A nucleic acid detection chip

Abstract

The present invention provides a nucleic acid detection chip, the nucleic acid detection chip includes a plurality of reaction units, each of the reaction units includes a first liquid storage part (1), a first passive valve (2), a second liquid storage part (3), a second passive valve (4), a sample injection buffer part (5) and a PCR detection part (6) which are connected and arranged in sequence from one end to the other end. The technical solution of the present application provides a high-throughput nucleic acid detection chip that does not require purification and can quickly release sample nucleic acid, and can quickly amplify and highly sensitively detect dozens of samples in a short time.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a nucleic acid detection chip. Background Art

[0002] Nucleic acid detection technology is currently the main means of direct detection of pathogens, and the lower limit of detection can reach dozens or even several copies. Compared with immunological-based detection methods, it can fundamentally detect whether the target sequence of the pathogen being detected exists, effectively reducing the presence of false negatives in the test results. During the detection process, sample preparation, namely the nucleic acid extraction step, is the most cumbersome step in the detection process, and this step is time-consuming and prone to cross-contamination between samples. Therefore, an integrated device that integrates sample preparation and detection systems and has both high-throughput detection and portability is a technical problem to be solved by those in this field.

[0003] Based on this problem, researchers have proposed and designed devices for simplifying sample preparation or nucleic acid detection. The device developed by HauVan Nguyen's team (Biosensors and Bioelectronics, 2021) is disc-shaped and can be used for multi-sample nucleic acid extraction and purification. Each unit includes two liquid storage chambers for injecting samples to be processed and reaction reagents respectively, and captures nucleic acid molecules through paper-based solid phase extraction technology. Finally, driven by directional centrifugal force, the adsorbed nucleic acid molecules are eluted and the waste liquid and purified nucleic acid are separated. The device developed by Juxin Yin's team (Lab On Chip, 2020) is based on nucleic acid purification magnetic bead technology. It includes 4 liquid storage tanks for storing various reagents during the purification process. The magnetic beads carrying nucleic acid molecules are dragged at the bottom of the chip by hand-held magnets to complete the reaction in the reagents. Driven by negative pressure, the purified nucleic acid solution flows into the digital PCR area for amplification reaction. The above device realizes the simultaneous extraction and purification of multiple samples or the integration of nucleic acid extraction, purification and detection for a single sample. However, the operation process relies on the injection method based on positive or negative pressure or requires a handheld magnet to manually complete the mixing reaction between reagents, which not only requires high professional skills of the operator but also makes the experimental results difficult to repeat. Therefore, the application of high-throughput nucleic acid extraction, purification and detection integrated devices in the field of pathogen detection still has problems that need to be solved. Summary of the invention

[0004] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a nucleic acid detection chip, which is purification-free, can quickly release sample nucleic acid, and has high throughput. It can quickly amplify and detect dozens of samples in a short period of time, and is combined with digital PCR detection with high sensitivity to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0006] The present invention provides a nucleic acid detection chip, which includes a plurality of reaction units, each of which includes a first liquid storage component, a first passive valve, a second liquid storage component, a second passive valve, an injection buffer component and a PCR detection component which are connected and arranged in sequence from one end to the other.

[0007] According to one embodiment, the first liquid storage parts of a plurality of reaction units are connected in sequence to form the same first sample inlet and first sample outlet.

[0008] According to one embodiment, the second liquid storage parts of a plurality of reaction units are connected in sequence to form the same second sample inlet and second sample outlet.

[0009] According to one embodiment, a plurality of reaction units are disposed on the same carrier and are radially distributed on the carrier.

[0010] According to one embodiment, the carrier is in a disc shape, any of the reaction units is arranged along the radius of the disc-shaped carrier, and one end of any of the reaction units is close to the center of the disc-shaped carrier.

[0011] According to one embodiment, the first liquid storage member includes a first liquid storage chamber, a first sample inlet channel and a first sample outlet channel; the first liquid storage chamber forms a first liquid storage cavity, and the first sample inlet channel and the first sample outlet channel are both connected to the liquid storage cavity of the first liquid storage chamber;

[0012] According to one embodiment, the second liquid storage member includes a second liquid storage chamber, a second sample inlet channel and a second sample outlet channel; the second liquid storage chamber forms a second liquid storage cavity, and the second sample inlet channel and the second sample outlet channel are both connected to the liquid storage cavity of the second liquid storage chamber.

[0013] According to one embodiment, the first liquid storage member further includes a first isolation column, which is disposed in the first liquid storage cavity and fixed to the top of the first liquid storage chamber.

[0014] According to one embodiment, the angle between the first sample inlet channel and the first sample outlet channel is not less than 90°.

[0015] According to one embodiment, the second liquid storage member further includes a second isolation column, which is disposed in the second liquid storage cavity and fixed to the top of the second liquid storage chamber.

[0016] According to one embodiment, the angle between the second sample inlet channel and the second sample outlet channel is not less than 90°.

[0017] According to one embodiment, the first passive valve and the second passive valve both include a first trap, a capillary connecting pipe and a second trap connected in sequence; the first trap and the second trap both include a pipe and a plurality of array-arranged trap columns disposed in the pipe.

[0018] According to one embodiment, the injection buffer comprises a buffer cavity and a spot sample pool disposed on the upper portion of the buffer cavity and connected to the buffer cavity; the second liquid storage member is connected to the buffer cavity.

[0019] According to one embodiment, the PCR detection element includes a detection chamber, the detection chamber is formed with a detection cavity, and the inner bottom surface of the detection chamber is provided with microcavities arranged in an array structure.

[0020] According to one embodiment, the microcavity opening has a regular hexagonal outline.

[0021] According to one embodiment, the nucleic acid detection chip also includes an air buffer, which includes an air buffer cavity and gas channels connected to both ends of the air buffer cavity, and the gas channels at both ends are respectively connected to the second liquid storage cavity and the buffer cavity.

[0022] According to one embodiment, the injection buffer component is connected to the PCR detection component through a pipeline, and the pipeline is connected to a buffer component.

[0023] The above technical solution of the present invention has the following beneficial effects:

[0024] The technical solution of the present application provides a high-throughput nucleic acid detection chip that is purification-free and can quickly release sample nucleic acids. It can quickly amplify and highly sensitively detect dozens of samples in a short period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is a schematic diagram of the structure of the nucleic acid detection chip of the present invention.

[0026] Figure 2 Shown is a schematic diagram of the structure of the reaction unit in the nucleic acid detection chip of the present invention.

[0027] Figure 3 Shown is a schematic diagram of the structure of the passive valve in the nucleic acid detection chip of the present invention.

[0028] Figure 4 Shown is a schematic diagram of the structure of the microcavity in the PCR detection element in the nucleic acid detection chip of the present invention.

[0029] Figure 1 to Figure 4 The accompanying drawings are denoted as follows:

[0030] 1 is a first liquid storage component;

[0031] 11 is a first liquid storage chamber;

[0032] 12 is the first injection channel;

[0033] 13 is the first sample outlet channel;

[0034] 14 is a first isolation column;

[0035] 2 is the first passive valve;

[0036] 21 is a first steam trap;

[0037] 22 is a capillary connecting pipeline;

[0038] 23 is a second steam trap;

[0039] 3 is a second liquid storage component;

[0040] 31 is a second liquid storage chamber;

[0041] 32 is a second injection channel;

[0042] 33 is the second sample outlet channel;

[0043] 34 is a second isolation column;

[0044] 4 is a second passive valve;

[0045] 5 is a sample injection buffer;

[0046] 51 is a buffer chamber;

[0047] 52 is a spotting pool;

[0048] 6 is the PCR test piece;

[0049] 7 is an air buffer;

[0050] 8 is a buffer member;

[0051] 9 is a carrier;

[0052] 100 is the first injection port;

[0053] 110 is the first sample outlet;

[0054] 120 is a second injection port;

[0055] 130 is the second sample outlet. DETAILED DESCRIPTION

[0056] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] See also Figures 1 to 4 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0058] like Figure 1 and Figure 2 As shown, a nucleic acid detection chip is provided in an embodiment of the present application, and the nucleic acid detection chip includes a plurality of reaction units, each of which includes a first liquid storage component 1, a first passive valve 2, a second liquid storage component 3, a second passive valve 4, an injection buffer component 5 and a PCR detection component 6 which are connected and arranged in sequence from one end to the other end.

[0059] In a preferred embodiment, the number of reaction units is 2 to 50. Figure 1 In the specific embodiment shown, there are 30 reaction units.

[0060] In a preferred embodiment, the first liquid storage member 1 and the second liquid storage member 2 are connected via a first passive valve 5 .

[0061] In a preferred embodiment, the second liquid storage component 3 and the injection buffer component 5 are connected via a second passive valve 4 .

[0062] In a preferred embodiment, the first liquid storage parts 1 of a plurality of reaction units are connected in sequence to form the same first sample inlet 100 and first sample outlet 110 .

[0063] In a preferred embodiment, the second liquid storage parts 2 of a plurality of reaction units are connected in sequence to form the same second sample inlet 120 and second sample outlet 130 .

[0064] In a preferred embodiment, a plurality of reaction units are disposed on the same carrier 9 and are radially distributed on the carrier 9 .

[0065] In a more preferred embodiment, the carrier is disc-shaped, any of the reaction units are arranged along the radius of the disc-shaped carrier, and one end of any of the reaction units is close to the center of the disc-shaped carrier. In a more specific embodiment, the distances from one end of the reaction units radially distributed on the disc-shaped carrier to the center of the carrier are consistent.

[0066] In the present application, the carrier can be a circular glass slide, and each reaction unit and its combination can be formed into a structural layer using PDMS material, and then the structural layer is bonded or glued to the circular glass slide. Alternatively, PMMA material can be directly used to form a disc-shaped carrier containing each reaction unit and its combination.

[0067] In a preferred embodiment, the first liquid storage member 1 includes a first liquid storage chamber 11, a first sample inlet channel 12 and a first sample outlet channel 13; the first liquid storage chamber 11 forms a first liquid storage cavity, and the first sample inlet channel 12 and the first sample outlet channel 13 are both connected to the liquid storage cavity of the first liquid storage chamber 11.

[0068] In a more preferred embodiment, the first liquid storage member 1 further comprises a first isolation column 14, which is disposed in the first liquid storage cavity and fixed to the top of the first liquid storage chamber 11. The isolation column is used to guide the liquid entering the liquid storage cavity to enter slowly and orderly, thereby avoiding the generation of bubbles and the enrichment of gas in the first liquid storage cavity. In a more preferred embodiment, the first isolation column 14 extends to the lower part of the liquid storage cavity but does not touch the bottom of the liquid storage cavity.

[0069] In a more preferred embodiment, the angle between the first sample inlet channel 12 and the first sample outlet channel 13 is not less than 90°. This minimizes the resistance on the flow channel such as when the liquid enters or flows out, thereby ensuring that the liquid fills the liquid storage cavity one by one. In a more preferred embodiment, the angle between the first sample inlet channel 12 and the first sample outlet channel 13 is an obtuse angle. Figure 1 and Figure 2 In the specific embodiment shown, the angle between the first sample inlet channel 12 and the first sample outlet channel 13 is 120°.

[0070] In a preferred embodiment, the second liquid storage member 3 includes a second liquid storage chamber 31, a second sample inlet channel 32 and a second sample outlet channel 33; the second liquid storage chamber 31 forms a second liquid storage cavity, and the second sample inlet channel 32 and the second sample outlet channel 33 are both connected to the second liquid storage cavity of the second liquid storage chamber 31.

[0071] In a more preferred embodiment, the second liquid storage member 3 further includes a second isolation column 34 , which is disposed in the second liquid storage cavity and fixed to the top of the second liquid storage chamber 31 .

[0072] In a more preferred embodiment, the angle between the second sample inlet channel 32 and the second sample outlet channel 33 is not less than 90°. This minimizes the resistance on the flow channel such as when the liquid enters or flows out, thereby ensuring that the liquid fills the liquid storage cavity one by one. In a more preferred embodiment, the angle between the second sample inlet channel 32 and the second sample outlet channel 33 is an obtuse angle. Figure 1 and Figure 2 In the specific embodiment shown, the angle between the second sample inlet channel 32 and the second sample outlet channel 33 is 120°.

[0073] In a more preferred embodiment, the first liquid storage members 1 of two adjacent reaction units are connected in sequence through the first sample outlet channel 13 and the first sample inlet channel 12, and form the same first sample inlet and first sample outlet.

[0074] In a more preferred embodiment, the second liquid storage members 3 of two adjacent reaction units are connected in sequence through the second sample outlet channel 33 and the second sample inlet channel 32, and form the same second sample inlet and second sample outlet.

[0075] In a Figure 3 In the preferred embodiment shown in the figure, the first passive valve 2 and the second passive valve 4 each include a first steam trap 21, a capillary connecting pipe 22 and a second steam trap 23 which are connected in sequence. Figure 3 In the preferred embodiment shown, the first trap 21 and the second trap 23 both include a pipeline and a plurality of array-arranged trap columns disposed in the pipeline. . The first trap 21 and the second trap 23 are both non-capillary diameters, and the structure of the capillary valve formed by the trap column and the capillary connecting pipeline with a sudden narrowing in the middle section can provide a pressure barrier for the injected aqueous reagent, so that the aqueous reagent cannot pass without force. The reaction unit in the present application introduces the first passive valve and the second passive valve, which, through the above characteristics, can control several units one by one in the detection chip without manual or mechanical control.

[0076] In a preferred embodiment, the injection buffer 5 includes a buffer cavity 51 and a spot sample pool 52 disposed on the upper portion of the buffer cavity and connected to the buffer cavity 51 ; the second liquid storage member 3 is connected to the buffer cavity 51 .

[0077] In a preferred embodiment, the nucleic acid detection chip further comprises an air buffer 7, wherein the air buffer 7 comprises an air buffer cavity and gas channels connected to both ends of the air buffer cavity, wherein the gas channels at both ends are respectively connected to the second liquid storage cavity and the buffer cavity 51. Figure 2 In the more specific embodiment shown, one end of the air buffer 7 is communicated with the upper part of the second liquid storage chamber, and the other end of the air buffer 7 is communicated with the buffer chamber 51. The air buffer 7 acts to fill the downstream chamber with reagents while the air is exchanged out during the centrifugal process when the reagents are driven to flow along the flow channel, which is beneficial to the flow and mixing of the liquid during the centrifugal force driving process.

[0078] In a preferred embodiment, the PCR detection element 6 includes a detection chamber, the detection chamber is formed with a detection cavity, and the inner bottom surface of the detection chamber is provided with microcavities arranged in an array structure. Figure 4 In the more specific embodiment shown, the opening profile of the microcavity is a regular hexagon. The regular hexagonal micropores arranged in an array form a honeycomb-like structure layout, which is used to form a honeycomb-shaped PCR amplification area. The design of the regular hexagonal microcavity facilitates the production of more microcavities per unit area in the processing technology. For the larger number of microcavities per unit area of ​​digital PCR, the detection unit can be divided into more small detection units, which can effectively improve the detection of a very small number of sample copies (theoretically, each microcavity contains only 1 copy number), which is very suitable for detecting the characteristics of the small number of pathogens in the samples of patients with early infection. In a preferred embodiment, the sampling buffer 5 is connected to the PCR detection component 6 by a pipeline, and a buffer 8 is connected to the pipeline.

[0079] By injecting a sample release agent into the first injection port of the first liquid storage part 1 and adding the sample to be processed to the spotting pool, the sample and the sample release agent are driven by clockwise centrifugal force to mix and release nucleic acid molecules. Then, the pre-prepared template-free PCR mixture is injected through the second injection port 12 of the second liquid storage part 3, and after centrifugation, it is mixed with the nucleic acid solution released in the previous step and sucked into the honeycomb microcavity structure through capillary force. Then, mineral oil is introduced and centrifuged so that the upper layer of the microcavity structure is sealed by mineral oil to reduce the evaporation of reagents during the PCR process. Finally, the device is placed in an in-situ PCR instrument and the relevant program is set. After the reaction is completed, the test results are observed under a fluorescence microscope.

[0080] Based on the nucleic acid detection chip described in this application, its working process includes the following steps:

[0081] 1) Preparation for testing: Figure 1 The first injection port 100, the first outlet port 110, the second injection port 120, the second outlet port 130 and Figure 2The sample spot pool 52 in the centrifugal plate is sealed with heat-resistant adhesive tape. The centrifugal platform switch is turned on, the disc-shaped carrier 9 is aligned with the center of the centrifugal platform and placed in the centrifugal plate, and the air pump is turned on to tightly suck the disc.

[0082] 2) Sample release stage: aspirate the sample release agent and open the tape at the first injection port 100 and the first outlet port 110, inject the reagent from the first injection port 100 until all the first liquid storage chambers 11 are filled, and then close the first injection port 100 and the first outlet port 110. Open the tape at the spot sample reservoir 52 and inject the sample solution into the reservoir, and then close the structure. Set the centrifugal device to +2000RPM for 40 seconds to drive the first liquid storage chamber 11 to contact the reagent at the spot sample reservoir 52 and leave it for 10 minutes to fully react.

[0083] 3) Nucleic acid amplification stage: The pre-configured PCR mixture without template is injected through the injection hole 120 to completely fill the second liquid storage chamber 31 of all reaction units and then the hole is closed. The reagent in the second liquid storage chamber 31 is driven by the centrifugal device set to +2000RPM for 40 seconds to mix with the sample mixture through the second passive valve 4 and the sample release agent and the sample and is distributed to the PCR microcavity at the bottom of the PCR detection element 6. Figure 4 Then, mineral oil is injected from the second injection port 120 to completely fill the second liquid storage chambers 31 of all reaction units, and then the second injection port 120 is closed. The centrifugal device is set to +2000RPM for 40 seconds to drive the mineral oil into the detection cavity of the PCR detection element 6 by centrifugal force, and the top of the microcavity is sealed with mineral oil to reduce the volatilization of the reagent during the amplification process. The disk is placed in the in-situ PCR instrument and the amplification program is run.

[0084] 4) Detection stage: When the amplification program is completed and the device is cooled to room temperature, the disk is placed under a fluorescence microscope to observe the amplification results.

[0085] The above technical solution of the present invention:

[0086] 1) Based on the rapid release of nucleic acid molecules by the sample release agent, the volume ratio between the reaction reagents is adjusted by designing the first liquid storage chamber 11 and the second liquid storage chamber 31 .

[0087] 2) The first passive valve 2 and the second passive valve 4 are designed to form a pressure barrier to prevent the reagent from entering other areas.

[0088] 3) The first injection channel 12 and the first outlet channel 13 arranged at an obtuse angle are used to connect the first liquid storage member 1 of all reaction units, and the second injection channel 32 and the second outlet channel 33 arranged at an obtuse angle are used to connect the second liquid storage member 3 of all reaction units; the reagent can be filled into multiple reaction units, such as 30 reaction units, with only one injection, and the turning angle formed by the pipeline at two adjacent reaction units greatly reduces the resistance encountered here during the reagent distribution process.

[0089] 4) Driven by centrifugal force, the reagents in the first liquid storage component 1 and the second liquid storage component 3 can break through the pressure barrier to enter the downstream structure and complete the reaction, and the injection of reaction reagents in different orders is used to regulate the reaction process to ensure sufficient reaction between the reagents.

[0090] 5) A hole of a certain height is provided at the PCR detection part 6, and the bottom is a digital PCR micro-cavity structure, which ensures that the mineral oil completely covers the micro-cavity during the reaction process, effectively reduces the evaporation of reagents during the amplification process, and has the function of absolute quantification of the sample. The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A nucleic acid detection chip, characterized in that: The nucleic acid detection chip comprises a plurality of reaction units, each of which comprises, from one end to the other, a first liquid storage component (1), a first passive valve (2), a second liquid storage component (3), a second passive valve (4), an injection buffer component (5) and a PCR detection component (6) which are connected and arranged in sequence; the first liquid storage components (1) of the plurality of reaction units are connected in sequence to form a first injection port (100) and a first outlet port (110); the second liquid storage components (3) of the plurality of reaction units are connected in sequence to form a second injection port (120) and a second outlet port (130); The PCR detection element (6) comprises a detection chamber, the detection chamber forms a detection cavity, and the inner bottom surface of the detection chamber is provided with microcavities arranged in an array structure; a plurality of reaction units are arranged on the same carrier (9), and are radially distributed on the carrier (9); the carrier (9) is disc-shaped, and any of the reaction units is arranged along the radius of the disc-shaped carrier, and one end of any of the reaction units is close to the center of the disc-shaped carrier.

2. The nucleic acid detection chip according to claim 1, characterized in that: The first liquid storage element (1) comprises a first liquid storage chamber (11), a first sample inlet channel (12) and a first sample outlet channel (13); the first liquid storage chamber (11) forms a first liquid storage cavity, and the first sample inlet channel (12) and the first sample outlet channel (13) are both connected to the liquid storage cavity of the first liquid storage chamber (11); And / or, the second liquid storage member (3) comprises a second liquid storage chamber (31), a second sample inlet channel (32) and a second sample outlet channel (33); the second liquid storage chamber (31) forms a second liquid storage cavity, and the second sample inlet channel (32) and the second sample outlet channel (33) are both connected to the second liquid storage cavity of the second liquid storage chamber (31).

3. The nucleic acid detection chip according to claim 2, characterized in that: The first liquid storage element (1) further comprises a first isolation column (14), wherein the first isolation column (14) is arranged in the first liquid storage cavity and fixed to the top of the first liquid storage chamber (11); And / or, the angle between the first sample inlet channel (12) and the first sample outlet channel (13) is not less than 90°; And / or, the second liquid storage member (3) further comprises a second isolation column (34), the second isolation column (34) being arranged in the second liquid storage cavity and fixed to the top of the second liquid storage chamber (31); And / or, the angle between the second sample inlet channel (32) and the second sample outlet channel (33) is not less than 90°.

4. The nucleic acid detection chip according to claim 1, characterized in that: The first passive valve (2) and the second passive valve (4) both comprise a first steam trap (21), a capillary connecting pipe (22) and a second steam trap (23) which are connected in sequence; the first steam trap (21) and the second steam trap (23) both comprise a pipe and a plurality of steam trap columns arranged in an array and disposed in the pipe.

5. The nucleic acid detection chip according to claim 1, characterized in that: The injection buffer component (5) comprises a buffer cavity (51) and a sample spotting pool (52) arranged on the upper part of the buffer cavity (51) and connected to the buffer cavity (51); the second liquid storage component is connected to the buffer cavity (51); and / or the injection buffer component (5) is connected to the PCR detection component (6) via a pipeline, and the pipeline is connected to a buffer component (8).

6. The nucleic acid detection chip according to claim 5, characterized in that: The nucleic acid detection chip further comprises an air buffer (7), wherein the air buffer (7) comprises an air buffer cavity and gas channels connected to two ends of the air buffer cavity, wherein the gas channels at the two ends are respectively connected to the second liquid storage cavity and the buffer cavity (51).

7. The nucleic acid detection chip according to claim 1, characterized in that: The microcavity opening profile is a regular hexagon.

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