CN209741124U - Array chip device for digital nucleic acid molecule micro-quantification - Google Patents
Array chip device for digital nucleic acid molecule micro-quantification Download PDFInfo
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Abstract
The utility model relates to a detection device in a plurality of fields of life science, medicine and the like, in particular to an array chip device for digital nucleic acid molecule micro-quantification; the reaction unit comprises a substrate and a cover sealing plate, wherein the cover sealing plate is sealed on the substrate, the back surface of the cover sealing plate is of a planar structure, and the reaction unit is protruded on the substrate; or a groove is arranged on the substrate, and the reaction unit is embedded in the groove; after the cover plate is covered on the substrate, a sample channel is formed between the cover plate and the substrate; the sealing plate is provided with a sample inlet and a sample outlet; the sample inlet is connected with a sample adding system, and the sample outlet is connected with a vacuum system. The array chip device can distribute trace liquid samples to hundreds of independent reaction units in a short time only by vacuumizing through the sample outlet and carrying out positive pressure sample injection through the sample inlet, thereby greatly improving the experimental speed and remarkably improving the trace efficiency of the samples.
Description
Technical Field
The invention belongs to detection devices in multiple fields of life science, medicine and the like, and particularly relates to an array chip device for digital nucleic acid molecule micro-quantification.
Background
In recent years, new technological breakthroughs are continuously made in the field of life science. In 1973, Qianjin, Taiwan scientist, discovered a stable Taq DNA polymerase. The invention of the polymerase chain reaction in 1985, Mullis in the United states, is a molecular biology technology for amplifying specific DNA fragments, which can be regarded as a special DNA replication in vitro, i.e., a simple DNA amplification method, and means the real birth of the PCR technology. The polymerase chain reaction is always one of the core technologies in the field of molecular biology, has been applied to the research fields of molecular sequencing, gene expression analysis, gene mutation research, early disease molecular diagnosis, single nucleotide polymorphism, drug screening and the like, and plays an important role. In particular, in the early molecular diagnosis of cancer, pathogenic infectious diseases and other diseases, a quantitative nucleic acid detection technique based on nucleic acid amplification can amplify DNA in vitro by PCR until the concentration of the target DNA is sufficient for detection, as long as a single-point DNA can be isolated. The method has the obvious advantages of high analysis speed, high sensitivity, high flux and short diagnosis time, is one of the main application technologies for early diagnosis of diseases, and has wide development space.
The technology of polymerase chain reaction has been developed for over thirty years. The fluorescent quantitative PCR technique (qPCR) was first reported and described in 1993. In 1995, the qPCR instrument was first invented and marketed by PerkinElmer corporation of America. The qPCR technology is that a double-fluorescence labeling probe which is complementary with a target sequence, namely a molecular beacon, is added into a common PCR reaction system, a PCR product is reported by a molecular beacon fluorescence signal, the strength of the molecular beacon is continuously enhanced along with the PCR amplification process and is in direct proportion to the quantity of the PCR product, the fluorescence intensity is measured by a specific fluorescence spectrum analyzer, the change of the logarithmic phase, the linear phase and the platform phase of the PCR amplification can be dynamically and quantitatively observed in real time, and the change is compared with a standard curve of a standard positive quantitative gradient sample, so that the initial copy quantity of an object to be detected is calculated. Therefore, the qPCR technique is a relative nucleic acid quantification method, the sensitivity and accuracy of which are limited, and nucleic acids cannot be counted at the macromolecular level, single molecule detection cannot be achieved, and absolute quantification is not achieved.
Vogelstein and Klstein proposed the concept of digital PCR (dPCR) by dividing a sample into tens to tens of thousands, assigning to different reaction units each containing one or more copies of a target molecule (DNA template), PCR amplifying the target molecule in each reaction unit, respectively, and statistically analyzing the fluorescent signal of each reaction unit after the amplification is finished. Different from qPCR, digital PCR does not depend on CT value, so that the method is not influenced by amplification efficiency, the average concentration (content) of each reaction unit is calculated by direct counting or a Poisson distribution formula after amplification is finished, the error can be controlled within 5%, and absolute quantitative analysis can be realized by digital PCR without reference to a standard sample and a standard curve.
The digital PCR technology is proposed so far, and the related technology and industrialization are rapidly developed. 2011, zhejiang university provides an integrated flow path chip device for digital nucleic acid amplification, which processes a sample channel in a reaction group, then etches thousands of pL-to-nL reaction cells at the bottom of the sample channel, and sets sample inlets and sample outlets at two ends of the reaction group. Since the reaction cell has a very small volume, the sample is introduced into the reaction cell by the gravity of the sample or the pressure of the inlet and outlet of the sample after the sample is injected, which results in a low probability of the sample entering the reaction cell and thus in a low utilization rate of the reaction cell. Even if thousands of small holes are etched, in practical application, the small holes used effectively do not meet the requirement of the digital PCR for reflecting the total number of units, so that the commercialization is difficult.
Disclosure of Invention
The invention aims to provide an array chip device for digital nucleic acid molecule micro-quantization, which has reasonable structural design, can perform negative pressure sampling through vacuumizing a sample outlet or perform positive pressure sampling through a sample inlet, obviously improves the micro-quantization efficiency of samples and improves the accuracy of experiments.
The above purpose is realized by the following technical scheme: an array chip device for digital nucleic acid molecule micro-quantization comprises a substrate, a sealing cover plate and a plurality of reaction units, wherein the sealing cover plate is sealed on the substrate, the back surface of the sealing cover plate is of a planar structure, and the reaction units are protruded on the substrate; or a groove is arranged on the substrate, and the reaction unit is embedded in the groove; after the cover plate is covered on the substrate, a sample channel is formed between the cover plate and the substrate; the cover plate is provided with a sample inlet and a sample outlet; the sample inlet is connected with a sample adding system, and the sample outlet is connected with a vacuum system; and the sample inlet, the sample channel and the sample outlet are communicated.
Furthermore, the sample adding system is provided with a sample and generates positive pressure, and under the action of the positive pressure, the sample moves towards the direction of the sample outlet in the sample channel.
Further, the vacuum system generates negative pressure, and the sample moves towards the sample outlet in the sample channel under the action of the negative pressure.
Further, one reaction unit is one reaction factor or a plurality of reaction factors; if the reaction unit has a plurality of reaction factors, the plurality of reaction factors form a triangle, a circle or a polygon.
Further, the substrate is made of glass.
Furthermore, the cover plate is made of glass materials, and the sample inlet and the sample outlet are sealed after the cover plate covers the substrate.
In the invention, the array chip device comprises a substrate, a cover plate and a plurality of reaction units; after the cover plate is covered on the base plate, a sample channel is formed between the base plate and the cover plate; the sealing plate is provided with a sample inlet and a sample outlet; the sample inlet is connected with a sample adding system, and the sample outlet is connected with a vacuum system; the sample inlet, the sample channel and the sample outlet are communicated; the sample adding system is provided with a sample and generates positive pressure, and the sample moves towards the direction of the sample outlet in the sample channel under the action of the positive pressure; therefore, the array chip device can distribute trace liquid samples to hundreds of independent reaction units in a short time only by vacuumizing the sample outlet to carry out negative pressure or positive pressure sample injection through the sample inlet, thereby greatly improving the experimental speed and remarkably improving the trace efficiency of the samples.
Drawings
FIG. 1 is a schematic diagram showing the structure of an array chip device for digital nucleic acid molecule micro-quantification according to the present invention;
FIG. 2 is a schematic structural view showing another embodiment of an array chip device for the digital nucleic acid molecule micro-quantification according to the present invention;
FIG. 3 shows a schematic diagram of a reaction set of the present invention before sample application;
FIG. 4 shows a schematic diagram of a reaction set of the present invention after sample application.
Detailed Description
in the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, 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 in specific cases to those skilled in the art.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
FIG. 1 is a schematic diagram of an array chip device for digital nucleic acid molecule quantification according to the present invention; the array chip device comprises a substrate 2, a cover plate 5 and a plurality of reaction units 1, wherein the cover plate 5 is covered on the substrate 2 in a sealing mode, the back face of the cover plate 5 is of a planar structure, and the reaction units 1 protrude from the substrate 2.
Or a groove is arranged on the substrate 2, and the reaction unit 1 is embedded in the groove; FIG. 2 is a schematic structural diagram of another embodiment of an array chip device for digital nucleic acid molecule quantification according to the present invention; after the cover plate 5 is covered on the base plate 2, a sample channel 6 is formed between the cover plate 5 and the base plate 2; the sample inlet 7 and the sample outlet 4 are arranged on the sealing plate 5; the sample inlet 7 is connected with a sample adding system 8, and the sample outlet 4 is connected with a vacuum system 3; the sample inlet 7, the sample channel 6 and the sample outlet 4 are communicated.
As a preferred technical scheme, the sample adding system 8 is provided with a sample and generates positive pressure, and under the action of the positive pressure, the sample moves towards the direction of the sample outlet 4 in the sample channel; the vacuum system 3 generates negative pressure, and the sample moves towards the sample outlet 4 in the sample channel under the action of the negative pressure.
In the invention, one reaction unit 1 is a reaction factor 11 or a plurality of reaction factors 11; when the reaction unit 1 protrudes on the substrate 2, the reaction unit 1 is composed of a columnar structure; if the reaction unit is a plurality of reaction factors 11, the plurality of reaction factors 11 form a triangular, circular or polygonal shape; exemplarily, as shown in fig. 3, which is a schematic structural diagram of a reaction unit of the present invention before sample application, and as shown in fig. 4, which is a schematic structural diagram of a reaction unit of the present invention after sample application, it can be seen that one reaction unit 1 includes three reaction factors 11, and in the shape of a triangle formed by the three reaction factors 11, sample solution is filled between the three reaction factors 11. If the reaction unit 1 is embedded in the groove, the pit-like structure of the groove can be a round pit or a square pit.
Exemplarily, the substrate 2 is made of glass material, the reaction unit 1 is a cylindrical structure, in the present invention, the substrate 2 uses 2mm thick glass as material, the reaction unit 1 is made by standard photolithography and etching technology, the reaction unit 1 is a cylindrical structure with a diameter of 10um, 20um, 30um, or 100um, etc., and a height of 50um,100um,200um, or 300um, etc., if the reaction group is triangular, it can be triangular arrangement with a side length of 100um,200um, or 300 um.
According to different columnar structures and arrangement forms, the reaction unit 1 can realize volumes from pL to nL; hydrophilic treatment is performed on the columnar structure of the reaction unit 1, and superhydrophobic treatment is performed in a sample channel other than the reaction unit 1.
the cover plate 5 is made of glass with the thickness of 1mm, and the surface connected with the sample channel is subjected to super-hydrophobic treatment. The cover plate 5 is a flat plate, and the diameters of the positions of the sample inlet 7 and the sample outlet 4 are both 1 mm. According to the invention, a cover plate 5 and a substrate 2 are sealed by adopting an air plasma processing bonding method, a sample channel 6 is formed between grooves of the cover plate 5 and the substrate 2, and a high-density integrated reaction unit 1 is sealed in the sample channel, wherein the cover plate 1 is made of a glass material, after the cover plate 5 is covered on the substrate 2, a sample inlet 7 and a sample outlet 4 are sealed, and a gummed paper tape is torn when the tape needs to use the sample inlet and the sample outlet.
When the sample feeding system 8 and the vacuum system 3 are used, the sample feeding system 8 and the vacuum system 3 can be used independently or used together, positive pressure generated by the sample feeding system 8 pushes a sample to move forwards in a sample channel, and negative pressure generated by the vacuum system 3 pulls the sample to move forwards in the sample channel. A certain amount of sample solution enters the sample channel 6 and the reaction group 9 on the substrate 2 through the sample inlet 7, after the sample injection is finished, mineral oil is injected into the sample channel 6 through the sample inlet 7, and under the action of positive pressure at the end of the sample inlet 7 or negative pressure at the end of the sample outlet 4, the mineral oil and the sample solution continuously flow forwards until the mineral oil is filled in the whole sample channel 6.
Due to the super-hydrophobicity of the sample channel and the hydrophilicity of the reaction unit 1, the spacing region between the reaction unit 1 and the reaction unit 1, namely the gap of the sample channel 6, can be occupied by the mineral oil, and the sample solution adsorbed only due to the hydrophilicity of the reaction unit 1 is left at the reaction unit 1, so that the solutions of different reaction units are spaced, namely, the samples are separated into thousands of reaction units of pL to nL grade, and the mineral oil covers the periphery and the top of the reaction unit 1, so that the evaporation of the solution in the subsequent reaction process can be prevented. And finally, sealing the sample inlet 7 and the sample outlet 4 by heat-resistant glue or curing glue.
When nucleic acid is amplified, the chip is placed on a hot plate of a PCR instrument, heat transfer grease and the like are added between the chip and the hot plate, the heat conductivity of the hot plate and the chip is increased, amplification is carried out according to a standard PCR amplification process, and because fluorescent markers which are only combined with double-stranded DNA, such as Gel and SYBR series, are added in a sample solution, a reaction unit which carries out nucleic acid amplification presents the color of the fluorescent markers, marks the fluorescence markers as positive, marks the fluorescence markers without the color of the fluorescent markers as negative, and then the initial quantity of the nucleic acid template can be accurately quantified by counting the positive reaction unit for statistical analysis.
Therefore, in the present invention, the shape of the reaction group may be formed by various arrangements such as a single arrangement, two arrangements, a triangular arrangement, a rhombic arrangement, a polygonal arrangement, and the like of a triangular pillar-shaped, a polygonal pillar-shaped, a cylindrical pillar-shaped, and a special-shaped pillar-shaped protrusion structure, and the reaction unit may be subjected to a hydrophilic treatment and a superhydrophobic treatment on a sample on the substrate except for the reaction unit. Firstly, injecting a mixture of a sample and a reagent, then injecting mineral oil, and pushing the mixture of the sample and the reagent to advance in a channel by utilizing positive pressure or negative pressure, wherein the mixture of the sample and the reagent is gradually attached to each reaction unit of a hydrophilic group in the advancing process due to the hydrophilicity of the reaction units and the hydrophobic property of the sample channel, and the mineral oil is filled in the sample channel so as to isolate the reaction units; therefore, the invention only needs to vacuumize through the sample outlet to carry out negative pressure or sample introduction through the sample inlet to carry out positive pressure sample introduction, and the trace liquid samples can be distributed to hundreds of independent reaction units in a short time through the flushing and covering of the mineral oil, thereby greatly improving the experimental speed. In addition, the distribution of the sample and the reagent is finished in the chip and is not in contact with the outside, so that the external pollution and the cross contamination can be effectively prevented.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (6)
1. an array chip device for digital nucleic acid molecule micro-quantization is characterized in that the array chip device comprises a substrate, a cover plate and a plurality of reaction units, wherein the cover plate is covered on the substrate, the back surface of the cover plate is of a planar structure, and the reaction units are protruded on the substrate; or a groove is arranged on the substrate, and the reaction unit is embedded in the groove; after the cover plate is covered on the substrate, a sample channel is formed between the cover plate and the substrate; the cover plate is provided with a sample inlet and a sample outlet; the sample inlet is connected with a sample adding system, and the sample outlet is connected with a vacuum system; and the sample inlet, the sample channel and the sample outlet are communicated.
2. The array chip device for digital nucleic acid molecule quantification of claim 1, wherein the sample application system is provided with a sample and generates a positive pressure, and the sample moves in the sample channel toward the sample outlet under the action of the positive pressure.
3. The array chip device for digital nucleic acid molecule micro-quantification as claimed in claim 1, wherein the vacuum system generates negative pressure, and the sample moves in the sample channel toward the sample outlet under the action of the negative pressure.
4. The array chip device for the digital nucleic acid molecule micro-quantification according to claim 2 or 3, wherein one reaction unit is one reaction factor or a plurality of reaction factors; if the reaction unit has a plurality of reaction factors, the plurality of reaction factors form a triangle, a circle or a polygon.
5. The array chip device for the digital nucleic acid molecule quantification of claim 4, wherein the substrate is made of glass.
6. The array chip device for digital nucleic acid molecule quantification of claim 5, wherein the cover plate is made of glass, and the sample inlet and the sample outlet are sealed after the cover plate is covered on the substrate.
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| CN201822146777.5U CN209741124U (en) | 2018-12-20 | 2018-12-20 | Array chip device for digital nucleic acid molecule micro-quantification |
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| CN201822146777.5U CN209741124U (en) | 2018-12-20 | 2018-12-20 | Array chip device for digital nucleic acid molecule micro-quantification |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304072A (en) * | 2020-02-28 | 2020-06-19 | 宁波胤瑞生物医学仪器有限责任公司 | Oil seal device for digital PCR chip |
| CN112934277A (en) * | 2021-01-25 | 2021-06-11 | 重庆大学 | Rapid low-consumption sample filling method for microfluidic chip |
| CN113030062A (en) * | 2021-03-29 | 2021-06-25 | 国家纳米科学中心 | Electricity-spectrum signal detection device, system device and detection method |
| CN113583800A (en) * | 2020-04-30 | 2021-11-02 | 京东方科技集团股份有限公司 | Detection chip, use method thereof and reaction system |
-
2018
- 2018-12-20 CN CN201822146777.5U patent/CN209741124U/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304072A (en) * | 2020-02-28 | 2020-06-19 | 宁波胤瑞生物医学仪器有限责任公司 | Oil seal device for digital PCR chip |
| CN111304072B (en) * | 2020-02-28 | 2023-05-02 | 宁波胤瑞生物医学仪器有限责任公司 | Oil seal device for digital PCR chip |
| CN113583800A (en) * | 2020-04-30 | 2021-11-02 | 京东方科技集团股份有限公司 | Detection chip, use method thereof and reaction system |
| CN112934277A (en) * | 2021-01-25 | 2021-06-11 | 重庆大学 | Rapid low-consumption sample filling method for microfluidic chip |
| CN113030062A (en) * | 2021-03-29 | 2021-06-25 | 国家纳米科学中心 | Electricity-spectrum signal detection device, system device and detection method |
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Address after: 510663 Room 601 and 701, building C, No.288, Shenzhou Road, Huangpu District, Guangzhou City, Guangdong Province Patentee after: Guangzhou bolutang Biotechnology Co.,Ltd. Address before: 510000 room A233, building 13, No. 232, Waihuan East Road, Xiaoguwei street, Panyu District, Guangzhou City, Guangdong Province Patentee before: GUANGZHOU BIOLIGHT EQUIPMENT Co.,Ltd. |
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Denomination of utility model: An Array Chip Device for Digital Nucleic Acid Molecular Traceability Effective date of registration: 20230426 Granted publication date: 20191206 Pledgee: China Construction Bank Corporation Guangzhou Development Zone Branch Pledgor: Guangzhou bolutang Biotechnology Co.,Ltd. Registration number: Y2023980039333 |