flow direction inside the chip.

[0023] Figure 4 This is a schematic flowchart of the detection method based on the integrated centrifugal microfluidic chip for nucleic acid extraction and detection of the present invention.

[0024] Figure 5 These are the primer sequences for each target used in Example 1.

[0025] Figure 6 The nucleic acid amplification reagents freeze-dried within the chip in Example 1 can be stored at 4°C for at least 28 days.

[0026] Figure 7 The chip shown in Example 1 can present various nucleic acid amplification results in different formats (the primers in the nucleic acid reaction cell from left to right are H1N1, FluB, RSVA, N, S, Orf1al, ACTB, and blank control; A in the figure is colorimetric method, B is fluorescence method, and C is agarose gel electrophoresis).

[0027] Figure 8 These are the experimental results of chip sensitivity detection in Example 1.

[0028] Figure 9 This is the chip-specific detection result of Example 1.

[0029] Figure 10 The results are from clinical samples tested using the chip described in Example 1.

[0030] The reference numerals in the attached figures are explained as follows:

[0031] 100-Microfluidic chip substrate; 11-Sample chamber; 12-Eluting chamber; 13-Nucleic acid extraction chamber; 14-Transfer chamber; 15-Nucleic acid collection chamber; 16-First waste liquid chamber; 21-Paraffin chamber; 211-Columnar structure; 22-Dispensing chamber; 23-Nucleic acid amplification reaction chamber; 24-Second waste liquid chamber; 31-Siphon channel; 32-First channel; 33-Exhaust valve; 40-Through hole; 200-Encapsulation film Detailed Implementation

[0032] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail with reference to the following specific embodiments and accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0033] like Figure 1A and Figure 1B As shown, the integrated centrifugal microfluidic chip for nucleic acid extraction and detection of the present invention includes a microfluidic chip substrate 100, which includes a nucleic acid extraction unit and a nucleic acid detection unit. The nucleic acid extraction unit is used to extract nucleic acids from the sample. The nucleic acid extraction unit stores dried nucleic acid amplification reagents, such as nucleic acid amplification reagents other than primers and targets, such as nucleic acid amplification enzymes, dNTPs, dyes, etc. (e.g., LavaLAMP). TM The RNA component kit (Biosearch Technologies, UK) includes a nucleic acid detection unit for detecting nucleic acids extracted by the nucleic acid extraction unit. This unit stores a dried primer set. The dried nucleic acid amplification reagent and dried primers can be obtained by any suitable method, such as freeze-drying, air-drying, or other similar methods. This invention's integrated centrifugal microfluidic chip for nucleic acid extraction and detection, by storing pre-prepared dried nucleic acid amplification reagents and primer sets, enables rapid extraction of nucleic acids from clinical samples and simultaneous detection of multiple targets, significantly improving the efficiency of nucleic acid extraction and detection.

[0034] In this patent, "connected" and "connected" include both direct and indirect connections between two units (rooms or structures). "Indirect connection" refers to the presence of other units (rooms or structures) along the pathway between two connected or interconnected units.

[0035] In alternative implementations, such as Figure 1A and Figure 1B As shown, the nucleic acid extraction unit may include a sample chamber 11, an elution chamber 12, a nucleic acid extraction chamber 13, a nucleic acid collection chamber 15, and a first waste liquid chamber 16, etc.

[0036] Sample chamber 11 is used to add samples and cleaning solution for nucleic acid purification.

[0037] Elution chamber 12 is used to add nucleic acid elution buffer.

[0038] The nucleic acid extraction chamber 13, connected to the sample chamber 11 and the elution chamber 12, is used to extract nucleic acids from the sample. Glass fiber filter paper can be placed inside the nucleic acid extraction chamber 13 to adsorb nucleic acids from the sample. The shape of the glass fiber filter paper corresponds to the shape of the nucleic acid extraction chamber 13, for example, it can be a circle with a diameter of 3 mm. Two layers of glass fiber filter paper can be used. The pore size of the filter paper can be 0.7-2.0 μm; an appropriate pore size can be selected according to the type of nucleic acid being extracted to achieve rapid and efficient RNA purification. For example, but not limited to, Whatman... TM The company's GF / F glass microfiber filter paper has a pore size of 0.7 μm. The inclusion of a microfiber filter (i.e., glass fiber filter paper) in the chip of this invention provides a simple solution compared to the complexities associated with nucleic acid extraction methods using magnetic beads, silica gel columns, Chelex resin, or glass beads. This paper microfiber filter can be easily prepared for large-scale production.

[0039] The nucleic acid collection chamber 15, connected to the nucleic acid extraction chamber 13, stores dried nucleic acid amplification reagents for amplification reactions with nucleic acids entering from the extraction chamber 13. The chip of this invention pre-stores dried nucleic acid amplification reagents, which, due to their high stability, address the challenges of storage, transportation, and availability in COVID-19 infection and influenza diagnostic kits.

[0040] The first waste liquid chamber 16 is connected to the nucleic acid extraction chamber 13 and is used to store the waste liquid flowing through the nucleic acid extraction chamber 13.

[0041] In an optional embodiment, the nucleic acid extraction unit further includes a transfer chamber 14. The transfer chamber 14 is disposed between the nucleic acid extraction chamber 13, the nucleic acid collection chamber 15, and the first waste liquid chamber 16. One side of the transfer chamber 14 communicates with the nucleic acid extraction chamber 13, the nucleic acid collection chamber 15, and the first waste liquid chamber 16. Nucleic acid extracted from the nucleic acid extraction chamber 13 and waste liquid extracted through the transfer chamber 14 enter the nucleic acid collection chamber 15 and the first waste liquid chamber 16, respectively. The other side of the transfer chamber 14 communicates with the second waste liquid chamber 24 of the nucleic acid detection unit through a first channel 32. An exhaust valve 33 is provided in the first channel 32 to balance the gas pressure inside the chip during centrifugation, ensuring smooth liquid flow.

[0042] In an optional implementation, the nucleic acid detection unit may include a paraffin chamber 21, a dispensing structure, and a nucleic acid amplification reaction structure.

[0043] The paraffin chamber 21 stores solid paraffin. The side of the paraffin chamber 21 closest to the dispensing structure can be a row of evenly arranged columnar structures 211, resembling a fence. This effectively confines the paraffin within the paraffin chamber during storage without affecting its subsequent melting and entry into the dispensing structure and nucleic acid amplification reaction structure.

[0044] The dispensing structure includes multiple dispensing chambers 22 and a second waste liquid chamber 24. The dispensing structure may include 3-12 dispensing chambers 22. An appropriate number of dispensing chambers 22 can be selected depending on the specific circumstances, such as the type of virus being tested. The figure shows an example with 8 chambers. Those skilled in the art should understand that the chip of this invention is not limited to including 8 dispensing chambers 22. The dispensing structure is connected to the nucleic acid collection chamber 15 via a siphon channel 31. Under centrifugal force, the mixed solution in the nucleic acid collection chamber 15 enters the dispensing structure through the siphon channel 31 and is then distributed to each dispensing chamber 22. The remaining liquid enters the second waste liquid chamber 24.

[0045] The nucleic acid amplification reaction structure includes multiple nucleic acid amplification reaction chambers 23, each of which is connected to a dispensing chamber 22. The dispensing chambers 22 are connected to the nucleic acid amplification reaction chambers 23 via thin, shallow channels. These thin, shallow channels allow the mixed solution from the nucleic acid collection chamber 15 to be dispensed into the respective dispensing chambers at low rotation speeds and to be filled into the corresponding nucleic acid amplification reaction chambers 23 at high rotation speeds. The nucleic acid amplification reaction chambers 23 store dried primer sets; each chamber 23 stores different primers to enable multiplex detection of viruses, such as respiratory viruses.

[0046] Each dispensing chamber 22 is connected to the paraffin chamber 21. After the solid paraffin melts, it can enter each dispensing chamber 22 to cut off the communication channels between multiple nucleic acid amplification reaction chambers 23.

[0047] In an optional embodiment, a water-absorbing polymer may be provided in the first waste liquid chamber 16 and the second waste liquid chamber 24 to adsorb the stored waste liquid.

[0048] In alternative implementations, such as Figure 2 As shown, the microfluidic chip substrate 100 can be a rounded rectangle with a through-hole 40 at its center for fixing the chip in a centrifuge. The microfluidic chip substrate 100 contains two identical functional modules, symmetrically distributed radially on the substrate 100 with the through-hole 40 as the center. Each functional module includes the aforementioned nucleic acid extraction unit and nucleic acid detection unit. Therefore, the chip of this invention can simultaneously detect two independent samples, significantly improving the efficiency of nucleic acid extraction and detection.

[0049] The microfluidic chip substrate 100 of the present invention is further covered with an encapsulation film 200 to seal the microchannels on the microfluidic chip substrate 100, thereby forming a double-layer structure. Through-holes are also provided at corresponding positions to the through-holes 40 on the microfluidic chip substrate 100 in the encapsulation film 200. The encapsulation film 200 can be pressure-sensitive adhesive (PSA) films. This invention integrates nucleic acid extraction and detection using a simple double-layer structure. Compared to a complex three-layer structure, this significantly reduces the manufacturing cost of centrifuged microfluidic chips.

[0050] The chip of this invention can directly process and detect clinical samples, simultaneously detecting multiple targets from two independent samples. It boasts high nucleic acid extraction and detection efficiency and is widely applicable to various detection devices, such as integrated devices for colorimetric, fluorescence, and immunochromatographic analysis. It includes colorimetric and fluorescence detectors for real-time monitoring of any color changes and / or peak emission of the fluorescence signal during the reaction process.

[0051] The following describes the detection method based on the aforementioned integrated nucleic acid extraction and detection centrifugal microfluidic chip. To clearly illustrate the detection method, Figure 3 and Figure 4 Orange indicates fluids, and blue indicates paraffin. For example... Figure 3 As shown, the nucleic acid extraction unit extracts nucleic acid from the sample under centrifugal force; and the extracted nucleic acid enters the nucleic acid detection unit for detection under centrifugal force.

[0052] Specifically, such as Figure 4 As shown, the sample and lysis buffer are first added to the sample chamber. The microfluidic chip is then rotated in the first rotational direction (e.g., counterclockwise). Under centrifugal force, the sample in the sample chamber enters the nucleic acid extraction chamber. The rotation speed can be 3000-6000 rpm / min, and the duration can be 20-60 s. The nucleic acids in the sample are adsorbed onto the filter paper in the nucleic acid extraction chamber, and the waste liquid enters the waste liquid chamber through the transfer chamber.

[0053] Next, cleaning solution is added to the sample chamber, and the microfluidic chip is rotated in the first rotation direction. Under centrifugal force, the cleaning solution passes sequentially through the nucleic acid extraction chamber and the transfer chamber, and then enters the waste liquid chamber. This step is to clean impurities adsorbed on the filter paper.

[0054] Subsequently, eluent is added to the elution chamber, and the microfluidic chip is rotated in a second direction (e.g., clockwise). Under centrifugal force, the eluent passes through the nucleic acid extraction chamber, eluting the nucleic acid adhering to the filter paper. This eluent then enters the nucleic acid collection chamber via the transfer chamber, where it undergoes a nucleic acid amplification reaction with the dried nucleic acid amplification reagent stored there. The rotation speed for this step can be 3000-6000 rpm / min, and the duration can be 60-90 seconds. The entire nucleic acid extraction step can be completed within 5 minutes.

[0055] Subsequently, the microfluidic chip rotates in a second direction at a first rotational speed (low rotation speed). Under the action of centrifugal force, the mixed solution in the nucleic acid collection chamber enters each dispensing chamber of the dispensing structure through a siphon channel, while excess liquid enters the second waste chamber. The first rotational speed can be 600-1500 rpm / min, and the duration can be 60-90 s.

[0056] Next, the microfluidic chip is rotated in a second direction at a second rotational speed (high speed). Under the action of centrifugal force, the mixed solution in each dispensing chamber enters its corresponding nucleic acid amplification reaction chamber. The second rotational speed can be 4000-5000 rpm / min, and the duration can be 60-90 s.

[0057] Next, the paraffin chamber is heated at a specific point until the solid paraffin stored inside melts. Then, the microfluidic chip is rotated in a second rotational direction. Under centrifugal force, the molten paraffin enters each dispensing chamber and seals its corresponding nucleic acid amplification reaction chamber, preventing cross-contamination and volatilization during the reaction. The heating temperature and time of the paraffin can be selected appropriately according to the type of paraffin, for example, but not limited to, a heating temperature of 70-90℃ and a heating time of 3-5 minutes. The rotation speed in this step can be 4000-5000 rpm / min, and the duration can be 30-60 seconds.

[0058] Finally, the microfluidic chip is placed in a suitable heating device to perform the nucleic acid amplification reaction, and then the results are evaluated. The heating temperature can be appropriately selected based on the reaction system; for example, using LavaLAMP. TM The RNA component kit (Biosearch Technologies, UK) reaction system needs to be heated at 65-70℃ for 30-60 minutes. Then, the fluorescence intensity or colorimetric changes during each reaction time are detected to determine whether the virus has caused the infection.

[0059] The present invention is further described below through specific examples. However, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents, materials, and instruments used in the following embodiments and comparative examples are commercially available.

[0060] Example 1

[0061] This embodiment utilizes Figure 1A , Figure 1B and Figure 2 The chip shown simultaneously detects and diagnoses four respiratory viruses—influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2—using loop-mediated isothermal amplification (LAMP) technology. Figure 5 As shown, the marker genes of influenza A, influenza B, and respiratory syncytial virus, as well as the N, S, and Orf1ab genes of SARS-CoV-2, were detected, while the seventh target, the ACTB gene, was an internal reference gene for human samples.

[0062] This chip utilizes a glass cellulose membrane to extract and purify nucleic acids from clinical samples. The glass cellulose membrane used in this embodiment is a Whatman brand. TM Grade GF / F glass microfiber filters with a pore size of 0.7 μm were used. Glass cellulose membranes were fabricated into 3 mm diameter discs using a perforator. Two pre-cut circular glass cellulose membranes were placed in each nucleic acid extraction chamber of the chip for subsequent experimental use.

[0063] The nucleic acid amplification reagents required for the chip are pre-stored using a freeze-drying method. LavaLAMP is used in this example. TM The RNA component kit (Biosearch Technologies, UK) was used to mix 45 μL of LavaLAMP RNAMaster Mix and 3.6 μL of Green Fluorescent Dye, which was then added to the nucleic acid collection chamber on the chip. After incubation at -80°C for 10 hours, the mixture was freeze-dried at -85°C for 12 hours. Figure 6 As shown, compared with the undried nucleic acid amplification reagent (the green line in the figure is the fluorescence curve of the liquid nucleic acid amplification reagent group, and the red line is the fluorescence curve of the lyophilized nucleic acid amplification reagent group), the reagent stored at 4℃ for 28 days still maintained a considerable nucleic acid amplification capacity and had a certain degree of stability.

[0064] The primers required for each target within the chip are pre-stored using an air-drying method. The primer sequences used in this embodiment are as follows: Figure 5 As shown, each target contains 5 or 6 primers, namely F3, B3, FIP, BIP, LF, and LB. In this embodiment, each nucleic acid amplification reaction chamber in the chip is pre-filled with one type of primer for a specific target, while the eighth reaction chamber is left unfilled as a blank control. Each reaction chamber is filled with 0.2 μM F3 and B3, 1.6 μM FIP and BIP, and 0.4 μM LF and LB primer solutions. The chip is then incubated at room temperature for 2 hours until the primers are completely dry.

[0065] The paraffin in this chip is produced by directly adding 90μL of molten paraffin into the paraffin chamber and allowing it to solidify through natural cooling.

[0066] The superabsorbent polymer (SAP) used in this chip is prepared by co-blending polyvinyl alcohol (type 1788) with sodium polyacrylate. This method yields a large SAP sheet, which is then punched into smaller pieces and placed directly into the chip's waste liquid chamber. This material has superabsorbent properties, thus absorbing and retaining liquid within the waste liquid chamber.

[0067] After all the above parts are prepared, the chip is encapsulated using a PSA film. In this embodiment, the PSA film used is MicroAmp Optical Adhesive Film (ThermoFisher).

[0068] In this embodiment, the chip prepared above is used for detection. Step 1: 18 μL of nasopharyngeal swab sample is thoroughly mixed with 72 μL of lysis buffer, and then the entire mixture is added to the sample chamber of the chip. The microfluidic chip is rotated in reverse at 4000 rpm / min for 60 s. Under centrifugal force, the sample in the sample chamber passes through the nucleic acid extraction chamber, where nucleic acid molecules are adsorbed onto the glass fiber filter paper. The sample then passes through the transfer chamber and enters the first waste liquid chamber. Step 2: 90 μL of washing solution is added to the sample chamber. The microfluidic chip is rotated in reverse at 4000 rpm / min for 90 s. Under centrifugal force, the washing solution passes through the nucleic acid extraction chamber and the transfer chamber, entering the first waste liquid chamber. This step is to clean impurities adsorbed on the filter paper. Step 3: Add 90 μL of eluent to the elution chamber and rotate the microfluidic chip clockwise at 4000 rpm / min for 60 seconds. Under centrifugal force, the eluent in the elution chamber passes through the nucleic acid extraction chamber, eluting nucleic acid molecules attached to the filter paper. The eluent then enters the nucleic acid collection chamber via the transfer chamber, where it fuses with the nucleic acid amplification reagent stored there for subsequent nucleic acid amplification. The nucleic acid extraction step is completed within 5 minutes. Step 4: Rotate the microfluidic chip clockwise at 1000 rpm / min for 60 seconds, followed immediately by rotation at 4000 rpm / min for 60 seconds. At low speeds, the mixed solution in the nucleic acid collection chamber is siphoned into eight dispensing chambers, while excess liquid enters the second waste chamber. At high speeds, the mixed solution in the dispensing chambers enters its corresponding nucleic acid amplification reaction chamber. Step 5: Heat the paraffin chamber at 90°C for 3 minutes until the solid paraffin melts. Then, rotate the microfluidic chip clockwise at 4000 rpm for 30 seconds. The melted paraffin then enters the eight dispensing chambers and seals their corresponding nucleic acid amplification reaction chambers to prevent cross-contamination and evaporation during the reaction. Step 6: Place the chip in the appropriate heating device and heat at 70°C for 45 minutes to perform the nucleic acid amplification reaction. After the reaction is complete, detect the color or fluorescence intensity. Alternatively, the PSA membrane in the reaction chamber area can be removed for agarose gel electrophoresis detection of the solution in each reaction chamber.

[0069] like Figure 7 As shown, the chip in this embodiment presents results in multiple formats, including colorimetric, fluorescence, and agarose gel electrophoresis. The primers added to the eight nucleic acid amplification reaction chambers in this chip, from left to right, are H1N1, FluB, RSVA, N, S, Orf1al, ACTB, and a blank control. In this experiment, the chip was used to detect a clinical sample as a negative control. Amplification at 70°C for 45 minutes revealed… Figure 7Image A shows that only the reagent in the reaction cell containing the ACTB primer changed color from red to yellow, while the reagents in other reaction cells remained red. This indicates that the sample originated from a human and was free of influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2 infection. The same negative sample showed the following fluorescence results: Figure 7 (As shown in image B) Only the ACTB reaction cell exhibited a significantly higher fluorescence intensity than the other reaction cells. Furthermore, the experimental results could be further verified by agarose gel electrophoresis after removing the PSA membrane from the chip. Figure 7 Image C shows that only the ACTB reaction chamber contained nucleic acid amplification products in this sample. This result demonstrates that the chip can provide multiple result presentation formats, making the experimental results more reliable.

[0070] Next, the sensitivity of the chip was tested, such as... Figure 8 As shown, taking H1N1 and FluB as examples, experiments were conducted using H1N1 and FluB plasmids as samples, respectively. Fluorescence intensity was detected by fluorescence method, revealing that the chip can detect samples with a concentration of 10 copies / μL, exhibiting good sensitivity.

[0071] Secondly, the specificity of the chip was tested. Similarly, the primers in the nucleic acid reaction chamber, from left to right, were H1N1, FluB, RSVA, N, S, Orf1al, ACTB, and a blank control. Using the plasmids corresponding to the H1N1, FluB, RSVA, N, S, and Orf1al genes as samples, the specificity of the chip was determined by detecting their fluorescence intensity. Figure 9 As shown in Figure A, when testing samples containing the H1N1 plasmid, only the fluorescence intensity in the reaction cell containing the primer corresponding to H1N1 was significantly higher than that in other reaction cells. This indicates that only H1N1 underwent nucleic acid amplification, and the H1N1 plasmid did not react with primers in other reaction cells, demonstrating good specificity. Similarly, tests using plasmids of other genes showed that primers for all targets exhibited good specificity. Therefore, this chip can be further used for the testing of clinical samples.

[0072] Finally, the chip prepared in this embodiment was used to test 53 positive samples for influenza A and COVID-19, and the results were compared with those of the PCR gold standard. The chip's detection results showed 100% specificity and 98% accuracy (e.g., ...). Figure 10 (As shown).

[0073] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A centrifugal microfluidic chip integrating nucleic acid extraction and detection, characterized in that, It includes a microfluidic chip substrate, wherein the microfluidic chip substrate includes a nucleic acid extraction unit and a nucleic acid detection unit; The nucleic acid extraction unit is used to extract nucleic acid from the sample, and the nucleic acid extraction unit stores dried nucleic acid amplification reagents; The nucleic acid detection unit is used to detect the nucleic acid extracted by the nucleic acid extraction unit, and the nucleic acid detection unit stores a dried primer set.

2. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 1, characterized in that, The nucleic acid extraction unit includes: The sample chamber is used for adding samples and cleaning solutions; Elution chamber, used for adding nucleic acid elution buffer; A nucleic acid extraction chamber, connected to the sample chamber and the elution chamber, is used to extract nucleic acids from the sample; A nucleic acid collection chamber, connected to the nucleic acid extraction chamber, stores the dried nucleic acid amplification reagent for amplification reactions with nucleic acids entering from the nucleic acid extraction chamber; and The first waste liquid chamber is connected to the nucleic acid extraction chamber and is used to store the waste liquid flowing through the nucleic acid extraction chamber.

3. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 2, characterized in that, The nucleic acid extraction chamber contains glass fiber filter paper with a pore size of 0.7-2.0 μm.

4. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 1 or 2, characterized in that, The nucleic acid detection unit includes: A paraffin chamber, containing solid paraffin wax; The dispensing structure includes multiple dispensing chambers and a second waste liquid chamber; and The nucleic acid amplification reaction structure includes multiple nucleic acid amplification reaction chambers, each of which is connected to a dispensing chamber. The nucleic acid amplification reaction chambers store the dried primer set for nucleic acid detection. Each dispensing chamber is connected to the paraffin chamber, and the solid paraffin, after melting, can enter each dispensing chamber to cut off the communication channels between the multiple nucleic acid amplification reaction chambers.

5. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 4, characterized in that, The packaging structure includes 3-12 packaging chambers.

6. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 4, characterized in that, The nucleic acid collection chamber and the dispensing structure are connected by a siphon channel.

7. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 4, characterized in that, The nucleic acid extraction unit further includes: The transfer chamber has one side connected to the nucleic acid extraction chamber, the nucleic acid collection chamber, and the first waste liquid chamber. The nucleic acid extracted by the nucleic acid extraction chamber and the waste liquid extracted by the nucleic acid extraction chamber through the transfer chamber enter the nucleic acid collection chamber and the first waste liquid chamber respectively. The other side is connected to the second waste liquid chamber. Preferably, an exhaust valve is provided in the channel connecting the transfer chamber and the second waste liquid chamber.

8. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 2 or 4, characterized in that, The first waste liquid chamber and / or the second waste liquid chamber are equipped with a water-absorbing polymer for adsorbing the stored waste liquid.

9. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to any one of claims 1-8, characterized in that, The microfluidic chip substrate is a rounded rectangle with a through hole at its center. The microfluidic chip substrate contains two identical functional modules, which are symmetrically distributed radially on the substrate with the through hole as the center. Each functional module contains the nucleic acid extraction unit and the nucleic acid detection unit.

10. The integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to claim 9, characterized in that, Also includes: An encapsulation film is used to seal the upper surface of the microfluidic chip substrate; Preferably, the encapsulation film is a pressure-sensitive adhesive film.

11. A detection method based on the integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to any one of claims 1-10, characterized in that, include: The nucleic acid extraction unit extracts nucleic acid from the sample under centrifugal force. as well as The extracted nucleic acid is introduced into the nucleic acid detection unit for detection under centrifugal force.

12. The detection method according to claim 11, characterized in that, include: The sample and lysis buffer are added to the sample chamber, and the microfluidic chip is rotated in the first rotation direction. Under the action of centrifugal force, the sample in the sample chamber enters the nucleic acid extraction chamber, and the nucleic acid in the sample is adsorbed on the filter paper of the nucleic acid extraction chamber. The waste liquid enters the waste liquid chamber through the transfer chamber. A cleaning solution is added to the sample chamber, and the microfluidic chip is rotated in the first rotation direction. Under the action of centrifugal force, the cleaning solution passes sequentially through the nucleic acid extraction chamber and the transfer chamber and enters the waste liquid chamber. Eluent is added to the elution chamber, and the microfluidic chip is rotated in the second rotation direction. Under the action of centrifugal force, the elution will pass through the nucleic acid extraction chamber, elute the nucleic acid attached to the filter paper, and enter the nucleic acid collection chamber through the transfer chamber to carry out a nucleic acid amplification reaction with the dried nucleic acid amplification reagent stored in the nucleic acid collection chamber. The microfluidic chip is rotated in a second direction at a first rotation speed. Under the action of centrifugal force, the mixed solution in the nucleic acid collection chamber enters each dispensing chamber of the dispensing structure through a siphon channel, and the excess liquid enters the second waste liquid chamber. The microfluidic chip is then rotated at a second rotation speed in a second rotation direction, and under the action of centrifugal force, the mixed solution in each dispensing chamber enters the corresponding nucleic acid amplification reaction chamber. The paraffin chamber is heated at a fixed point. After the solid paraffin stored in the paraffin chamber melts, the microfluidic chip is rotated in a second rotation direction. Under the action of centrifugal force, the melted paraffin enters each dispensing chamber and seals its corresponding nucleic acid amplification reaction chamber. After placing the microfluidic chip in a corresponding heating device to perform a nucleic acid amplification reaction, a judgment is made. Wherein, the first rotation direction is opposite to the second rotation direction, and the first rotation speed is less than the second rotation speed.

13. The application of the integrated centrifugal microfluidic chip for nucleic acid extraction and detection according to any one of claims 1-10 in the field of colorimetric, fluorescence or immunochromatographic analysis.

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