CN108865821B - Nucleic acid isothermal amplification chip integrated with thermal cracking and use method - Google Patents
Nucleic acid isothermal amplification chip integrated with thermal cracking and use method Download PDFInfo
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- CN108865821B CN108865821B CN201810745533.0A CN201810745533A CN108865821B CN 108865821 B CN108865821 B CN 108865821B CN 201810745533 A CN201810745533 A CN 201810745533A CN 108865821 B CN108865821 B CN 108865821B
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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
Abstract
The invention relates to an integrated thermal cracking nucleic acid isothermal amplification chip, which comprises a separable cracking cell chip body, a detection cell chip body, a connecting piece and a buckle structure, and is used with a matched detector; during pretreatment, the chip body of the cracking pool and the chip body of the detection pool are in different temperature areas, and the pretreatment high temperature does not influence the reagents prestored behind. When the cracking cell chip body and the detection cell chip body are folded, the spine part of the second micro-flow channel on the detection cell chip body is inserted into the sealing sheet of the cracking cell chip body, so that the first micro-flow channel is communicated with the second micro-flow channel; the chip body of the cracking cell and the chip body of the detection cell are clamped through the clamping structure, so that liquid leakage is avoided, and the problems of mismatching, confusion and the like caused by the fact that the connecting piece is connected and cannot be completely separated during separation are solved. The whole process can not cause the failure of the detection reagent and the interference to the experimental result, thereby improving the detection efficiency.
Description
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a nucleic acid isothermal amplification chip integrating thermal cracking and a using method thereof.
Background
Microfluidic chips are an emerging technology for manipulating and detecting chemical or biological samples on a chip. The microfluidic chip can integrate operation units of sample preparation, reaction, separation, detection and the like on a chip with a few square centimeters, and automatically complete the analysis process by controlling and controlling the fluid in the microchannel network. The micro-fluidic chip technology has the characteristics of small sample volume, high integration level and easy realization of automatic control and high-throughput analysis, so that the biochemical reaction operation on the micro-fluidic chip is more convenient and rapid than the conventional analysis sample pretreatment, and the cost is low.
The existing microfluidic chip is basically integrated, but in practical application, different temperatures are often used for pretreatment, reaction, detection and the like of a sample, reagents are often pre-stored in the microfluidic chip, the reagents are not high in temperature, and the high temperature is likely to be used for pretreatment, so that the design of the integrated chip has many difficulties. Considering that different temperature can set different temperature control areas for the matched instrument, different parts of the same chip are placed in different areas of the instrument through separation, so that the failure of the subsequent thermolabile reaction detection reagent during pretreatment can be avoided, but the chip is generally required to be integrated for the convenience of actual use and the sealing property during reagent storage.
Isothermal nucleic acid amplification techniques are a generic term for a class of molecular biology techniques that amplify a specific DNA or RNA at a specific temperature. Compared with the PCR technology, the isothermal amplification of nucleic acid greatly simplifies the requirements of instruments, greatly shortens the reaction time and can meet the requirements of rapidness, simplicity and convenience. The nucleic acid isothermal amplification technology is combined with a microfluidic chip, partial products such as a Boo respiratory tract pathogenic bacterium nucleic acid detection kit exist, but pretreatment and the like of a nucleic acid template are carried out outside, the integration level of the chip is low, and manual operation is complicated. In addition, there is another report that the magnetic bead method is used for extracting nucleic acid and integrating the nucleic acid into a chip, but no actual product is found, and the magnetic bead method is high in cost. The existing nucleic acid template rapid extraction methods, such as a glass bead method and a nucleic acid directional releasing agent method, have simpler steps, and the extracted template is completely suitable for rapid detection, but the extraction process needs high-temperature heating, and the detection reagent cannot tolerate high temperature, and if the reagent is prestored on a chip, the reagent will lose efficacy, which is a difficulty in designing a nucleic acid rapid detection chip.
Disclosure of Invention
In view of the above problems, the present invention provides an isothermal nucleic acid amplification chip with integrated thermal cracking, wherein the chip is designed to be separable and used with a matched detector, during pretreatment, different parts of the chip are in different temperature regions, and the pretreatment high temperature does not affect the reagents pre-stored in the back. The chip can be folded integrally or separated into different parts.
The embodiment of the invention provides an integrated thermal cracking nucleic acid isothermal amplification chip, which comprises:
the device comprises a cracking cell chip body, wherein a sample inlet hole, a cracking cell, a first micro-flow channel and a sealing sheet for sealing the outlet end of the first micro-flow channel are arranged on the cracking cell chip body;
the detection cell chip body is provided with a second micro-flow channel, a detection cell and a micro-pump interface; a sharp thorn part is arranged at the inlet end of the second micro-flow channel;
the connecting piece is used for connecting the chip body of the cracking cell and the chip body of the detection cell into a whole;
the clamping structure is used for clamping the cracking cell chip body and the detection cell chip body together; when the cracking cell chip body and the detection cell chip body are clamped together, the outlet end of the first microflow channel is communicated with the inlet end of the second microflow channel.
In one embodiment, one end of the connecting piece is connected to the chip body of the lysis cell, and the other end of the connecting piece is connected to the chip body of the detection cell;
the connecting piece is a telescopic pull rod or an elastic rib.
In one embodiment, the snap structure includes: the clamping pin and the clamping groove are matched with the clamping pin;
the clamping pins are arranged on the chip body of the cracking cell, and the clamping grooves are arranged on the chip body of the detection cell;
or
The clamping foot is arranged on the detection cell chip body, and the clamping groove is arranged on the cracking cell chip body.
In one embodiment, a chip fixing member is disposed on the chip body of the lysis cell and/or the chip body of the detection cell.
In one embodiment, the chip body of the lysis cell is further provided with a vent hole for connecting the lysis cell.
In one embodiment, the lysis cell chip body and the detection cell chip body are made of any one of the following materials:
glass, quartz, polycarbonate, polymethyl methacrylate, and polydimethylsiloxane.
In a second aspect, the embodiments of the present invention further provide a method for using an integrated thermally cracked nucleic acid isothermal amplification chip, including:
the method comprises the following steps: sample adding: adding the liquid to be detected into a cracking pool of the chip body of the cracking pool through the sample inlet hole, and sealing the sample inlet hole by using a breathable hydrophobic membrane or a cover;
step two: placing a chip: putting the chip into a specific area of a corresponding detector for cracking treatment;
step three: folding the chip: the detector automatically aligns and folds the separated chip body of the cracking cell and the chip body of the detection cell, the spine part of the chip body of the detection cell is inserted into the sealing sheet of the chip body of the cracking cell, and the first microflow channel is communicated with the second microflow channel and is clamped tightly by the clamping structure;
step four: and the detector micropump sucks the liquid in the cracking pool to the detection pool or transfers the liquid in the cracking pool to the detection pool through centrifugation, and corresponding reaction detection is carried out.
Compared with the prior art, the embodiment of the invention has the beneficial effects that: the chip comprises a separable cracking cell chip body and a detection cell chip body, and is used together with a matched detector, when in pretreatment, the cracking cell chip body and the detection cell chip body are in different temperature areas, and the pretreatment high temperature does not influence the reagent prestored at the back. The chip can be folded integrally or separated into different parts. Through the separated design, different parts of the chip, the chip body of the cracking pool and the chip body of the detection pool are placed in different areas of the instrument, so that the failure of the rear heat-labile reaction detection reagent caused during pretreatment can be avoided, and the detection efficiency is improved.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of the separation of an integrated thermally cleaved nucleic acid isothermal amplification chip according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a closed structure of FIG. 1 according to an embodiment of the present invention;
FIG. 3 is an elevation view of the chip interface in the direction A-A of FIG. 1;
FIG. 4 is an enlarged view of a portion of the interface of FIG. 1 in accordance with an embodiment of the present invention;
FIG. 5 is a schematic diagram of the separation of another integrated thermal-cleavage isothermal nucleic acid amplification chip according to the present invention;
FIG. 6 is a schematic diagram of the closed structure of FIG. 5 according to an embodiment of the present invention;
wherein: 1-a cracking cell chip body, 11-a sample inlet hole, 12-a cracking cell, 13-a first micro-flow channel, 14-a sealing sheet, 15-an air vent, 2-a detection cell chip body, 21-a second micro-flow channel, 22-a detection cell, 23-a micro-pump interface, 24-a spine part, 3-a connecting piece, 4-a buckle structure, 41-a clamping pin, 42-a clamping groove and 5-a chip fixing piece.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The embodiment of the invention provides an integrated thermal cracking nucleic acid isothermal amplification chip, which is shown in a figure 1-2 and comprises: the device comprises a cracking cell chip body 1, a detection cell chip body 2, a connecting piece 3 and a buckle structure 4;
the cracking cell chip body 1 is provided with a sample inlet hole 11, a cracking cell 12, a first micro-flow channel 13 and a sealing sheet 14, and the sealing sheet 14 is used for sealing the outlet end of the first micro-flow channel 13;
the detection cell chip body 2 is provided with a second micro-flow channel 21, a detection cell 22 and a micro-pump interface 23; the inlet end of the second micro-flow channel 21 is provided with a spike part 24;
the two chip bodies are connected together through the connecting piece 3, so that the marking function can be achieved, and the two chip bodies belong to the same chip; a buckle structure is arranged on an interface at the joint of the chip body of the cracking pool and the chip body of the detection pool; when the cracking cell chip body and the detection cell chip body are clamped together, the sharp part at the inlet end of the second micro-flow channel pierces the sealing sheet at the outlet end of the first micro-flow channel, so that the first micro-flow channel is communicated with the second micro-flow channel.
In this embodiment, for example, for a chip requiring temperature zone control, the integrated thermal-cracking isothermal nucleic acid amplification chip provided by the present invention can be used. For example, the isothermal nucleic acid amplification detection can be performed by using the integrated thermal cracking isothermal nucleic acid amplification chip of the present invention, wherein the temperature of the template extraction is usually 70-100 ℃ (operation in the chip body of the lysis cell), and the detection temperature is generally lower than 70 ℃ (operation in the chip body of the detection cell). If the detection reagent is also at a high temperature of 70 ℃ or higher during the template extraction process (detection cell chip body), the failure is likely to occur, resulting in erroneous results.
Therefore, the integrated thermal cracking nucleic acid isothermal amplification chip disclosed by the invention is designed into a split type as shown in FIG. 1, and can be connected into a whole through a connecting piece, and a chip body of a cracking cell, namely a microfluidic channel port of a pretreatment part, is sealed by a sealing sheet; referring to fig. 2, when the sealing sheet is folded (the detection cell chip body), the spike part of the second microfluidic channel port of the reaction detection part is inserted into the sealing sheet of the pretreatment part (the cleavage cell chip body), as shown in fig. 3, so that the first microfluidic channel is communicated with the second microfluidic channel, that is: make the micro-flow channel intercommunication of pretreatment part and reaction detection part, the schizolysis cell chip body passes through the buckle structure chucking with the detection cell chip body, and the weeping is prevented, is linked by the connecting piece when separately and is unlikely to separate completely and cause the problem of mismatching, mixing up etc..
Namely: when the liquid to be detected is extracted at high temperature, the chip body of the detection cell can be far away from the high-temperature environment; and when the extracting solution is detected after the extraction is finished, the separate cracking cell chip body and the detection cell chip body can be folded into a whole, and the detection is finished in a low-temperature environment. The whole process can not cause the invalidation of the liquid to be detected and can not cause the interference to the experimental result, thereby improving the detection efficiency.
Furthermore, one end of the connecting piece 3 is connected to the chip body of the cracking cell, the other end is connected to the chip body of the detection cell, and the connecting piece can be a telescopic pull rod or an elastic rib; the form of the connecting piece is not limited to the above two modes, and other connecting pieces can play a role in marking, which indicates that the two connecting modes or the connecting pieces belong to the chip, and both the connecting modes and the connecting pieces belong to the protection scope of the invention.
Referring to fig. 4, it is an enlarged schematic view of an interface between the chip body of the lysis cell and the chip body of the detection cell in fig. 1, wherein the fastening structure 4 includes a fastening pin 41 and a fastening groove 42 adapted to the fastening pin 41; when the clamping pin 41 is arranged on the chip body 1 of the cracking cell, the clamping groove 42 is arranged on the chip body 2 of the detection cell; or when the clamping pin 41 is arranged on the detection cell chip body 2, the clamping groove 42 is arranged on the lysis cell chip body 1.
After the liquid is waited to detect in the extraction of the schizolysis cell chip body, when needing to carry out follow-up detection, through buckle structure 4 with the schizolysis cell chip body 1 with detect cell chip body 2 joint together, the spine portion 24 that detects cell chip body 2 simultaneously inserts the gasket 14 of the schizolysis cell chip body 1, realizes that first miniflow passageway 13 and second miniflow passageway 21 are linked together, guarantees to wait to detect the liquid and can not leak.
Further, referring to fig. 5-6, in order to facilitate fixing the integrated thermal-cracked nucleic acid isothermal amplification chip on a detector, or to facilitate clamping the chip, a chip holder 5 may be disposed on the cracking cell chip body 1, or a chip holder 5 may be disposed on the detection cell chip body 2; or chip fixing pieces 5 are arranged on the chip body 1 of the cracking cell and the chip body 2 of the detection cell at the same time.
In this embodiment, the chip fixing member 5 may be a fixing hole as shown in fig. 5, a fixing groove, or a protruding column, and in this embodiment, the shape, size, and position of the chip fixing member are not limited, and any member may be used as long as it is used in cooperation with a detector or a clamping tool to perform a fixing function.
Further, as shown in FIGS. 5 to 6, a vent hole 15 for connecting the lysis cell 12 may be provided in the lysis cell chip body 1 in order to facilitate the filling of the liquid to be detected from the inlet hole 11. The integrated thermal cracking nucleic acid isothermal amplification chip provided by the embodiment of the invention can be made of any one of glass, quartz, polycarbonate, polymethyl methacrylate and polydimethylsiloxane. In addition, the overall shape of the chip is not limited, and the chip can be designed into various shapes according to specific requirements; for example, when the micro-fluidic chip for controlling liquid inlet by the micro-pump is shown in fig. 1-2, the micro-fluidic chip is in a round corner rectangle shape; for another example, the centrifugal separable microfluidic chip, as shown in fig. 5-6, is approximately fan-shaped when folded. In addition, according to the specific detection function of the chip, a plurality of lysis cells or detection cells can be arranged on the chip, which is not limited in the embodiment of the present invention.
In a second aspect, the invention also provides a method for using the integrated thermally cleaved nucleic acid isothermal amplification chip:
example 1:
taking a micro-fluidic chip with a micro pump controlling liquid feeding as an example, the chip can be designed into an isothermal amplification chip integrating thermal cracking nucleic acid, as shown in fig. 1-2, the overall shape of the chip is a rounded rectangle.
The using method comprises the following steps:
1. sample adding: adding the liquid to be detected into the microfluidic chip cracking pool through the sample inlet hole, and sealing the sample inlet hole by using a breathable hydrophobic membrane or a cover;
2. placing a chip: putting the micro-fluidic chip into a specific area of a corresponding detector for cracking treatment;
3. folding the chip: the detector automatically aligns and folds the separated chip part, a sharp thorn part at the front end of a microfluidic channel of the reaction detection part is inserted into a sealing sheet of the pretreatment part, the channel is communicated, and the chip is clamped tightly;
4. and the detector micropump sucks the liquid in the cracking pool to the detection pool to perform corresponding reaction detection.
Example 2:
taking a centrifugal separable microfluidic chip as an example, the chip can be designed into an integrated thermal cracking isothermal nucleic acid amplification chip provided by the present invention, and as shown in fig. 5 to 6, the overall shape of the chip is approximately a sector.
The use steps are as follows:
1. sample adding: adding the liquid to be detected into the microfluidic chip cracking pool from the sample inlet hole, and sealing the sample inlet hole and the vent hole by using a breathable hydrophobic membrane or a cover;
2. placing a chip: putting the micro-fluidic chip into a specific area of a corresponding detector for cracking treatment;
3. folding the chip: the detector automatically aligns and folds the separated chips, the tip of the front end of the microfluidic channel of the reaction detection part is inserted into the sealing sheet of the pretreatment part, the channels are communicated, and the buckle is clamped tightly;
4. the detector transfers the liquid in the cracking pool to the detection pool by centrifugation for corresponding detection.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (7)
1. An integrated thermally cleaved nucleic acid isothermal amplification chip, comprising:
the device comprises a cracking cell chip body (1), wherein a sample inlet hole (11), a cracking cell (12), a first micro-flow channel (13) and a sealing sheet (14) are formed in the cracking cell chip body (1); two ends of the cracking cell (12) are respectively communicated with one first microflow channel (13); wherein the inlet end of one of the first microfluidic channels (13) is communicated with the sample inlet (11); the outlet end of the other first micro-flow channel (13) is provided with the sealing sheet (14);
the detection cell chip body (2), wherein a second micro-flow channel (21), a detection cell (22), a micro-pump interface (23) and a spike part (24) are arranged on the detection cell chip body (2); two ends of the detection cell (22) are respectively communicated with one second microfluidic channel (21); wherein the outlet end of one second micro-flow channel (21) is communicated with the micro-pump interface (23); the inlet end of the other second micro-flow channel (21) is provided with the sharp thorn part (24);
the connecting piece (3) is used for connecting the chip body (1) of the cracking cell and the chip body (2) of the detection cell into a whole;
the buckle structure (4) is used for clamping the cracking cell chip body (1) and the detection cell chip body (2) together; when the cracking cell chip body (1) and the detection cell chip body (2) are clamped together, the outlet end of the first microflow channel (13) provided with the sealing sheet (14) is communicated with the inlet end of the second microflow channel (21) provided with the spine part (24).
2. An integrated thermal-cracking nucleic acid isothermal amplification chip according to claim 1, wherein the connecting member (3) is connected to the cracking cell chip body (1) at one end and connected to the detection cell chip body (2) at the other end;
the connecting piece (3) is a telescopic pull rod or an elastic rib.
3. The integrated thermal-cracking isothermal nucleic acid amplification chip according to claim 1, wherein the snap structure (4) comprises: the clamping device comprises a clamping pin (41) and a clamping groove (42) matched with the clamping pin (41);
the clamping pins (41) are arranged on the cracking cell chip body (1), and the clamping grooves (42) are arranged on the detection cell chip body (2);
or
The clamping foot (41) is arranged on the detection cell chip body (2), and the clamping groove (42) is arranged on the cracking cell chip body (1).
4. The integrated thermal cracking nucleic acid isothermal amplification chip according to claim 1, wherein a chip holder (5) is disposed on the chip body (1) of the cracking cell and/or the chip body (2) of the detection cell.
5. The integrated thermal-cracking nucleic acid isothermal amplification chip according to claim 1, wherein the cracking cell chip body (1) is further provided with a vent hole (15) for connecting the cracking cell (12).
6. The integrated thermal-cracking nucleic acid isothermal amplification chip according to any one of claims 1 to 5, wherein the material of the cracking cell chip body (1) and the detection cell chip body (2) is any one of the following materials:
glass, quartz, polycarbonate, polymethyl methacrylate, and polydimethylsiloxane.
7. A method of using the integrated thermally cleaved nucleic acid isothermal amplification chip according to any one of claims 1-6, comprising:
the method comprises the following steps: sample adding: adding the liquid to be detected into a cracking pool of the chip body of the cracking pool through the sample inlet hole, and sealing the sample inlet hole by using a breathable hydrophobic membrane or a cover;
step two: placing a chip: putting the chip into a specific area of a corresponding detector for cracking treatment;
step three: folding the chip: the detector automatically aligns and folds the separated chip body of the cracking cell and the chip body of the detection cell, the spine part of the chip body of the detection cell is inserted into the sealing sheet of the chip body of the cracking cell, and the first microflow channel is communicated with the second microflow channel and is clamped tightly by the clamping structure;
step four: and the detector micropump sucks the liquid in the cracking pool to the detection pool or transfers the liquid in the cracking pool to the detection pool through centrifugation, and corresponding reaction detection is carried out.
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| CN112414983B (en) * | 2020-11-05 | 2021-08-06 | 北京中科生仪科技有限公司 | Biological detection method based on excitation light source |
| CN114113567A (en) * | 2020-11-26 | 2022-03-01 | 深圳市理邦精密仪器股份有限公司 | Molecular diagnosis centrifugal test card |
| CN113174307B (en) * | 2021-01-15 | 2022-02-15 | 北京中科生仪科技有限公司 | Upper cover based on nucleic acid detection chip installation bin |
| CN113175466B (en) * | 2021-01-29 | 2022-06-28 | 北京中科生仪科技有限公司 | Fastening device |
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