CN110616138A - Chambered multi-index nucleic acid amplification micro-fluidic chip - Google Patents
Chambered multi-index nucleic acid amplification micro-fluidic chip Download PDFInfo
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- CN110616138A CN110616138A CN201910954457.9A CN201910954457A CN110616138A CN 110616138 A CN110616138 A CN 110616138A CN 201910954457 A CN201910954457 A CN 201910954457A CN 110616138 A CN110616138 A CN 110616138A
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 126
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 126
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 126
- 230000003321 amplification Effects 0.000 title claims abstract description 120
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 120
- 238000002347 injection Methods 0.000 claims abstract description 40
- 239000007924 injection Substances 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims description 32
- 238000003860 storage Methods 0.000 claims description 20
- 230000002209 hydrophobic effect Effects 0.000 claims description 12
- 239000002390 adhesive tape Substances 0.000 claims description 7
- 238000012545 processing Methods 0.000 claims description 7
- 238000004806 packaging method and process Methods 0.000 claims description 6
- 238000001746 injection moulding Methods 0.000 claims description 5
- 238000003754 machining Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 5
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 2
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 2
- 239000012994 photoredox catalyst Substances 0.000 claims description 2
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 2
- 239000004417 polycarbonate Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 31
- 238000000034 method Methods 0.000 abstract description 10
- 238000011901 isothermal amplification Methods 0.000 description 13
- 238000003752 polymerase chain reaction Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000588747 Klebsiella pneumoniae Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000007731 hot pressing Methods 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract
A multi-index nucleic acid amplification micro-fluidic chip with separated chambers comprises a nucleic acid amplification sample system sample injection pipeline layer and a nucleic acid amplification reaction chamber layer, wherein reaction chambers connected in series are arranged on a surface to be fixed of the nucleic acid amplification reaction chamber layer, and a secondary sample injection port and a sample injection exhaust port are sequentially arranged at the bottom end of each reaction chamber; the sample introduction pipeline layer of the nucleic acid amplification sample system is provided with a sample introduction pipeline on a to-be-fixed surface, the head end of the sample introduction pipeline is provided with a chip sample introduction port, and the tail end of the sample introduction pipeline is provided with a sample outlet, wherein the sample introduction pipeline is used for sequentially communicating the second-stage sample introduction port of the reaction chamber and the sample introduction exhaust port. The invention is a rapid detection tool with simple operation and low cost, and is suitable for rapid multi-index detection of various types of detection samples under various detection occasions. The whole process does not need external complex instruments (such as a thermal cycler, a pump and the like) for supporting, the chambered multi-index nucleic acid amplification detection can be realized within 1 hour, the final result can be directly interpreted, and the operation is simple, convenient and quick.
Description
Technical Field
The invention belongs to the field of biomedical detection and rapid disease diagnosis, and particularly relates to a chamber-divided multi-index nucleic acid amplification device based on a microfluidic chip.
Background
From the nineties of the last century, nucleic acids, one of the most fundamental substances in organisms, play an increasingly important role in modern biomedicine, characterized by: the detection specificity is strong, the operation is very easy, the technology is developed to be mature, the detection time is extremely short, and therefore, the method has great significance in the fields of infectious disease detection, food safety monitoring, environmental protection and the like. Therefore, rapid detection based on nucleic acids has been recognized as one of the important molecular detection means.
The rapid detection based on nucleic acid mainly comprises the following two modes: polymerase Chain Reaction (PCR) and isothermal amplification techniques. As a mature nucleic acid amplification technology, PCR has been a non-negligible technology in the field of biomedical detection and disease diagnosis through the recent decades, and involves a plurality of different reaction temperatures, a temperature cycler can be used to precisely control the temperature of each thermal cycle step. The nucleic acid isothermal amplification technology only needs single reaction temperature in the whole process, has simpler reaction temperature conditions, is simpler and more convenient than the PCR technology in both actual operation and instrument requirements, can release the dependence on excellent equipment, and shows good application prospect in clinical and on-site rapid diagnosis.
However, both PCR and isothermal amplification techniques are faced with multi-index detection, and it is necessary to avoid the mutual interference between primers. The mainstream way to avoid the mutual interference between the primers is to use a chamber-divided way. For example, a sliding chambered microfluidic chip for multiplex PCR can realize nanoscale multiplex PCR multi-index detection by sliding a top plate and a bottom plate with specific patterns. However, the precise alignment of the top and bottom plates requires extremely precise operations, which makes the whole process extremely complex; in addition, a platform for multiple PCR chambers is completed by an open hydrophobic microarray, but the manufacturing process of the chip is very complicated, multiple amplifications are needed to obtain enough products, and the amplifications are performed in an open environment, so that the whole amplification has the possibility of pollution. In addition, the disc-shaped centrifugal butterfly chip is also a method for successfully carrying out multi-index detection by using chambers, but the product cannot be recycled, so that the disc-shaped centrifugal butterfly chip can only be used for field detection, and large-volume high-throughput detection cannot be realized.
Disclosure of Invention
According to the background analysis, the invention aims to overcome the defects in the prior art and solve the problems of incomplete sample introduction of the chip, mutual crosstalk between primers, external pollution and the like in a nucleic acid amplification region by using an independent chamber as a reaction region based on a microfluidic chip.
In order to solve the problems, the invention provides a chambered multi-index nucleic acid amplification micro-fluidic chip which comprises a nucleic acid amplification sample system sample injection pipeline layer and a nucleic acid amplification reaction chamber layer, wherein the nucleic acid amplification sample system sample injection pipeline layer is fixedly connected with the nucleic acid amplification reaction chamber layer;
at least one row of reaction chambers connected in series is arranged on the surface to be fixed of the nucleic acid amplification reaction chamber layer, and the chip is provided with two rows of reaction chambers. The reaction chambers are respectively independent and have the same structure, and the bottom ends of the reaction chambers are sequentially provided with a secondary sample inlet and a sample outlet;
a sample introduction pipeline is arranged on the surface to be fixed of the sample introduction pipeline layer of the nucleic acid amplification sample system, a chip sample introduction port is arranged at the head end of the sample introduction pipeline, a sample outlet is arranged at the tail end of the sample introduction pipeline, and the chip sample introduction port and the sample outlet both penetrate through the sample introduction pipeline layer of the nucleic acid amplification sample system; the sample introduction pipeline is used for sequentially communicating a second-stage sample introduction port and a sample introduction exhaust port of the reaction chamber, and before the nucleic acid amplification sample system sample introduction pipeline layer and the nucleic acid amplification reaction chamber layer are fixedly connected, the sample introduction pipeline and the sample introduction exhaust port are required to be subjected to hydrophobic treatment.
Further, still be equipped with the liquid reserve tank on the micro-fluidic chip, the liquid reserve tank is equipped with upper and lower liquid reserve chamber, the sample introduction pipeline that nucleic acid amplification sample system sample introduction pipeline layer was located to lower liquid reserve chamber is close to out appearance mouth department, go up the liquid reserve chamber and locate nucleic acid amplification reaction chamber room layer with the corresponding position of liquid reserve chamber down.
Furthermore, the sample injection pipeline layer of the nucleic acid amplification sample system and the to-be-fixed surface of the nucleic acid amplification reaction chamber layer are respectively provided with at least three positioning holes for packaging and positioning.
Further, the surfaces to be fixed of the nucleic acid amplification sample system sample injection pipeline layer and the nucleic acid amplification reaction chamber layer are tightly packaged and bonded by using double-sided adhesive tapes, and each double-sided adhesive tape has the same structure as the nucleic acid amplification sample system sample injection pipeline layer and the nucleic acid amplification reaction chamber layer, which are cut by using a cutting die.
Furthermore, the sample injection pipeline layer of the nucleic acid amplification sample system and the surface to be fixed of the nucleic acid amplification reaction chamber layer are bonded together by hot pressing.
Furthermore, the bottom end of the reaction cavity is provided with a second-stage sample inlet in the previous time, the back side of the bottom end is provided with a sample inlet and outlet, and the sample inlet and outlet is smaller than the second-stage sample inlet.
Furthermore, the nucleic acid amplification sample system sample injection pipeline layer and the nucleic acid amplification reaction chamber layer are obtained by processing materials such as glass, PMMA, PC, PDMS and the like in a precise injection molding or machining mode.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention is a rapid detection tool with simple operation and low cost, and is suitable for rapid multi-index detection of various types of detection samples under various detection occasions.
(2) The nucleic acid amplification sample system sample injection pipeline layer and the nucleic acid amplification reaction chamber layer are obtained by processing various materials such as PMMA, PC and the like in a precise injection molding or machining mode, and the detection cost can be greatly reduced.
(3) The hydrophobic treatment is carried out on the sample introduction pipeline, so that the sample introduction integrity of the reaction chamber can be greatly improved, and the nucleic acid amplification system is stable and uniform in sample introduction. In addition, the cavity layer is divided by utilizing the operation of forward pumping or reverse oil passing, so that the problems of incomplete sub-cavity sample introduction, mutual interference among primers, external pollution and the like which possibly occur in the amplification process can be completely avoided.
(4) The chip sample injection process provided by the invention can be completely and manually injected by using a pipette or a dropper besides injection of an injection pump, the whole process does not need external complex instruments (such as a thermal cycler, a pump and the like) for supporting, the chamber-divided multi-index nucleic acid amplification detection can be realized within 1 hour, the final result can be directly interpreted, and the operation is simple, convenient and quick.
Description of the drawings:
for ease of illustration, the invention is described in detail by the following detailed description and the accompanying drawings.
FIG. 1 is a diagram of a chip architecture;
FIG. 2 is a diagram of a reaction chamber layer structure;
FIG. 3 is a view showing a structure of a sample introduction channel layer;
in the figure, 1, a sample injection pipeline layer of a nucleic acid amplification sample system, 2, a nucleic acid amplification reaction chamber layer, 3, a reaction chamber, 4, a secondary sample injection port, 5, a sample injection exhaust port, 6, a sample injection pipeline, 7, a chip sample injection port, 8, a sample outlet, 9, a liquid storage tank, 10 and a positioning hole.
The specific implementation mode is as follows:
the following detailed description of the present invention will be made in conjunction with the accompanying drawings, but the following examples are merely illustrative of preferred embodiments, which are provided to assist understanding of the present invention, and are not to be construed as limiting the present invention.
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout.
The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.
The first embodiment is as follows:
as shown in fig. 1 to 3, the following technical solutions are adopted in the present embodiment: a multi-index nucleic acid amplification micro-fluidic chip with chambers comprises a nucleic acid amplification sample system sample injection pipeline layer 1 and a nucleic acid amplification reaction chamber layer 2, wherein the nucleic acid amplification sample system sample injection pipeline layer 1 is fixedly connected with the nucleic acid amplification reaction chamber layer 2, the to-be-fixed surfaces of the nucleic acid amplification sample system sample injection pipeline layer 1 and the nucleic acid amplification reaction chamber layer 2 are provided with at least three positioning holes 10 for packaging and positioning;
at least two rows of reaction chambers 3 connected in series are arranged on the surface to be fixed of the nucleic acid amplification reaction chamber layer 2, the reaction chambers 3 have the same independent structure, and the bottom end of each reaction chamber 3 is sequentially provided with a secondary sample inlet 4 and a sample inlet and outlet 5;
a sample inlet pipeline 6 is arranged on the surface to be fixed of the sample inlet pipeline layer 1 of the nucleic acid amplification sample system, a chip sample inlet 7 is arranged at the head end of the sample inlet pipeline 6, a sample outlet 8 is arranged at the tail end of the sample inlet pipeline, and the chip sample inlet 7 and the sample outlet 8 both penetrate through the sample inlet pipeline layer 1 of the nucleic acid amplification sample system; sample introduction pipeline 6 will reaction chamber 3's second grade introduction port 4 and advance kind gas vent 5 communicate in proper order, before nucleic acid amplification sample system sample introduction pipeline layer 1 and nucleic acid amplification reaction chamber layer 2 fixed connection sample introduction pipeline 6 and advance kind gas vent 5 all need carry out hydrophobic treatment.
The micro-fluidic chip is also provided with a liquid storage tank 9, the liquid storage tank 9 is provided with an upper liquid storage cavity and a lower liquid storage cavity, the lower liquid storage cavity is arranged at the position close to the sample outlet 8 of the sample introduction pipeline 6 of the nucleic acid amplification sample system sample introduction pipeline layer 1, and the upper liquid storage cavity is arranged at the position corresponding to the lower liquid storage cavity of the nucleic acid amplification reaction cavity layer 2.
The surfaces to be fixed of the nucleic acid amplification sample system sample injection pipeline layer 1 and the nucleic acid amplification reaction chamber layer 2 are tightly packaged and bonded by using double-sided adhesive tapes, and each double-sided adhesive tape has a structure which is cut by using a cutting die and is the same as the nucleic acid amplification sample system sample injection pipeline layer 1 and the nucleic acid amplification reaction chamber layer 2.
3 bottom of reaction chamber is equipped with second grade introduction port 4 the first time, and introduction gas vent 5 is located to the bottom rear side, introduction gas vent 5 is less than far away second grade introduction port 4.
The nucleic acid amplification sample system sample injection pipeline layer 1 and the nucleic acid amplification reaction chamber layer 2 are both obtained by processing PMMA and PC materials in a precise injection molding or machining mode.
Example 2
As shown in fig. 1 to 3, the following technical solutions are adopted in the present embodiment: a multi-index nucleic acid amplification micro-fluidic chip with chambers comprises a nucleic acid amplification sample system sample injection pipeline layer 1 and a nucleic acid amplification reaction chamber layer 2, wherein the nucleic acid amplification sample system sample injection pipeline layer 1 is fixedly connected with the nucleic acid amplification reaction chamber layer 2, the to-be-fixed surfaces of the nucleic acid amplification sample system sample injection pipeline layer 1 and the nucleic acid amplification reaction chamber layer 2 are provided with at least three positioning holes 10 for packaging and positioning;
at least two rows of reaction chambers 3 connected in series are arranged on the surface to be fixed of the nucleic acid amplification reaction chamber layer 2, the reaction chambers 3 have the same independent structure, and the bottom end of each reaction chamber 3 is sequentially provided with a secondary sample inlet 4 and a sample inlet and outlet 5;
a sample inlet pipeline 6 is arranged on the surface to be fixed of the sample inlet pipeline layer 1 of the nucleic acid amplification sample system, a chip sample inlet 7 is arranged at the head end of the sample inlet pipeline 6, a sample outlet 8 is arranged at the tail end of the sample inlet pipeline, and the chip sample inlet 7 and the sample outlet 8 both penetrate through the sample inlet pipeline layer 1 of the nucleic acid amplification sample system; sample introduction pipeline 6 will reaction chamber 3's second grade introduction port 4 and advance kind gas vent 5 communicate in proper order, before nucleic acid amplification sample system sample introduction pipeline layer 1 and nucleic acid amplification reaction chamber layer 2 fixed connection sample introduction pipeline 6 and advance kind gas vent 5 all need carry out hydrophobic treatment.
The micro-fluidic chip is also provided with a liquid storage tank 9, the liquid storage tank 9 is provided with an upper liquid storage cavity and a lower liquid storage cavity, the lower liquid storage cavity is arranged at the position close to the sample outlet 8 of the sample introduction pipeline 6 of the nucleic acid amplification sample system sample introduction pipeline layer 1, and the upper liquid storage cavity is arranged at the position corresponding to the lower liquid storage cavity of the nucleic acid amplification reaction cavity layer 2.
The sample injection pipeline layer 1 of the nucleic acid amplification sample system and the surface to be fixed of the nucleic acid amplification reaction chamber layer 2 are bonded together by hot pressing.
3 bottom of reaction chamber is equipped with second grade introduction port 4 the first time, and introduction gas vent 5 is located to the bottom rear side, introduction gas vent 5 is less than far away second grade introduction port 4.
The nucleic acid amplification sample system sample injection pipeline layer 1 and the nucleic acid amplification reaction chamber layer 2 are both obtained by processing PMMA and PC materials in a precise injection molding or machining mode.
Example 3
The embodiment is that the micro-fluidic chip for multi-index nucleic acid amplification detection in chambers is designed to carry out isothermal amplification detection on nucleic acid of escherichia coli, staphylococcus aureus and klebsiella pneumoniae. The invention designs a micro-fluidic chip for multi-index nucleic acid amplification detection in different chambers to establish an isothermal amplification detection system for escherichia coli, staphylococcus aureus and klebsiella pneumoniae nucleic acid and perform amplification reaction, and the steps comprise:
(1) and (3) hydrophobic treatment: carrying out hydrophobic treatment on the whole sample introduction channel of the sample introduction pipeline layer and the sample introduction exhaust port of the reaction chamber layer;
(2) pre-spotting primer: because the current detection reagent only detects three sites, in order to verify the feasibility of the multiplex amplification reaction under the existing conditions, each site is repeated for 4 times by a repeated sample application mode, and other 4 chambers do not need to point primers and are used as negative controls;
(3) packaging the chip: bonding the sample injection pipeline layer of the nucleic acid amplification sample system, the nucleic acid amplification reaction chamber layer and the packaging double-sided adhesive tape to form a complete nucleic acid isothermal amplification chip, manually injecting the prepared nucleic acid isothermal amplification system (60 muL of the reaction system is prepared from KSH400H mix21.6 mu L, LAMP mixed primers of 6.0 muL, Bst2.0 enzyme of 2.4 muL and a sample of 30 muL) into the nucleic acid isothermal amplification chip, injecting the nucleic acid isothermal amplification system from a chip sample injection port, advancing the sample injection pipeline, performing hydrophobic treatment on the sample injection pipeline, wherein the liquid surface advancing resistance is larger due to the existence of liquid surface tension, the liquid surface advancing resistance is small due to the non-treatment of the reaction chamber layer, so that the liquid flows into the reaction chamber without further advancing, after the liquid discharges the gas in the reaction chamber to fill the reaction chamber, the next fluid is fine due to the fine gas outlet and is subjected to hydrophobic treatment, the resistance of the gas outlet is larger than that of the sample introduction pipeline, so that the nucleic acid isothermal amplification system does not flow out of the gas outlet but continuously advances from the sample introduction pipeline until entering the next reaction chamber, and the chamber sample introduction is realized;
(4) dividing the reaction chamber:
a. injecting oil into the sample inlet pipeline from a sample outlet of a sample inlet pipeline layer of the nucleic acid amplification sample system, wherein the oil is not influenced by hydrophobic treatment, and the resistance of a sample inlet exhaust port is greater than that of the sample inlet pipeline, so that oil bodies directly circulate in the sample inlet pipeline, and all the nucleic acid isothermal amplification system in the sample inlet pipeline is discharged, so that the reaction chamber division in the attached drawing is realized;
b. pumping gas into the sample inlet pipeline from a sample inlet of a sample inlet pipeline layer of the nucleic acid amplification sample system, wherein the gas is not influenced by hydrophobic treatment, liquid in the reaction chamber is full of the gas, and the resistance in the reaction chamber is greater than that of the sample inlet pipeline, so that the gas directly circulates in the sample inlet pipeline, and the nucleic acid isothermal amplification system in the sample inlet pipeline is completely discharged, so that the reaction chamber in the attached drawing is divided;
s5: nucleic acid isothermal amplification: putting the chip into a flat PCR instrument for amplification;
s6: and (4) interpretation of results: after the reaction is complete, the signal is read by fluorescence.
The multiple amplification reaction is feasible on the chip, namely the chamber-divided multi-index nucleic acid amplification detection device can well solve the problem of mutual interference between primers during multi-index detection.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. The utility model provides a minute chamber multi-index nucleic acid amplification micro-fluidic chip which characterized in that: the micro-fluidic chip comprises a nucleic acid amplification sample system sample injection pipeline layer (1) and a nucleic acid amplification reaction chamber layer (2), wherein the nucleic acid amplification sample system sample injection pipeline layer (1) is fixedly connected with the nucleic acid amplification reaction chamber layer (2);
at least one row of reaction chambers (3) connected in series are arranged on the surface to be fixed of the nucleic acid amplification reaction chamber layer (2), the reaction chambers (3) have the same independent structure, and the bottom end of each reaction chamber (3) is sequentially provided with a secondary sample inlet (4) and a sample inlet and outlet (5);
a sample introduction pipeline (6) is arranged on a surface to be fixed of the nucleic acid amplification sample system sample introduction pipeline layer (1), a chip sample introduction port (7) is arranged at the head end of the sample introduction pipeline (6), a sample outlet (8) is arranged at the tail end of the sample introduction pipeline, and the chip sample introduction port (7) and the sample outlet (8) both penetrate through the nucleic acid amplification sample system sample introduction pipeline layer (1); sample introduction pipeline (6) will second grade introduction port (4) and introduction gas vent (5) of reaction chamber (3) communicate in proper order, before nucleic acid amplification sample system introduction pipeline layer (1) and nucleic acid amplification reaction chamber layer (2) fixed connection introduction pipeline (6) and introduction gas vent (5) all carry out hydrophobic processing, and reaction chamber (3) carry out hydrophilic processing.
2. The chambered multi-index nucleic acid amplification microfluidic chip of claim 1, wherein: still be equipped with liquid storage tank (9) on the micro-fluidic chip, liquid storage tank (9) are equipped with about, the sample pipeline (6) of nucleic acid amplification sample system inlet pipe way layer (1) are located and are close to out appearance mouth (8) department down in liquid storage tank, go up the liquid storage tank and locate nucleic acid amplification reaction chamber layer (2) with the corresponding position in liquid storage tank down.
3. The chambered multi-index nucleic acid amplification microfluidic chip of claim 1, wherein: the sample injection pipeline layer (1) of the nucleic acid amplification sample system and the to-be-fixed surface of the nucleic acid amplification reaction chamber layer (2) are provided with at least three positioning holes (10) for packaging and positioning.
4. The chambered multi-index nucleic acid amplification microfluidic chip of claim 1, wherein: the surfaces to be fixed of the nucleic acid amplification sample system sample injection pipeline layer (1) and the nucleic acid amplification reaction chamber layer (2) are tightly packaged and bonded by using double-sided adhesive tapes, and the double-sided adhesive tapes have the structures which are cut by using a cutting die and are the same as the nucleic acid amplification sample system sample injection pipeline layer (1) and the nucleic acid amplification reaction chamber layer (2).
5. The chambered multi-index nucleic acid amplification microfluidic chip of claim 1, wherein: the sample introduction pipeline layer (1) of the nucleic acid amplification sample system and the surface to be fixed of the nucleic acid amplification reaction chamber layer (2) are bonded together by hot pressure.
6. The chambered multi-index nucleic acid amplification microfluidic chip of claim 1, wherein: reaction chamber (3) bottom is equipped with second grade introduction port (4) the previous time, and introduction gas vent (5) are located to the bottom rear side, introduction gas vent (5) are less than far away second grade introduction port (4).
7. The chambered multi-index nucleic acid amplification microfluidic chip of claim 1, wherein: the nucleic acid amplification sample system sample injection pipeline layer (1) and the nucleic acid amplification reaction chamber layer (2) are obtained by processing materials such as glass, PMMA, PC, PDMS and the like in a precise injection molding or machining mode.
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| CN111013676A (en) * | 2019-12-17 | 2020-04-17 | 江苏圣极基因科技有限公司 | Liquid drop preparation method and micro-fluidic chip |
| CN111363664A (en) * | 2020-04-24 | 2020-07-03 | 吉林医药学院 | LAMP detection chip based on three-layer microchip detection and control method thereof |
| CN113025476A (en) * | 2020-06-17 | 2021-06-25 | 山东大学 | Double-layer micro-fluidic chip, kit and method for detecting novel coronavirus |
| WO2021189064A1 (en) * | 2020-03-16 | 2021-09-23 | Siemens Healthcare Diagnostics Inc. | Sample holders, pcr station assemblies, and methods of operating pcr testing systems |
| CN113969232A (en) * | 2021-11-12 | 2022-01-25 | 南通大学 | Digital micro-fluidic chip device for nucleic acid detection and use method |
| CN114471759A (en) * | 2022-01-27 | 2022-05-13 | 盖秩舶 | Micro-fluidic chip based on polytetrafluoroethylene and glass and preparation method thereof |
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| WO2022174472A1 (en) * | 2021-02-19 | 2022-08-25 | 杭州梓晶生物有限公司 | Fully-integrated multi-index nucleic acid test microfluidic chip |
| WO2023060849A1 (en) * | 2021-10-12 | 2023-04-20 | 江苏汇先医药技术有限公司 | Lamp-based microfluidic chip |
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