CN111363664B - LAMP detection chip based on three-layer microchip detection and control method thereof - Google Patents
LAMP detection chip based on three-layer microchip detection and control method thereof Download PDFInfo
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- CN111363664B CN111363664B CN202010330178.8A CN202010330178A CN111363664B CN 111363664 B CN111363664 B CN 111363664B CN 202010330178 A CN202010330178 A CN 202010330178A CN 111363664 B CN111363664 B CN 111363664B
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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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
- C12Q3/00—Condition responsive control processes
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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
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- 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 an LAMP detection chip based on a three-layer microchip, which comprises a main body, a first injection layer, a second load layer and a third reaction layer, wherein the main body comprises the three-layer microchip; the second layer of the load layer comprises a plurality of load areas, and the third layer of the reaction layer comprises a plurality of reaction areas for the formation of a system and the reaction. The invention discloses a control method of an LAMP detection chip based on a three-layer microchip.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to an LAMP detection chip based on three-layer microchip detection and a control method thereof.
Background
Loop-mediated isothermal amplification (LAMP) can amplify nucleic acid in a short time (usually within one hour) under isothermal (60-65 ℃) conditions, and is a simple, rapid, accurate and low-cost gene amplification method; compared with conventional PCR, the method does not need the processes of thermal denaturation of a template, temperature circulation, electrophoresis, ultraviolet observation and the like. The loop-mediated isothermal amplification method is a brand-new nucleic acid amplification method and has the characteristics of simplicity, rapidness and strong specificity. The technology can be comparable or even better than the PCR technology in indexes such as sensitivity, specificity, detection range and the like, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, and has detection cost far lower than fluorescent quantitative PCR.
The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip, and automatically completes the whole analysis process. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field where the disciplines of biology, chemistry, medicine, fluids, electronics, materials, machinery and the like are crossed.
However, since there are still great problems in the prior art, including: (1) The prior art can only carry out qualitative detection, can not carry out quantitative detection and can not accurately determine pathogen content; (2) The existing method has the defects that the reaction container has insufficient tightness, aerosol pollution is easy to cause after the reaction container is opened, and false positive is caused; (3) The sample and the reagent in the prior method need to be added respectively, and dosage errors are easy to be caused in the process of adding samples for a plurality of times; (4) The result judgment is complex, the reaction result is easy to generate errors through precipitation change judgment, and most of the situations still need electrophoresis detection.
Disclosure of Invention
Based on the technical problems, the invention designs and develops an LAMP detection chip based on three-layer microchip detection, and the invention aims to maintain the air tightness of a detection system without opening the detection chip and perform rapid quantitative detection without multiple sample addition.
The invention designs and develops a control method of an LAMP detection chip based on three-layer microchip detection, and aims to perform rapid quantitative detection without multiple sample addition under the condition that the air tightness of a detection system is kept through an oil seal sample.
The technical scheme provided by the invention is as follows:
a LAMP detection chip based on three-layer microchip detection, comprising:
a first injection layer comprising a first sample addition port, a first oil filling port, and a plurality of first waste pool vent holes;
the second loading layer comprises a second sample adding hole communicated with the first sample adding hole, a second oil adding hole communicated with the first oil adding hole and a plurality of loading areas correspondingly arranged with the first waste liquid pool exhaust hole;
the load area comprises a first oil distributing hole communicated with the second oil filling hole and a second waste liquid pool exhaust hole communicated with the first waste liquid pool exhaust hole;
the third reaction layer comprises a third sample adding hole communicated with the second sample adding hole and a plurality of reaction areas which are arranged corresponding to the load area;
wherein the reaction zone comprises:
a reagent microchamber, one end of which is communicated with the third sample adding hole;
a mixing chamber, one end of which is communicated with the other end of the reagent microchamber;
a second oil distribution hole communicating with one end of the mixing chamber and communicating with the first oil distribution hole;
a LAMP reaction zone, one end of which is communicated with the other end of the mixing chamber;
a waste liquid pool which is communicated with the other end of the LAMP reaction zone;
and the third waste liquid pool exhaust hole is arranged on the waste liquid pool and is communicated with the second waste liquid pool exhaust hole.
Preferably, the load zone further comprises:
and one end of each oil distributing channel is communicated with the second oil filling hole, and the other end of each oil distributing channel is communicated with the first oil distributing hole.
Preferably, the method further comprises:
the first air storage chambers are respectively and correspondingly arranged in the load area;
a plurality of second air-storing chambers which are respectively and correspondingly arranged in the reaction zones and are arranged on a channel which is communicated between the mixing chamber and the LAMP reaction zone;
wherein the first air storage chamber is communicated with the second air storage chamber.
Preferably, the mixing chambers are arranged in a spiral.
Preferably, any one of the LAMP reaction zones includes:
and the reaction microchambers are communicated and are arranged in a serpentine shape.
Preferably, the reaction microchamber is a plurality of reaction spaces separated by a protrusion of a rectangular parallelepiped structure having a length of 375 to 425 μm, a width of 100 to 150 μm, and a height of 69 to 79 μm.
Preferably, the interval between the rectangular parallelepiped structures is 80 to 120 μm.
The control method of the LAMP detection chip based on three-layer microchip detection is characterized by comprising the following steps of:
step one, performing an oil inlet process, wherein an oil phase is injected through the first oil filling hole, enters the second layer of load layer through the second oil filling hole, is divided into a plurality of first oil distributing holes connected with oil channels by the oil distributing channel, and enters the third layer of reaction layer from the first oil distributing holes;
the second oil distribution hole is used for receiving the oil phase of the second load layer, enters the main channel through the second oil distribution hole, flows through the mixing chamber, enters the LAMP reaction zone and fills the whole reaction micro-chamber, the redundant oil phase flows into the waste liquid pool, and the first oil inlet hole is sealed after the oil phase fills the reaction micro-chamber to ensure the pressure;
step two, carrying out a sample injection process, wherein a sample flows through the second sample injection hole through the first sample injection hole, directly enters the third sample injection hole, flows through the reagent micro chamber to wash away reagent dry powder in the reagent micro chamber, and enters the mixing chamber to be fully and uniformly mixed, at the moment, the first oil injection hole is in a sealed state, the sample continuously enters the LAMP reaction zone and replaces an oil phase in the reaction micro chamber, and redundant water phase flows into the waste liquid pool;
step three, the oil inlet process is carried out again, the oil phase is injected through the first oil filling hole, and flows through the second oil filling hole, the oil distribution channel, the first oil distribution hole, the oil inlet channel, the second oil distribution hole and the mixing chamber in sequence to reach the LAMP reaction area to cut the water phase, and the water phase is combined and sealed with the oil layer remained in the reaction micro-chamber to wrap and seal the reaction system;
and step four, LAMP reaction detection is carried out.
Compared with the prior art, the invention has the following beneficial effects:
the reaction and detection are integrated, a reaction container is not required to be opened, a reaction result can be obtained, the problem of aerosol pollution is avoided, the reagent is arranged in the chip in a dry powder mode, the operation is simpler and more convenient, the detection time is greatly shortened, multiple sample adding is not required, and the dosage error is avoided.
Drawings
FIG. 1 is a schematic diagram of the structure of a LAMP detection chip for three-layer microchip detection according to the present invention.
Fig. 2 is a schematic diagram of the structure of the first injection layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 3 is a front view of the first injection layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 4 is a cross-sectional view of the first injection layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 5 is a schematic diagram showing the structure of the second layer of the load layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 6 is a front view of the second loading layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 7 is a cross-sectional view of the second supporting layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 8 is a schematic diagram showing the structure of the third reaction layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
Fig. 9 is a partial enlarged view at "a" in fig. 8.
FIG. 10 is a front view of the third reaction layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 11 is a schematic structural diagram of a reaction region in a third reaction layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
FIG. 12 is a cross-sectional view of the third reaction layer of the LAMP detection chip for three-layer microchip detection according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
As shown in fig. 1, the present invention provides a LAMP detection chip based on three-layer microchip detection, the main body of which comprises three-layer microchip, a first injection layer 100, a second load layer 200 and a third reaction layer 300; wherein the second supporting layer 200 comprises a plurality of supporting regions, and the third reactive layer 300 comprises a plurality of reactive regions for the formation of the system and the progress of the reaction.
As shown in fig. 2 to 4, the first injection layer 100 includes a first sample application port 110, a first fuel filler port 120, and a plurality of first waste pool vent holes 130; the first sample adding hole 110 is simultaneously communicated with the second sample adding hole 210 of the second load layer 200 and the third sample adding hole 310 of the third reaction layer 300, the first oil adding hole 120 is simultaneously communicated with the second oil adding hole 220 of the second load layer 200, and the first sample adding hole 110 and the first oil adding hole 120 are respectively used for injecting water phase and oil phase;
as shown in fig. 5 to 7, the second loading layer 200 includes a second sample application port 210, a second fuel filler port 220, and a plurality of loading zones; wherein the plurality of load regions include a plurality of oil distribution channels 250, a plurality of first oil distribution holes 240, a plurality of first air storage chambers 260, and a plurality of second waste liquid pool exhaust holes 230, which are gradually branched; the second loading layer 200 is gradually divided into a plurality of oil distribution channels 250 from a second oil filling hole 220, and is respectively connected to a plurality of first oil distribution holes 240 entering the third reaction layer 300, and a plurality of first air storage chambers 260 are additionally designed for storing the exhaust gas of the third reaction layer 300; wherein, the sample adding device specifically further comprises a second sample adding hole 210 communicated with the first sample adding hole 110, a second oil filling hole 220 communicated with the first oil filling hole 120, and a plurality of second waste liquid pool exhaust holes 230 respectively communicated with a plurality of first waste liquid pool exhaust holes 130 correspondingly;
as shown in fig. 8 to 12, the third reaction layer 300 is composed of a third sample addition hole 310 and a plurality of reaction regions, and the reaction regions are respectively arranged in one-to-one correspondence with the load regions; wherein, each reaction zone is composed of a reagent micro chamber 341, a second oil distributing hole 320, an oil inlet channel 350, a spiral mixer 342, a second gas storage chamber 360, an LAMP reaction zone 343, a waste liquid pool 370 and a third waste liquid pool exhaust hole 330; the LAMP reaction area 343 comprises a serpentine channel 343a and a plurality of reaction micro-chambers 343b, the reagent micro-chambers 341 store the primer, fluorescent detection reagent and other dry powder needed for reaction, the second oil distributing holes 320 of the third layer reaction layer 300 are communicated with the first oil distributing holes 240 of the second layer load layer 200, the spiral mixer 342 is used for uniformly mixing the sample and the reaction reagent, the second gas storage chamber 360 of the third layer reaction layer 300 is communicated with the first gas storage chamber 260 of the second layer load layer 200 and is used for storing the residual gas before the sample is added after the oil phase is injected, the serpentine channel 343a is connected with the plurality of reaction micro-chambers 343b to uniformly divide the reaction system into a plurality of parts, the waste liquid pond 370 is used for storing redundant waste liquid, the LAMP reaction area 343 contains a plurality of rows of serpentine channels 343a, and each row of the plurality of reaction micro-chambers 343b divide the total reaction system into a plurality of parts, so as to realize digitization.
In another embodiment, as shown in fig. 5 to 7, the second loading layer 200 includes 1 second sample well 210, 1 second oil filling hole 220, a separation channel 250 gradually branched into 8, 8 first oil separation holes 240, 8 first air storage chambers 260 and 8 second waste liquid pool exhaust holes 230, the second loading layer 200 is gradually separated into 8 separation channels 250 from one second oil filling hole 220, the second oil filling holes 220 are respectively connected to 8 first oil separation holes 240 entering the third layer reaction layer 300, and 8 first air storage chambers 260 are additionally designed for storing the waste gas of the third layer reaction layer 300.
In another embodiment, as shown in fig. 8, the third reaction layer 300 is composed of 8 reaction regions, and is disposed in one-to-one correspondence with the oil distribution channel 250, the first oil distribution hole 240, the first air storage chamber 260, and the second waste liquid pool vent hole 230 of the second load layer 200, respectively.
In another embodiment, as shown in fig. 11, the serpentine channels 343a are connected with 360 reaction micro-chambers 343b to uniformly divide the reaction system into 360 parts, the waste liquid pool 370 is used for storing redundant waste liquid, the LAMP reaction area 343 contains 12 rows of serpentine channels 343a, each row of 30 reaction micro-chambers 343b is divided into 360 parts, the total reaction system is divided into 360 parts, and the detection reagent is stored in the reagent micro-chambers 341 in the form of dry powder, so as to realize detection digitization.
In another embodiment, the spacing between each row of serpentine channels 343a is 100-150 μm; preferably, the spacing between each row of serpentine channels 343a is 125 μm.
In another embodiment, the reaction micro chamber 343b is a plurality of reaction spaces partitioned by protrusions of a rectangular parallelepiped structure having a length of 375-425 μm, a width of 100-150 μm, and a height of 69-79 μm; as a preferred embodiment, the rectangular parallelepiped structure has a length of 400 μm, a width of 120 μm and a height of 74. Mu.m.
In another embodiment, the spacing between the cuboid structures is 80-120 μm; as one preferable, the interval between the rectangular parallelepiped structures is 100 μm.
The invention provides a process of an LAMP detection chip based on three-layer microchip detection, which comprises four steps, wherein the first step of oil phase protection layer formation is used for oil feeding, the second step of reagent and sample entering, mixing and digital sample feeding, the third step of reaction system sealing and oil feeding, and the fourth step of LAMP reaction detection, and the method specifically comprises the following steps:
step one, an oil inlet process is carried out: the oil phase is injected into the oil phase through the connection between the first oil filling hole 120 of the first injection layer 100 and the oil source, enters the second loading layer 200 through the second oil filling hole 220, is divided into 8 oil channels by the oil dividing channel 250 to be connected with 8 first oil dividing holes 240, and enters the third reaction layer 300 through the second oil dividing holes 320 by the first oil dividing holes 240;
the second oil distributing hole 320 located in the third reaction layer 300 is configured to receive the oil phase of the second load layer 200, and enter the oil inlet channel 350 through the second oil distributing hole 320, flow through the spiral mixer 342, enter the LAMP reaction area 343, fill the whole reaction micro-chamber 343b through the serpentine channel 343a, and flow the redundant oil phase into the waste liquid pool 370, and seal the first oil inlet hole 120 to ensure pressure after the oil phase fills the reaction micro-chamber;
step two, carrying out a sample injection process: the sample flows through the first sample injection hole 110 of the first injection layer 100, flows through the second sample injection hole 210 of the second load layer 200, directly enters the third sample injection hole 310 of the third reaction layer 300, flows through the reagent micro chamber 341 to wash away the reagent dry powder in the reagent micro chamber 341, and enters the spiral mixer 342 to be fully mixed, at this time, in order to ensure the pressure, the first oil filling hole 120 is in a sealed state, so that part of waste gas exists from the third sample injection hole 310 to the oil inlet channel 350, in the process of flowing through the second gas storage chamber 360, the surplus waste gas is fully discharged, and is stored in the first gas storage chamber 260 of the second load layer 200, so that the influence of gas on the filling rate of the reaction micro chamber 343b is avoided, the sample continuously flows into the LAMP reaction region 343 after passing through the second gas storage chamber 360, replaces the oil phase in the reaction micro chamber 343b, and as the water phase is subjected to lipophilic treatment, a layer of oil film is reserved for wrapping and preventing evaporation of water, and the water phase of PDMS flows into the liquid pool 370;
step three, carrying out oil inlet process again: sealing the first sample adding hole 110 after the second step is completed, injecting an oil phase through the first oil filling hole 120 again, sequentially flowing through the second oil filling hole 220, the oil distributing channel 250, the first oil distributing hole 240, the oil inlet channel 350 and the spiral mixer 342 to reach the LAMP reaction area 343 to cut the water phase, combining with the oil layer remained in the reaction micro-chamber 343b for sealing, forming a complete oil film, and wrapping a sealed reaction system;
step four, carrying out LAMP reaction: the sample template, six specific primers, mix reagent, fluorescence detection reagent and the like required by the reaction are completely contained in the reagent micro-chamber 341 of each reaction zone, and the detection chip is reacted for 1 hour under the constant temperature condition of 65 ℃, so that the reaction result of the micro-chamber can be judged through fluorescence reaction; the fluorescence light spots of 360 microchambers are collected through a CCD, and the reaction results of different microchambers are analyzed through computer Image J software.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (5)
1. An LAMP detection chip based on three-layer microchip detection, which is characterized by comprising:
a first injection layer comprising a first sample addition port, a first oil filling port, and a plurality of first waste pool vent holes;
the second loading layer comprises a second sample adding hole communicated with the first sample adding hole, a second oil adding hole communicated with the first oil adding hole and a plurality of loading areas correspondingly arranged with the first waste liquid pool exhaust hole;
the load area comprises a first oil distributing hole communicated with the second oil filling hole and a second waste liquid pool exhaust hole communicated with the first waste liquid pool exhaust hole;
the third reaction layer comprises a third sample adding hole communicated with the second sample adding hole and a plurality of reaction areas which are arranged corresponding to the load area;
wherein the reaction zone comprises:
a reagent microchamber, one end of which is communicated with the third sample adding hole;
a mixing chamber, one end of which is communicated with the other end of the reagent microchamber;
a second oil distribution hole communicating with one end of the mixing chamber and communicating with the first oil distribution hole;
a LAMP reaction zone, one end of which is communicated with the other end of the mixing chamber;
a waste liquid pool which is communicated with the other end of the LAMP reaction zone;
a third waste liquid pool exhaust hole which is arranged on the waste liquid pool and is communicated with the second waste liquid pool exhaust hole;
the load zone further comprises:
one end of each of the plurality of oil distributing channels is communicated with the second oil filling hole, and the other end of each of the plurality of oil distributing channels is communicated with the first oil distributing hole;
the mixing chambers are spirally arranged;
any one of the LAMP reaction zones includes:
and the reaction microchambers are communicated and are arranged in a serpentine shape.
2. The LAMP detection chip based on three-layer microchip detection as claimed in claim 1, further comprising:
the first air storage chambers are respectively and correspondingly arranged in the load area;
a plurality of second air-storing chambers which are respectively and correspondingly arranged in the reaction zones and are arranged on a channel which is communicated between the mixing chamber and the LAMP reaction zone;
wherein the first air storage chamber is communicated with the second air storage chamber.
3. The LAMP detection chip based on three-layer microchip detection as claimed in claim 2, wherein the reaction microchamber is a plurality of reaction spaces partitioned by projections of a rectangular parallelepiped structure having a length of 375 to 425 μm, a width of 100 to 150 μm and a height of 69 to 79 μm.
4. The LAMP detection chip based on three-layer microchip detection according to claim 3, wherein the interval between said rectangular parallelepiped structures is 80-120 μm.
5. A control method of a LAMP detection chip based on three-layer microchip detection, characterized by using the LAMP detection chip based on three-layer microchip detection as set forth in any one of claims 1 to 4, comprising the steps of:
step one, performing an oil inlet process, wherein an oil phase is injected through the first oil filling hole, enters the second layer of load layer through the second oil filling hole, is divided into a plurality of first oil distributing holes connected with oil channels by the oil distributing channel, and enters the third layer of reaction layer from the first oil distributing holes;
the second oil distribution hole is used for receiving the oil phase of the second load layer, enters the main channel through the second oil distribution hole, flows through the mixing chamber, enters the LAMP reaction zone and fills the whole reaction micro-chamber, the redundant oil phase flows into the waste liquid pool, and the first oil filling hole is sealed after the oil phase fills the reaction micro-chamber to ensure the pressure;
step two, carrying out a sample injection process, wherein a sample flows through the second sample injection hole through the first sample injection hole, directly enters the third sample injection hole, flows through the reagent micro chamber to wash away reagent dry powder in the reagent micro chamber, and enters the mixing chamber to be fully and uniformly mixed, at the moment, the first oil injection hole is in a sealed state, the sample continuously enters the LAMP reaction zone and replaces an oil phase in the reaction micro chamber, and redundant water phase flows into the waste liquid pool;
step three, the oil inlet process is carried out again, the oil phase is injected through the first oil filling hole, and flows through the second oil filling hole, the oil distribution channel, the first oil distribution hole, the oil inlet channel, the second oil distribution hole and the mixing chamber in sequence to reach the LAMP reaction area to cut the water phase, and the water phase is combined and sealed with the oil layer remained in the reaction micro-chamber to wrap and seal the reaction system;
and step four, LAMP reaction detection is carried out.
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