CN116103139B - Totally enclosed full-integrated nucleating acid detection micro-fluidic chip - Google Patents
Totally enclosed full-integrated nucleating acid detection micro-fluidic chip Download PDFInfo
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- CN116103139B CN116103139B CN202310400361.4A CN202310400361A CN116103139B CN 116103139 B CN116103139 B CN 116103139B CN 202310400361 A CN202310400361 A CN 202310400361A CN 116103139 B CN116103139 B CN 116103139B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a fully-closed fully-integrated nucleic acid detection micro-fluidic chip, which relates to the field of biomedical detection and the field of micro-fluidic chips, and comprises a sample adding layer, a sample injection layer, an air passage layer, a reaction layer and a sealing layer which are sequentially sealed and attached from top to bottom; the top surface of the sample adding layer is integrated with a control component, a reagent component, a sample feeding component and a valve component; the surface of the reaction layer is provided with a waste liquid pool, an extraction pool and a detection area which are sequentially communicated; the sample adding layer, the sample injection layer and the air passage layer are provided with a central channel corresponding to the extraction tank and a valve control channel for the valve component to pass through; the surface of the sample injection layer is provided with a liquid cavity communicated with the central channel, a puncture body corresponding to the reagent component and the sample injection component is arranged in the liquid cavity, and the sample injection layer and the air passage layer are provided with air channels communicated with the central channel, the waste liquid pool and the detection area. The nucleic acid detection chip provided by the invention is simple to operate, is convenient to carry, meets the requirements of different application scenes by multi-index detection, and is particularly suitable for the field of in-vitro diagnosis POCT (point of care testing).
Description
Technical Field
The invention relates to the field of biomedical detection and the field of microfluidic chips, in particular to a fully-closed type full-integrated nucleic acid detection microfluidic chip.
Background
Nucleic acid detection is one of the important methods for in vitro diagnosis, and has an important role in diagnosis and prognosis of diseases, and besides, is widely used in other fields, such as public health, food safety, forensic identification, and the like. Current disease diagnostic procedures based on nucleic acid analysis include sample collection, transport, and laboratory nucleic acid detection. The standard nucleic acid detection steps in the laboratory are divided into sample pretreatment, nucleic acid extraction and purification, nucleic acid amplification and detection, and the whole operation process involves reagent and sample exposure, so strict requirements are put on the operation environment, on one hand, the operation needs to be completed in a professional partition experiment, and on the other hand, the operation needs to be completed by a professional operator. These limitations have hampered the popularity of nucleic acid detection in different settings, areas, such as secondary and lower community hospitals. The full-integration nucleic acid detection system has the capability of 'sample in-out', can effectively solve the problem of high laboratory construction and personnel investment, and can further realize POCT (Point-of-Care Testing, bedside detection, also called instant detection) application.
The micro-fluidic chip technology accurately controls fluid through a micro-channel, a reaction chamber and other functional components, integrates basic operation units of sample preparation, reaction, separation, detection and the like of chemical, biological and medical analysis on a chip with the size of a few centimeters, and automatically completes the whole analysis process. Compared with the traditional analysis technology, the microfluidic chip and the matched instrument thereof have the advantages of miniaturization, low consumption and full automation, greatly reduce the dependence on hardware equipment and professionals, and are particularly suitable for various scenes with imperfect medical facilities and scenes needing on-site rapid detection.
From the technical advantage aspect, the nucleic acid detection system based on the microfluidic chip can effectively solve the pain point problem in the traditional method, but the implementation of the full-set nucleic acid detection flow on one microfluidic chip still faces a plurality of challenges, and the currently proposed microfluidic chip is more in realizing part of functions and does not completely meet the actual detection requirement, so that the method is difficult to replace the traditional method to popularize.
The requirements of the full-integrated nucleic acid detection micro-fluidic chip capable of realizing rapidness, convenience, accuracy and cheapness comprise: 1. the method comprises the whole process and reagents of sample splitting, nucleic acid extraction purification and amplification detection, an operator only needs to add a sample, the sample enters and exits, and the whole process is automatic; 2. the whole chip is completely sealed, sample leakage or aerosol pollution can cause the infection of operators and the abnormality of detection results, and the most effective avoiding mode is to isolate the whole chip inner environment from the whole chip outer environment, so that zero air holes are truly realized; 3. the cost is low, the disposable microfluidic chip is used as a consumable, and the material cost is low, which is a great premise of application popularization.
Therefore, the fully-closed fully-integrated nucleic acid detection micro-fluidic chip with high practicability is provided, and has important significance in the fields of disease diagnosis and monitoring, animal epidemic diseases, food safety and the like.
Disclosure of Invention
In view of the above, the present invention provides a fully-enclosed fully-integrated nucleic acid detection micro-fluidic chip, which aims to solve the above technical problems.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a fully-closed fully-integrated nucleic acid detection micro-fluidic chip comprises a sample adding layer, a sample injection layer, an air passage layer, a reaction layer and a sealing layer which are sequentially and hermetically attached from top to bottom;
the top surface of the sample adding layer is integrated with a control component, a reagent component, a sample feeding component and a valve component;
the surface of the reaction layer is provided with a waste liquid pool, an extraction pool and a detection area which are sequentially communicated;
the sample adding layer, the sample injection layer and the air passage layer are provided with a central channel corresponding to the extraction tank and a valve control channel for the valve assembly to pass through; the central channel controls the on-off of the gas circuit and the fluid channel through the up-and-down movement of the control component, and the valve component controls the on-off of the fluid channel between the waste liquid pool and the extraction pool and between the extraction pool and the detection zone through the up-and-down movement;
the sample layer surface offer with the liquid chamber of central channel intercommunication, the liquid intracavity have with reagent subassembly with sample subassembly corresponds the puncture body, sample layer with set up on the air flue layer intercommunication the central channel the waste liquid pond with the gas passage in detection zone.
Through the technical scheme, the invention provides the fully-closed fully-integrated nucleic acid detection micro-fluidic chip, reagents required by the full flow of nucleic acid detection are pre-embedded and poured on the chip, and the chip is provided with a gas internal circulation structure, so that under a fully-sealed state, a special instrument is matched, sample cracking, nucleic acid extraction and purification, amplification and real-time detection can be automatically completed, and the 'sample inlet-outlet' is truly realized; the nucleic acid detection chip is simple to operate, convenient to carry, and particularly suitable for the field of in-vitro diagnosis POCT (point of care testing), and the multi-index detection meets the requirements of different application scenes.
Preferably, in the fully-enclosed fully-integrated nucleic acid detection microfluidic chip, the control assembly includes a threaded control rod, a slider, and a piston; the slide block is fixed on the sample adding layer, the control rod is in threaded connection with the slide block, and the piston is fixed at the bottom end of the control rod.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the reagent assemblies are cylinders capable of penetrating through the bottom film.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the sample injection assembly includes a barrel, a screwed barrel cover screwed with the top of the barrel, and a sealing film fixed at an opening at the bottom of the barrel.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the valve-controlled channel includes a through hole formed on the sample-adding layer, a threaded through hole of the sample-adding layer, and a valve channel formed on the air channel layer.
Preferably, in the fully-closed fully-integrated nucleic acid detecting micro-fluidic chip, the outer side wall of the valve assembly is provided with threads.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the detection area comprises a double-fluid-bath communicated with the extraction tank, the double-fluid-bath is communicated with the amplification tank through a sample inflow channel, the amplification tank is communicated with an air hole through an air channel, and the air hole is covered with a waterproof and breathable film.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the bottom surface of the extraction tank is slope-shaped, and the side far away from the channel is higher than the side near the channel so as to play a role in diversion.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the air channel layer is provided with a gas chamber corresponding to the air hole, the top of the gas chamber is communicated with the central channel, the inner wall of the bottom of the gas chamber is fixed with a sealing cover through a connecting rib, the sealing cover corresponds to the air hole, and the gas chamber is correspondingly provided with a pressing valve rod.
Preferably, in the fully-closed fully-integrated nucleic acid detection microfluidic chip, the sample injection layer and the air passage layer are provided with a gas passage which is communicated with the central passage and the waste liquid pond.
Compared with the prior art, the invention discloses a fully-closed fully-integrated nucleic acid detection micro-fluidic chip, which has the following beneficial effects:
1. and (3) fully sealing design: in the nucleic acid detection process, the problem of aerosol pollution caused by leakage of a sample or an amplified product directly affects the accuracy of subsequent detection and even causes biological pollution to operators, and the existing molecular POCT semi-open system does not really achieve zero pollution. In order to balance the pressure change caused by the inflow of liquid into the extrusion gas, the upper end of the waste liquid pool is communicated with a gas chamber at the upper end of the control component through a gas flow passage, the other side of the chamber is also communicated with the gas chamber on the air hole of the amplification pool, and the control component piston moves up and down to balance the pressure of the gas chamber at the upper end of the control component.
2. Nucleic acid analysis full-flow integrated design: from sample cracking, extraction and purification to qPCR, in-vitro diagnosis is considered to be gold standard for diagnosis of infectious diseases and the like, the traditional operation has extremely high requirements on experimental environment and personnel profession, the molecular POCT technology based on the microfluidic technology has outstanding advantages in the aspects of shortening analysis time, saving labor expenditure and the like, but the technology only realizes partial standard nucleic acid analysis function due to complex fluid operation and high technical barriers, and the diagnosis value is greatly discounted. The invention adopts a simple piston assembly through a multi-layer structure design, which can be used as a liquid-driven control assembly and a fluid control valve assembly, thereby realizing quantitative and sequential transfer of the whole flow reagents and samples of nucleic acid analysis.
3. Sealing cover design of amplification pool: the invention is characterized in that a hydrophobic and breathable film is arranged on a gas exhaust hole in an amplification pool, a sample is stopped from continuing to sample injection after the amplification pool is filled with the sample, so that the quantification is realized, nucleic acid is exponentially increased in the amplification process, and the reaction volume is a high risk stage of aerosol pollution.
4. And (3) designing an independent reagent barrel: the fully integrated POCT nucleic acid detection process relates to a multi-step liquid reagent such as cleaning liquid, eluent and the like, the inner chamber of a microfluidic chip is directly filled with liquid for sealing storage, the leakage liquid-flowing problem in the transportation process is difficult to avoid, or a normally closed valve is needed to design sealing liquid, the structure is complicated, the processing difficulty is high is undoubtedly brought, the liquid reagent is pre-packaged by adopting an independent reagent tube, the liquid reagent is connected with a chip main body through a buckle without using a valve structure, the stable storage of the liquid reagent is realized, and when the liquid reagent is started, only the reagent tube is needed to be pressed, and a bottom film is penetrated by a puncture needle arranged on the chip, so that the reagent is released.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a microfluidic chip according to the present invention;
FIG. 2 is an exploded view of the structure of the microfluidic chip according to the present invention;
FIG. 3 is a schematic diagram of a sample loading layer structure of the microfluidic chip according to the present invention;
FIG. 4 is a schematic diagram of a sample injection layer structure in the microfluidic chip according to the present invention;
FIG. 5 is a schematic diagram of the structure of an airway layer in the microfluidic chip according to the present invention;
FIG. 6 is a schematic diagram of a reaction layer structure in the microfluidic chip according to the present invention;
fig. 7 is a schematic diagram of a structure of a lysis solution cartridge in the microfluidic chip provided by the invention.
Wherein:
1-a sample adding layer; 2-a sample injection layer; 3-airway layer; 4-a reaction layer; 5-sealing layer; a 6-reagent assembly; 7-a sample injection assembly; 8-a control assembly; a 9-valve assembly; 10-pressing down the valve rod;
21-piercing body; 22-fluid channel; 23-threaded through holes; 24-a first gas channel; 25-a second gas channel; 26-a first gas penetration hole;
31-sealing cover; 32-connecting ribs; 33-a gas chamber; 34-a second gas penetration hole; 35-valve passage; 36-a third gas passage;
41-an extraction tank; 42-a waste liquid pool; 43-valve position; 44-microchannel; 45-a double dissolving tank; 46-sample inflow channel; 47-air holes; 48-airway; 49-amplification cell;
61-a cylinder of cleaning liquid; 62-a second cleaning liquid cylinder; 63-eluent cartridge;
71-lysing the cartridge; 711-sealing film; 712-a barrel; 713-a screw-on cap;
81-a control lever; 82-a slider; 83-a piston;
91-a first valve assembly; 92-a second valve assembly.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1 to 6, the embodiment of the invention discloses a fully-closed fully-integrated nucleic acid detection micro-fluidic chip, which comprises a sample adding layer 1, a sample feeding layer 2, an air passage layer 3, a reaction layer 4 and a sealing layer 5 which are sequentially and hermetically attached from top to bottom;
the top surface of the sample adding layer 1 is integrated with a control component 8, a reagent component 6, a sample feeding component 7 and a valve component 9;
the surface of the reaction layer 4 is provided with a waste liquid pool 42, an extraction pool 41 and a detection zone which are sequentially communicated;
the sample adding layer 1, the sample injecting layer 2 and the air passage layer 3 are provided with a central channel corresponding to the extraction tank 41 and a valve control channel for the valve assembly 9 to pass through; the central channel drives the inflow and outflow of reagents and samples in the amplification cell through the up-and-down movement of the control assembly 8, and the valve assembly 9 controls the on-off of fluid channels between the waste liquid cell 42 and the extraction cell 41 and between the extraction cell 41 and the detection area through the up-and-down movement;
the surface of the sample layer 2 is provided with a liquid cavity communicated with the central channel, the liquid cavity is internally provided with a puncture body 21 corresponding to the reagent component 6 and the sample component 7, and the sample layer 2 and the air channel layer 3 are provided with gas channels communicated with the central channel, the waste liquid pool 42 and the detection area.
Specifically, a control assembly 8 is fixed at the center clamping groove of the sample adding layer 1, and the control assembly 8 consists of a threaded control rod 81, a sliding block 82 and a piston 83. The slide block 82 is fixed in the central clamping groove of the sample adding layer 1, the control rod 81 rotates in situ under the action of manual or instrument, and the piston 83 moves upwards or downwards under the transmission action of the slide block 82.
Further, a reagent component 6 and a sample injection component 7 are arranged on one side of the surrounding control component 8, the reagent component 7 consists of reagent cylinders filled with different reagents, the reagent cylinders comprise a first cleaning liquid cylinder 61, a second cleaning liquid cylinder 62 and an eluent cylinder 63, and a pierceable bottom film is sealed at the bottom.
Referring to fig. 7, the sample injection assembly 7 is composed of a sealing membrane 711, a barrel 712 and a screwed barrel cover 713, and is filled with a lysis solution.
Further, a valve assembly 9 and a depression bar 10 are provided around the other side of the control assembly 8, the valve assembly 9 including a first valve assembly 91 and a second valve assembly 92.
The center of the sample introduction layer 2 is a circular through hole, and the circular through hole is used as a part of up-and-down moving channel of the piston 83 of the control assembly 8, and is respectively connected with the third gas channel 36 and the first gas through hole 26 through the upper layer first gas channel 24 and the second gas channel 25.
Further, a plurality of puncture bodies 21 are arranged around the central through hole of the sample adding layer 2 and at the positions corresponding to the upper and lower parts of the reagent assembly 6 and the sample feeding assembly 7, and under the action of pressure, the puncture bodies 21 puncture the bottom film of the reagent cartridge to release the internal reagent. The bottom of the sample adding layer 2 is provided with a fluid channel 22 which is respectively communicated with the liquid cavity and the central through hole. The other side of the central through hole is provided with a threaded through hole 23 which is matched with the threads on the valve assembly 9 at the position corresponding to the upper and lower positions of the valve assembly 9.
The air passage layer 3 is composed of a plurality of groups of through holes including a central circular through hole, through which a piston 83 part as the control unit 8 moves up and down, and around which a second gas through hole 34, a valve passage 35, and a gas chamber 33 are provided, and the upper layer of the gas chamber 33 communicates with the third gas passage 36.
Further, the inner wall of the gas chamber 33 is fixed with a sealing cover 31 through the connecting ribs 32, the pressing rod 10 is pressed down to the sealing cover 31 along the through hole under the pressure effect, the connecting ribs 32 are broken to release the sealing cover 31 in the continuous pressing process, and the pressing rod 10 continuously pushes the sealing cover 31 to the position to be sealed under the pressing effect.
The reaction layer 4 comprises an extraction tank 41 and a waste liquid tank 42, which are connected by a micro-channel 44, a valve position 43 is arranged in the middle of the micro-channel 44, the micro-channel 44 is sealed when the first valve component 91 reaches the valve position 43, and the extraction tank 41 and the waste liquid tank 42 are blocked.
Further, the other side of the extraction tank 41 is communicated with the double-dissolving tank 45 through a micro-channel 44, a valve position 43 of a second component 92 is arranged on the micro-channel 44, and one side, close to the valve position 43, of the bottom surface of the extraction tank 41 is higher than the other side, so that the liquid diversion effect is achieved.
Further, the sample in the extraction cell 41 is uniformly dispersed to a plurality of amplification cells 49 through the sample inflow channel 48 under the action of the control module 8, and the gas in the amplification cells 49 and the channels is supplied from the gas holes 47 to the gas chamber 33.
Further, the air hole 47 is provided with a waterproof air-permeable film, which is air-permeable and water-impermeable.
Specifically, in the process of detecting sample nucleic acid, the specific working flow of the chip of the invention is as follows:
firstly, the cylinder cover 713 of the sample injection assembly 7 is opened, the liquid sample is quantitatively added and then is sealed, the chip is put into a matched instrument, the operation is started by one key after the parameters are set, and the following actions are automatically completed on the instrument:
s1, sample splitting: pressing the sample injection assembly 7, the corresponding puncture body 21 on the sample addition layer 2 punctures the sealing film 711 of the sample-containing cracking sample cylinder 71, the cracking liquid is released to flow into the extraction tank 41 through the micro-channel, the control assembly 8 can be pulled upwards appropriately to realize the complete transfer of the cracking liquid, for fully cracking the sample, the ultrasonic module can be arranged at the bottom of the extraction tank 41 to improve the cracking efficiency, the cracked nucleic acid is captured by the magnetic beads pre-embedded in the extraction tank 41, after full capture, the electromagnet at the side edge of the amplification tank 41 is opened to adsorb the magnetic beads, the first valve assembly 91 is opened, the control assembly 8 is pushed to send the cracking liquid into the waste liquid tank 42 through the micro-channel 44, and part of gas in the waste liquid tank 42 is transferred to the upper chamber of the piston 83 of the control assembly 8 from the second gas through hole 34 above under the pressure.
S2, nucleic acid extraction and purification: pressing the upper part of a cylinder 61 of cleaning liquid, penetrating the bottom film by the corresponding penetrating body 21 on the sample adding layer 2, releasing the cleaning liquid inside and flowing into the extracting pool 41, properly pulling the control component 8 upwards to realize the whole transfer of the cleaning liquid, releasing the cleaning liquid for a plurality of times by the stroke of the control component 8, applying ultrasonic energy to the bottom of the extracting pool 41, cleaning impurities except nucleic acid in the extracting pool 41, opening an electromagnet on the side of the amplifying pool 41, adsorbing magnetic beads, opening the first valve component 91, pushing the control component 8 to send the cleaning liquid into the waste liquid pool 42 through the micro-channel 44, and transferring part of gas in the waste liquid pool 42 from the second gas through hole 34 above to a cavity above the piston 83 of the control component 8 under the pressure effect. The cleaning liquid in the cleaning liquid second cylinder 62 is released in the same manner, and the cleaning is completed.
S3, eluting and sampling: pressing the upper part of the eluent barrel 63, the corresponding puncture body 21 on the sample adding layer 2 punctures the bottom film of the eluent barrel 63 containing the sample, the eluent is released to flow into the extraction tank 41 through the micro-channel, the control component 8 can be pulled upwards appropriately to realize the complete transfer of the lysate, ultrasonic energy is applied to the bottom of the extraction tank 41, the temperature of the extraction tank is controlled at about 50 ℃, and the elution efficiency is improved. The electromagnet at the side of the amplification cell 41 is opened to adsorb magnetic beads, the second valve assembly 92 is opened, the control assembly 8 is pushed to transfer eluent to the eight parallel amplification cells 49 through the sample inflow channel 46, the gases in the amplification cells 49 and the channels are transferred to the upper gas chamber 33 through the gas holes 47, and internal circulation is realized through the third gas channel 36 and the first gas channel 24.
S4, PCR amplification and real-time detection: after the sample fills the amplification pool 49, the pressure-reducing rod 10 is pushed to move downwards, the sealing cover 31 is released from the inside of the gas chamber 33 under the pressure action to reach the position above the air hole 47, the sealing cover presses the waterproof and breathable film on the air hole 47 under the extrusion action of the pressure-reducing rod 10, the amplification system is sealed, the circulation temperature control is started, and the amplification and fluorescence real-time detection are started.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. The fully-closed fully-integrated nucleic acid detection micro-fluidic chip is characterized by comprising a sample adding layer (1), a sample injecting layer (2), an air passage layer (3), a reaction layer (4) and a sealing layer (5) which are sequentially and hermetically attached from top to bottom;
the top surface of the sample adding layer (1) is integrated with a control component (8), a reagent component (6), a sample feeding component (7) and a valve component (9);
the surface of the reaction layer (4) is provided with a waste liquid pool (42), an extraction pool (41) and a detection zone which are sequentially communicated;
the sample adding layer (1), the sample injection layer (2) and the air passage layer (3) are provided with a central channel corresponding to the extraction tank (41) and a valve control channel for the valve assembly (9) to pass through; the central channel controls the on-off of a gas path and a fluid channel through the up-and-down movement of the control component (8), and the valve component (9) controls the on-off of the fluid channel between the waste liquid pool (42) and the extraction pool (41) and between the extraction pool (41) and the detection zone through the up-and-down movement;
the surface of the sample injection layer (2) is provided with a liquid cavity communicated with the central channel, a puncture body (21) corresponding to the reagent component (6) and the sample injection component (7) is arranged in the liquid cavity, and the sample injection layer (2) and the air channel layer (3) are provided with a gas channel communicated with the central channel, the waste liquid pool (42) and the detection area;
the detection area comprises a double-dissolving pool (45) communicated with the extraction pool (41), the double-dissolving pool (45) is communicated with an amplification pool (49) through a sample inflow channel (46), the amplification pool (49) is communicated with an air hole (47) through an air channel (48), and the air hole (47) is covered with a waterproof and breathable film;
the air passage layer (3) is provided with a gas chamber (33) corresponding to the air hole (47), the top of the gas chamber (33) is communicated with the central channel, the inner wall of the bottom of the gas chamber (33) is fixedly provided with a sealing cover (31) through a connecting rib (32), the sealing cover (31) corresponds to the air hole (47), and the gas chamber (33) is correspondingly provided with a pressing valve rod (10);
the control assembly (8) comprises a threaded control rod (81), a sliding block (82) and a piston (83); the sliding block (82) is fixed on the sample adding layer (1), the control rod (81) is in threaded connection with the sliding block (82), and the piston (83) is fixed at the bottom end of the control rod (81).
2. The fully enclosed fully integrated nucleic acid detection microfluidic chip of claim 1, wherein the reagent assemblies (6) are cartridges that pierce the carrier film.
3. The fully-enclosed fully-integrated nucleic acid detection microfluidic chip according to claim 1, wherein the sample injection assembly (7) comprises a barrel (712), a screwed barrel cover (713) screwed with the top of the barrel (712), and a sealing film (711) fixed at the bottom opening of the barrel (712).
4. The fully-enclosed fully-integrated nucleic acid detection microfluidic chip according to claim 1, wherein the valve-controlled channel comprises a through hole formed in the sample-adding layer (1), a threaded through hole (23) of the sample-injecting layer (2), and a valve channel (35) formed in the air channel layer (3).
5. The fully enclosed fully integrated nucleic acid detection microfluidic chip of claim 4, wherein the valve assembly (9) outer sidewall has threads.
6. The fully-enclosed fully-integrated nucleic acid detection microfluidic chip according to claim 1, wherein the bottom surface of the extraction tank (41) is sloped, and the side far from the channel is higher than the side near the channel.
7. The fully-enclosed fully-integrated nucleic acid detection microfluidic chip according to claim 1, wherein the sample introduction layer (2) and the air passage layer (3) are provided with a gas passage communicating the central passage and the waste liquid tank (42).
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| US20120077260A1 (en) * | 2009-03-30 | 2012-03-29 | Fraunhofer Usa, Inc. | Reservoir-buffered mixers and remote valve switching for microfluidic devices |
| CN115151630A (en) * | 2020-09-22 | 2022-10-04 | 京东方科技集团股份有限公司 | Nucleic acid extraction micro-fluidic chip, nucleic acid extraction device and extraction method |
| CN113122613B (en) * | 2021-04-09 | 2023-06-09 | 四川微康朴澜医疗科技有限责任公司 | Totally-enclosed fluorescent quantitative PCR microfluidic detection chip |
| CN113088446B (en) * | 2021-04-30 | 2022-07-05 | 华中科技大学 | Full-automatic nucleic acid rapid detection device and detection method |
| CN115466670A (en) * | 2022-09-20 | 2022-12-13 | 上海健康医学院 | Micro-controlled flow chip for nucleic acid extraction and detection |
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| CN115612593A (en) * | 2022-10-09 | 2023-01-17 | 北京梓晶生物科技有限公司 | Full-integrated nucleic acid detection micro-fluidic chip and nucleic acid detector with same |
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| An integrated nucleic acid detection method based on a microfluidic chip for collection and culture of rice false smut spores;Ning Yang et al.;《Lab on a Chip》;第22卷;第4894-4904页 * |
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