CN113652332A - Microchannel blockage-based nucleic acid on-site rapid detection device and detection method thereof - Google Patents
Microchannel blockage-based nucleic acid on-site rapid detection device and detection method thereof Download PDFInfo
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Abstract
The invention discloses a micro-channel blockage-based nucleic acid on-site rapid inspection device and a preparation method and a detection method thereof, belonging to the technical field of integrated micro-fluidic chip manufacturing and rapid on-site qualitative detection of nucleic acid amplification. The invention solves the problems that the existing nucleic acid detection needs to use a special detector, the field pollution-free and safety are difficult to ensure, and a POCT method needs to be efficiently and accurately visualized. The invention is based on nucleic acid specific amplification, relies on a microfluidic chip technology, establishes a visual POCT method which relies on distinguishing the characteristic that a nucleic acid precipitate blocks a micro channel, can obtain an accurate result at the initial stage of infection aiming at virus nucleic acid detection, is suitable for the conditions of variable temperature amplification and constant temperature amplification, does not need complex nucleic acid detection equipment, is connected to the microfluidic chip after closed heating amplification at the working temperature of a corresponding nucleic acid amplification reagent, and is driven by negative pressure at room temperature, so that the detection result can be visually judged through the color development effect of the chip.
Description
Technical Field
The invention relates to a micro-channel blockage-based nucleic acid on-site rapid inspection device and a detection method thereof, which are compatible with various existing non-constant-temperature or constant-temperature amplification reagents and belong to the technical field of integrated micro-fluidic chip manufacturing and rapid on-site qualitative detection of nucleic acid amplification.
Background
At present, nucleic acid detection is widely applied to the field of clinical medicine, and compared with a colloidal gold chromogenic detection method for detecting an antibody of a patient, the nucleic acid detection has the capability of detecting in the early stage of infection, and can obtain precious time lead when being applied to epidemic prevention detection. Since the amount of nucleic acid in the sample to be detected is very limited, amplification of a trace amount of nucleic acid is required. The nucleic acid amplification method proposed at the earliest is Polymerase Chain Reaction (PCR), which is also the most widely used nucleic acid amplification method at present. However, PCR requires highly trained personnel to operate sophisticated temperature cycling equipment to effect denaturation, annealing and extension of nucleic acids, which limits the possibilities for convenient applications. With the generation of a series of nucleic acid isothermal amplification methods, the complexity of the instrument is reduced, the reaction process is simple and efficient, and the miniaturization and field arrangement of detection equipment are facilitated. However, there is still a great challenge to Point-of-Care Testing (POCT) for nucleic acids in the field.
Most of the micro-fluidic chip nucleic acid detection systems used in the current market, such as PCR detection technology of a constant-temperature amplification method, use fluorescence detection, require professional detection equipment to interpret results, and background fluorescence prolongs the interpretation time of negative samples. For the isothermal amplification technology, the amplification temperature change is simplified, such as Loop-mediated isothermal amplification (LAMP), the product detection method comprises agarose electrophoresis detection, magnesium pyrophosphate turbidity detection, fluorescent quantitative detection, fluorescent visual inspection and the like, the uncovering of the product in the lipoglucose electrophoresis detection is easy to cause pollution, the fluorescent quantitative detection needs professional instruments and equipment, the visual resolution of the magnesium pyrophosphate turbidity is not obvious, and the wide application of the product is limited by using the professional instruments.
How to popularize the easily-used nucleic acid POCT detection technology in the global scope, especially in developing areas and less developed areas, faces a series of problems of how to overcome field pollution, reduce cost, remove complex instruments, simplify operation and the like. The micro-fluidic chip provides a possible solution, integrates a plurality of reaction steps into a micro-fluidic chip with the square centimeter, and realizes the miniaturization of a detection instrument, the simplification of sample pretreatment, the high efficiency and the precision of experimental operation and the economy of detection cost.
Disclosure of Invention
The invention aims to solve the problems and provides a micro-channel blockage-based nucleic acid on-site rapid inspection device and a detection method thereof.
The technical scheme of the invention is as follows:
a nucleic acid on-site rapid inspection device based on micro-channel blockage is compatible with the existing non-constant temperature amplification or constant temperature amplification method, and comprises a micro-fluidic chip 1 and an accessory device, wherein the micro-fluidic chip 1 consists of a substrate provided with a micron-sized channel and a cover plate bonded with the substrate, and the accessory device comprises a soft cover test tube 2, a sample inlet tube, an injection tube and a negative pressure source 3;
one end of the micro-fluidic chip 1 is communicated with the soft-cap test tube 2 through a conduit, and the other end of the micro-fluidic chip 1 is connected with a negative pressure source 3;
the micro-channel comprises an inlet section 4, a bent channel 5, a precipitation blocking section 6 and a color development cavity 8 which are sequentially communicated;
the inlet section 4 is provided with a liquid inlet, and the soft-cap test tube 2 is communicated with the liquid inlet of the inlet section 4 by a conduit; the two ends of the precipitation blocking section 6 are respectively provided with an anti-backflow structure 9, particulate matter is filled between the two anti-backflow structures 9, the precipitation blocking section 6 is also provided with an assembly opening 7, and the particulate matter is filled in the precipitation blocking section 6 through the assembly opening 7; the color developing cavity 8 is provided with an outlet 10, a conduit is arranged on the outlet 10, and the other end of the conduit is communicated with the negative pressure source 3.
Further, the number of the precipitation-clogging sections 6 is 1 to 3.
Further, the detection is carried out by utilizing the principle that nucleic acid amplified by alcohol precipitation blocks the porous medium in the micro-channel, the total width of the section of the channel of the precipitation blocking section 6 is not more than 500 μm, the height of the channel is not less than 30 μm, and the height of the channel is not more than 150 μm.
Further, the precipitation blocking section 6 is a porous medium channel capable of trapping nucleic acid and ethanol precipitate.
Further, the diameter of the particulate matter packed in the precipitation blocking section 6 is 5 to 30 μm.
Further, the soft-cap test tube 2 is a 200 μ L test tube equipped with a pierceable sealing cap.
The use method of the micro-channel blockage-based nucleic acid on-site rapid inspection device comprises the following steps:
adding a detection sample and an isothermal amplification reaction system or a non-isothermal amplification reaction system into a soft-covered test tube 2, carrying out vortex mixing, carrying out isothermal heating amplification, inserting a sample injection tube filled with a colored indicator from a pierceable sealing cover of the soft-covered test tube 2 after amplification is finished, and shearing off the tail of the sample injection tube;
step two, inserting an injection tube filled with alcohol solution from the pierceable sealing cover of the soft-covered test tube 2, pushing the alcohol solution into the soft-covered test tube 2, pulling out the injection tube, and vibrating and mixing uniformly;
inserting one end of the conduit into the bottom of the soft-cap test tube 2 through the pulling-out position of the alcohol solution injection tube, and communicating the other end of the conduit with the liquid inlet of the inlet section 4; meanwhile, the color developing cavity 8 is communicated with the negative pressure source 3 through the outlet 10 by using another conduit;
and step four, applying negative pressure to the color development cavity 8 by adopting the negative pressure source 3, standing for 10min, and observing the micro-channel and the color development cavity 8 to obtain a detection result.
Further limiting, the non-isothermal amplification reaction system is a Polymerase Chain Reaction (PCR) system.
Further limiting, the isothermal amplification reaction system is a nucleic acid sequence dependent amplification NASBA system, a loop-mediated isothermal amplification reaction LAMP system, a rolling circle amplification RCA system, a transcription-mediated amplification TMA system or a strand displacement amplification SDA system and the like.
Further defined, the alcohol solution has an ethanol concentration of not less than 50% by volume.
Further limiting, 20 microliter of colored indicator is sealed in the sampling tube, and the tail part of the sampling tube can be cut off to be communicated with the atmosphere; the syringe is filled with 30. mu.l of an alcohol solution and can be injected into the test tube.
The preparation method of the micro-channel blockage-based nucleic acid on-site rapid inspection device adopts PDMS as a substrate and is manufactured by using a soft lithography processing mode; the specific operation process is as follows: PDMS is bonded with glass through glue evening, exposure, development, pouring, stripping and punching to complete the manufacturing.
The preparation method of the micro-channel blockage-based nucleic acid on-site rapid inspection device adopts PMMA as a substrate and is manufactured by a processing mode of a hot molding method; the specific operation process is as follows: the method comprises the steps of exposure, development, fixation, water bleaching, postbaking, chromium corrosion, glass corrosion, photoresist removal and chromium removal, wherein the chromium plate glass is used for manufacturing a template, the processed PMMA substrate and the template are placed on a workbench of a tablet press for mould pressing, and finally the PMMA substrate and the template are bonded to complete the manufacturing.
The invention has the following beneficial effects: the invention relates to a visual POCT (point of care testing) technology based on nucleic acid amplification, which is used for establishing a visual rapid detection method for distinguishing micro channels blocked by nucleic acid precipitates by relying on a microfluidic chip technology. Particularly, the device is in a negative pressure and closed cover state in the detection process, and simultaneously, the high-concentration ethanol solution is used for directly inactivating the reagent, so that better safety in the field test process of the amplification reaction and subsequent pollution-free treatment are ensured in the detection process.
Drawings
FIG. 1 is a schematic view of a detection device according to the present application;
FIG. 2 is a schematic structural view of a microchannel;
FIG. 3 is a schematic diagram of a microchannel having two precipitation-blocking sections;
FIG. 4 is a diagram showing the results of nucleic acid detection.
1-microfluidic chip, 2-soft cap test tube, 3-negative pressure source, 4-inlet section, 5-bending channel, 6-sediment blocking section, 7-assembly port, 8-color development cavity, 9-anti-backflow structure and 10-outlet.
Detailed Description
The test methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and apparatus used, unless otherwise specified, are conventional in the art and are commercially available to those skilled in the art.
Embodiment mode 1: LAMP is taken as an example for explanation
A nucleic acid on-site rapid inspection device based on micro-channel blockage is shown in figure 1 and comprises a micro-fluidic chip 1 and an accessory device, wherein the micro-fluidic chip 1 is composed of a substrate provided with a micro-channel and a cover plate bonded with the substrate, and the material of the micro-fluidic chip 1 can be selected from PDMS, PMMA, glass and the like. Wherein, the accessory device comprises a soft cover test tube 2, a sample inlet tube, a syringe, a constant temperature water bath and a negative pressure source 3 (a common medical injector); the microchannel includes inlet section 4, crooked passageway 5, deposits blocking section 6 and colour development chamber 8, inlet section 4, crooked passageway 5, deposit blocking section 6 and colour development chamber 8 communicate in proper order, inlet section 4 is equipped with liquid inlet, including detecting sample, amplification reaction liquid and ethanol solution in the mixed solution, this access connection PTFE pipe insert is equipped with the soft lid test tube 2 bottoms of the liquid that awaits measuring, inlet section 4 connects one section crooked passageway for strengthen the liquid mixture and flow steadily. As shown in fig. 2, the precipitation blocking section 6 is a slender structure to facilitate blocking, the channel width is 300 μm, the height is 30 μm, backflow prevention structures 9 are respectively arranged at two ends of the precipitation blocking section 6, particulate matters (such as silica, PMMA microspheres, etc.) with the diameter of 5-20 μm are filled between the two backflow prevention structures 9, the precipitation blocking section 6 is further provided with an assembly port 7, the particulate matters are filled in the precipitation blocking section 6 through the assembly port 7, pre-filling of the channel blocking section is realized, the assembly port 7 is closed after the microspheres are compactly arranged, and assembly of the precipitation blocking section 6 is completed. At this time, the precipitation blocking section 6 allows water and the post-reaction solution of the negative sample to pass through, whereas the post-reaction solution of the positive sample is difficult to pass through due to the large amount of ethanol precipitation. The color developing cavity 8 is provided with an outlet 10, a conduit is arranged on the outlet 10, and the other end of the conduit is communicated with the negative pressure source 3. Microcolumns with equal size are designed in the color development cavity 8 and can support the PDMS film; the color development cavity 8 is used for observing whether the colored indicating liquid flows through the channel or not, and the color development of the color development cavity 8 detects that the sample is negative, otherwise, the sample is positive. The device utilizes the nucleic acid structure that exists in a large number in the reaction solution of positive amplification, produces the nucleic acid deposit after mixing with ethanol, deposits and attaches to and forms the jam on the good stereoplasm granule of pre-assembly in the passageway, makes solution through speed decline remarkably or even not pass through completely, and then judges from the condition of passing through of color development indicating liquid and obtains whether amplification reaction takes place, and it is positive to detect the sample promptly.
The number of the precipitation-clogging sections 6 is 1 to 3, as shown in FIG. 3.
The specific use method of the device comprises the following steps:
firstly, adding a prepared 20-microliter LAMP reaction system into a soft-covered test tube 2, then adding a detection sample, carrying out vortex mixing for 10s, putting the mixture into a constant-temperature water bath kettle at 65 ℃ to ensure that all reagents are submerged below the water surface, and carrying out water bath heating amplification for about 20 minutes (specifically according to the characteristics of the reagents). In particular, different amplification reagents differ only in the above-described thermal amplification process. For example, in the case of PCR, an amplification apparatus capable of heating cyclically is used instead of the constant-temperature water bath. For TMA nucleic acid amplification technology, the sample to be detected and the amplification reaction solution are all taken out by a pipette and added into a soft-covered test tube containing the amplification enzyme, mixed for 10s by vortex, and then put into a water bath at 42 ℃ for heating amplification.
And after the heating amplification of the detection sample in the soft-covered test tube 2 is finished, cooling for 1min at room temperature. A sample tube filled with 20 mu L of oily colored indicator is taken, the test tube is inserted from the soft cover of the soft-covered test tube 2, the tail part is cut off, the atmosphere is communicated, and the oily indicator plays a role in sealing a reaction system besides indication. Taking a syringe containing 20 μ L of alcohol solution, inserting the tube from the soft cover of the soft-covered tube 2, slowly pushing the liquid in, pulling out the syringe, and mixing by shaking. A liquid supply tube with a 30mm long flat needle head is slowly inserted into the position of the alcohol solution injection tube through the pulling-out position to the bottom of the soft-cap test tube 2, and then the other end of the liquid supply tube is connected with a liquid inlet of the inlet section 4. A negative pressure source 3 plus an 8mL evacuated volume was used at the outlet 10 of the color development chamber 8 to drive the in-chip liquid flow. As a large amount of magnesium pyrophosphate precipitates are generated in the LAMP nucleic acid amplification reaction process, the diameters of the precipitates are within the range of 2-7 mu m. Meanwhile, nucleic acid precipitates can be formed by mixing ethanol with the solution after the amplification reaction, and the precipitates have larger diameters, so that the blockage of positive detection results can be obviously enhanced. When the reaction system mixed with ethanol flows through the blocked channel section, a plurality of nucleic acid precipitates are generated in the reaction liquid of the positive sample under the action of the ethanol and are accumulated on particles in the channel, so that the flow rate in the channel is remarkably reduced, and the colored oily fluid of the negative sample can rapidly pass through the blocked section and enter the color development cavity. And finally, judging the detection result by depending on whether the upstream oily colored indicating liquid reaches the color development cavity or not, and making an accurate judgment by visual observation. In the method, the negative sample can be controlled to flow through the channel to develop color within 5 minutes, and the positive sample does not pass through and does not develop color for not less than 10 minutes, so that the nucleic acid detection result can be visually judged within 30 minutes. The standard tests in the laboratory were all correctly passed in 20 out of 10 groups (10 negative and positive) and the test results are shown in FIG. 4.
The detection device and the operation are simple, and the field implementation is easy. Amplification can be carried out in a liquid supply tube or a detection tube, and the driving force for the flow in the chip can be either negative pressure operation as described above or positive pressure driving flow at the tip of the liquid supply tube. And in the detection process, a high-concentration ethanol solution is used for directly inactivating the reagent, so that the safety of the field test process of the amplification reaction is further ensured, and the subsequent field harmless treatment is facilitated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A nucleic acid on-site rapid inspection device based on micro-channel blockage is characterized by being compatible with the existing non-constant temperature amplification or constant temperature amplification method, the device comprises a micro-fluidic chip (1) and an auxiliary device, the micro-fluidic chip (1) consists of a substrate provided with a micron-sized channel and a cover plate bonded with the substrate, and the auxiliary device comprises a soft-cover test tube (2), a sample inlet tube, an injection tube and a negative pressure source (3);
one end of the micro-fluidic chip (1) is communicated with the soft cover test tube (2) through a conduit, and the other end of the micro-fluidic chip (1) is connected with a negative pressure source (3);
the micro-channel comprises an inlet section (4), a bent channel (5), a sediment blocking section (6) and a color developing cavity (8) which are sequentially communicated;
the inlet section (4) is provided with a liquid inlet, and the soft-cap test tube (2) is communicated with the liquid inlet of the inlet section (4) by a conduit; two ends of the precipitation blocking section (6) are respectively provided with an anti-backflow structure (9), particulate matter is filled between the two anti-backflow structures (9), the precipitation blocking section (6) is also provided with an assembly port (7), and the particulate matter is filled in the precipitation blocking section (6) through the assembly port (7); the color developing cavity (8) is provided with an outlet (10), a conduit is arranged on the outlet (10), and the other end of the conduit is communicated with the negative pressure source (3).
2. The on-site rapid nucleic acid testing device based on micro-channel blockage according to claim 1, wherein the number of the precipitation blockage sections (6) is 1-3.
3. The device for the on-site rapid detection of nucleic acid based on microchannel blockage according to claim 1, wherein the detection is performed by using the principle that nucleic acid amplified by alcohol precipitation blocks a porous medium in a microchannel, and the total channel section width of the precipitation blocking section (6) is not more than 500 μm, the channel height is not less than 30 μm, and the channel height is not more than 150 μm.
4. The on-site rapid nucleic acid testing device based on microchannel blockage according to claim 1, wherein the precipitation blockage section (6) is a porous medium channel.
5. The device for the on-site rapid detection of nucleic acid based on microchannel blockage according to claim 1, wherein the diameter of the granular material filled in the precipitation blockage section (6) is 5-30 μm.
6. The device for the rapid field test of nucleic acid based on the blockage of the micro-channel as claimed in claim 1, wherein the soft-cap test tube (2) is a 200 μ L test tube equipped with a pierceable sealing cap.
7. The method for using the micro-channel blockage-based nucleic acid on-site rapid inspection device as claimed in claim 1, which is characterized by comprising the following steps:
adding a detection sample and an isothermal amplification reaction system or a non-isothermal amplification reaction system into a soft-covered test tube (2), performing vortex mixing, performing heating amplification, inserting a sample injection tube filled with a colored indicator into a pierceable sealing cover of the soft-covered test tube (2) after amplification is finished, and shearing off the tail of the sample injection tube;
step two, inserting an injection tube filled with alcohol solution from the pierceable sealing cover of the soft-cover test tube (2), pushing the alcohol solution into the soft-cover test tube (2), pulling out the injection tube, and vibrating and mixing uniformly;
inserting one end of the conduit into the bottom of the soft-cap test tube (2) through the pulling-out position of the alcohol solution injection tube, and communicating the other end of the conduit with the liquid inlet of the inlet section (4); meanwhile, the color developing cavity (8) is communicated with the negative pressure source (3) through the outlet (10) by using another catheter;
and step four, applying negative pressure to the color development cavity (8) by adopting a negative pressure source (3), standing for 10min, and observing the micro-channel and the color development cavity (8) to obtain a detection result.
8. The use method of the micro-channel blockage-based nucleic acid on-site rapid inspection device as claimed in claim 7, wherein the non-isothermal amplification reaction system is a Polymerase Chain Reaction (PCR) system; the isothermal amplification reaction system is a nucleic acid sequence dependent amplification NASBA system, a loop-mediated isothermal amplification reaction LAMP system, a rolling circle amplification RCA system, a transcription-mediated amplification TMA system or a strand displacement amplification SDA system.
9. The use method of the device for the rapid on-site inspection of nucleic acid based on micro-channel blockage according to claim 8, wherein the alcohol solution has an alcohol concentration of not less than 50% by volume.
10. The use method of the micro-channel blockage-based nucleic acid on-site rapid inspection device as claimed in claim 7, wherein the sample introduction tube is internally sealed with 20 μ L of colored indicator, and the tail part can be cut off to be communicated with the atmosphere; the syringe is filled with 30 mul of alcohol solution and enters the soft-cap test tube (2) by injection.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1932924A1 (en) * | 2006-11-22 | 2008-06-18 | FUJIFILM Corporation | Nucleic acid amplification method using microchip and microchip, and nucleic acid amplification system using the same |
| CN107904161A (en) * | 2017-12-15 | 2018-04-13 | 上海交通大学医学院附属仁济医院 | It is a kind of to visualize micro-fluidic chip of detection pathogen nucleic acid and preparation method thereof and detection method immediately |
| CN108126765A (en) * | 2017-11-27 | 2018-06-08 | 深圳华炎微测医疗科技有限公司 | ELISA detects micro-fluidic chip and ELISA detection micro-fluidic chip systems and their application |
| CN108160125A (en) * | 2017-11-27 | 2018-06-15 | 深圳华炎微测医疗科技有限公司 | Biochemistry detection micro-fluidic chip and biochemistry detection micro-fluidic chip system and their application |
| CN109425733A (en) * | 2017-08-25 | 2019-03-05 | 国家纳米科学中心 | Flexible biological probe, its application method and application apparatus |
-
2020
- 2020-07-29 CN CN202010741313.8A patent/CN113652332A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1932924A1 (en) * | 2006-11-22 | 2008-06-18 | FUJIFILM Corporation | Nucleic acid amplification method using microchip and microchip, and nucleic acid amplification system using the same |
| CN109425733A (en) * | 2017-08-25 | 2019-03-05 | 国家纳米科学中心 | Flexible biological probe, its application method and application apparatus |
| CN108126765A (en) * | 2017-11-27 | 2018-06-08 | 深圳华炎微测医疗科技有限公司 | ELISA detects micro-fluidic chip and ELISA detection micro-fluidic chip systems and their application |
| CN108160125A (en) * | 2017-11-27 | 2018-06-15 | 深圳华炎微测医疗科技有限公司 | Biochemistry detection micro-fluidic chip and biochemistry detection micro-fluidic chip system and their application |
| CN107904161A (en) * | 2017-12-15 | 2018-04-13 | 上海交通大学医学院附属仁济医院 | It is a kind of to visualize micro-fluidic chip of detection pathogen nucleic acid and preparation method thereof and detection method immediately |
Non-Patent Citations (1)
| Title |
|---|
| WONHWI NA 等: "Rapid molecular diagnosis of infectious viruses in microfluidics using DNA hydrogel formation", BIOSENSORS AND BIOELECTRONICS, 19 February 2018 (2018-02-19), pages 9 - 13, XP085366347, DOI: 10.1016/j.bios.2018.02.040 * |
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