CN110628567A - Ultrahigh-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip - Google Patents
Ultrahigh-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip Download PDFInfo
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Abstract
The invention discloses an ultrahigh-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip, which comprises a micropore array chip and a microfluidic packaging structure, wherein the micropore array chip is arranged in the microfluidic packaging structure; the micropore array chip is provided with at least one micropore array area on a substrate, the micropore array area is provided with a plurality of micropores, the micropores have the size and the shape which can only accommodate single cells in one micropore, and at least one DNA probe is modified on the inner wall of each micropore. The invention designs the chip with the millipore of hundred thousand and million orders, and the target nucleic acid molecule in the cell is captured by modifying the DNA probe in the millipore, so that the capture of the single cell of hundred thousand and million orders can be realized, the in-situ lysis and the nucleic acid amplification can be further realized, and the chip foundation can be improved for the real-time fluorescence quantitative analysis of the ultrahigh-flux single cell nucleic acid molecule.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to an ultrahigh-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip.
Background
The growth, development, differentiation, aging and pathological changes of the body are all related to the differential expression of genes. The development and metastasis of tumors are also related to the mutation and differential expression of genes, and cells in the center of tumor tissues, cells around the tumor tissues, cells of metastatic foci and the like also cause different functional characteristics due to differences of genome and transcription expression profiles, thus influencing and determining the treatment results of tumors and the like.
Traditional gene expression studies typically measure the expression of a gene at the mRNA level. Expression at the mRNA level is usually achieved by real-time fluorescent quantitative PCR (RT-PCR). Current fluorescent quantitative RT-PCR (RT-qPCR) can only observe the result of multicellular averaging at the level of a cell population. At the level of the cell population, the final result, which is actually an average of many cells, often loses information about cellular heterogeneity and critical information about the functional diversity of single cells.
Along with the development of single cell analysis technology, a single cell multi-gene detection system is developed. The method generally comprises the steps of manufacturing an independent unit by utilizing a microfluidic channel, independently isolating single cells in the independent unit, carrying out cDNA amplification, capturing hundreds to thousands of single cells at most each time, and then carrying out nucleic acid amplification detection by matching with an instrument.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a chip for real-time fluorescence quantitative analysis of ultra-high flux single-cell nucleic acid molecules, aiming at the above-mentioned deficiencies in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: an ultrahigh-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip comprises a micropore array chip and a microfluidic packaging structure, wherein the micropore array chip is arranged in the microfluidic packaging structure;
the micropore array chip is provided with at least one micropore array area on a substrate, the micropore array area is provided with a plurality of micropores, the micropores have the size and the shape which can only accommodate single cells in one micropore, and at least one DNA probe is modified on the inner wall of each micropore.
Preferably, the inner surface of the microwell is subjected to hydrophilic treatment, and the other surfaces of the microwell array chip except the inner surface of the microwell are subjected to hydrophobic treatment.
Preferably, the microwell array region comprises 10 microwells arranged in line on the substrate, and the number of microwells included in a single microwell array region is not less than 106And (4) respectively.
Preferably, the micropores are through holes and have a regular polygonal structure.
Preferably, the shape of the micropores is a regular hexagon, and the diameter of a circumscribed circle thereof is 1 to 100 μm.
Preferably, the microfluidic packaging structure comprises a bottom substrate and an upper cover plate, an upper chip groove is formed in the middle of the lower surface of the upper cover plate, and a sample inlet runner groove and a sample outlet runner groove which are communicated with the upper chip groove are symmetrically formed in two sides of the upper chip groove; a lower chip groove is formed in the middle of the upper surface of the bottom substrate, and an auxiliary runner groove with the same structure as the sample outlet runner groove is formed in one side of the lower chip groove;
the upper cover plate is connected with the bottom substrate in a laminating manner, the upper chip groove and the lower chip groove are aligned to form a chip mounting groove for accommodating the micro-pore array chip, the sample injection channel groove is aligned to form a sample injection channel between the upper surface of the bottom substrate, and the sample injection channel groove and the secondary channel groove are aligned to form a sample injection channel.
Preferably, the upper cover plate is provided with a sample inlet, a total liquid outlet, a buffer liquid inlet and a buffer liquid outlet; advance kind runner groove and go out kind runner groove and set up along width direction the lower surface both sides of upper apron, introduction port, total liquid outlet set up along width direction the both sides of upper apron, buffer solution entry and buffer solution export set up along length direction the both sides of upper apron.
Preferably, the sample inlet channel groove, the sample outlet channel groove and the secondary channel groove have the same structure, are all tree-shaped branched structure channels, and are provided with a tree root node port and a plurality of sub-node ports;
a root node port of the sample injection runner groove is communicated with the sample injection port, and a sub-node port of the sample injection runner groove is communicated to one side of the upper chip groove close to the sample injection port;
a sub-node port of the sample outlet runner groove is communicated with one side of the upper chip groove close to the main liquid outlet, and a root node port of the sample outlet runner groove is communicated with the main liquid outlet;
a sub-node port of the secondary runner groove is communicated with one side of the lower chip groove close to the main liquid outlet, and a root node port of the secondary runner groove is communicated with the main liquid outlet;
and two ends of the lower chip groove along the length direction are respectively communicated with the buffer solution inlet and the buffer solution outlet.
Preferably, the number of the sub-node ports of the flow channel of the tree-like branched structure is the same as the number of the micro-pore array regions on the micro-pore array chip.
Preferably, V-shaped diversion trenches are arranged at the sub-node ports of the flow channel of the tree-shaped branched structure, and the number and the positions of the V-shaped diversion trenches correspond to the micropore array regions on the micropore array chip one by one; the sharp-end of the V-shaped diversion trench is communicated with the sub-node port, and the other end of the V-shaped diversion trench is communicated with the side part of the micropore array area.
The invention has the beneficial effects that:
the invention can realize the capture of single cells of hundreds of thousands of orders and millions of orders by designing the chip with the micropores of hundreds of thousands of orders and millions of orders and modifying the DNA probe in the micropores to capture target nucleic acid molecules in cells, further realize in-situ lysis and nucleic acid amplification, and can improve the chip foundation for the real-time fluorescence quantitative analysis of ultrahigh-flux single cell nucleic acid molecules;
according to the invention, the micro-pore array chip is subjected to hydrophilic and hydrophobic treatment, so that the micro-pore capillary force can be increased, single cells can be favorably absorbed into micro-pores, and single cell capture is realized;
the microfluidic packaging structure can realize uniform distribution of samples by designing the flow channel of the multi-branch tree-shaped branched structure, and is fully distributed in the whole micropore array area, so that efficient uniform sample introduction is realized, the flow of each tail end branch is basically consistent, and the liquid level is stably pushed.
Drawings
FIG. 1 is a schematic diagram of the exploded structure of the ultra-high throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip of the present invention;
FIG. 2 is a schematic view of the structure of the lower surface of the upper cover plate according to the present invention;
FIG. 3 is a schematic structural view of the upper cover plate of the present invention with a micro well array chip disposed on the lower surface thereof;
FIG. 4 is a schematic structural view of a secondary channel groove on the upper surface of the underlying substrate according to the present invention;
fig. 5 is a schematic structural diagram of an integrated flow channel in the microfluidic packaging structure of the present invention.
Description of reference numerals:
1-a microwell array chip; 2-microfluidic packaging structure; 10-a microwell array region; 20-bottom substrate; 21-upper cover plate; 22-upper chip slot; 23-sample introduction runner groove; 24-a sample outlet runner groove; 25-secondary flow channel slots; 26-lower chip slot; 27-sample inlet; 28 — a total liquid outlet; 29-buffer inlet; 30-buffer outlet; 31-tree root node port; 32-child node port; 33-V type guiding gutter.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
As shown in fig. 1-5, the ultrahigh-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip of the present embodiment includes a micro-pore array chip 1 and a micro-fluidic packaging structure 2, wherein the micro-pore array chip 1 is disposed in the micro-fluidic packaging structure 2;
the micro-pore array chip 1 is provided with at least one micro-pore array area 3 on a substrate, the micro-pore array area 3 is provided with a plurality of micro-pores, the micro-pores have the size and the shape which can only accommodate single cells in one micro-pore, and at least one DNA probe is modified on the inner wall of each micro-pore.
Wherein, the substrate of the micropore array chip 1 can adopt materials such as polymer, Si and the like, and a micropore structure with the diameter of 1-100 mu m can be formed by etching. And the micropores are through holes and are in regular polygon structures. Only a single cell can be accommodated in a single well, and in this embodiment, the well has a shape of a regular hexagon, and a diameter of a circumscribed circle thereof is 1 to 100. mu.m. And the number of microwells included in a single microwell array region 3 is not less than 106And single micropore array region 3 can achieve single cell capture of hundreds of thousands of orders. Furthermore, 10 micropore array regions 3 arranged in a row are arranged on the micropore array chip 1, so that the micropore array chip 1 can achieve ultra-high-flux single cell capture with the total amount of millions. Of course, the number of the micropores of the single micropore array region 3 and the number of the micropore array regions 3 on the micropore array chip 1 can be expanded, and the capture of tens of millions of ultrahigh-flux single cells can be expanded. The DNA probe is used for capturing target nucleic acid molecules, and the detection of a plurality of gene loci can be realized by modifying a plurality of DNA probes.
Wherein, the inner surface of the micro-hole is treated by hydrophilicity, and the other surfaces of the micro-hole array chip 1 except the inner surface of the micro-hole are treated by hydrophobicity. The capillary force of the micropores can be increased through the hydrophilic treatment of the inner surfaces of the micropores, so that the single cells can be favorably absorbed into the micropores; through hydrophobic treatment of the surface, the surface liquid residue can be reduced. The processed micropore array chip 1 can suck a sample through the capillary force of micropores, and single cell capture and in-situ observation are realized.
In a preferred embodiment, the hydrophilic and hydrophobic processing method of the micro-well array chip 1 is as follows:
1) hydrophilic treatment: immersing the whole microporous array chip 1 in a hydrophilic reagent for reaction for a certain time, wherein the hydrophilic reagent can adopt at least one of hydrogen peroxide, ammonia water, acetic acid, concentrated sulfuric acid, hydrochloric acid or sodium hydroxide;
2) filling micropores: filling the micropores of the micropore array chip 1 with fillers to isolate the inner walls of the micropores; the filler can be at least one of water, trichloroethylene, n-hexane, silicone oil, liquid paraffin, solid paraffin, fluorinated oil, blue film or PDMS;
3) and (3) hydrophobic treatment: immersing the filled micro-pore array chip 1 in a hydrophobic reagent for reaction for a certain time, wherein the hydrophobic reagent can adopt at least one of perfluorodecyl trimethoxy silane, octadecyl trichlorosilane, octadecyl trimethoxy silane, hexadecyl triethoxysilane, hexyl triethoxysilane or octyl trichlorosilane;
4) removing the filler in the micropores: and (3) carrying out ultrasonic cleaning on the micropore array chip 1 by adopting a cleaning reagent, removing the filler in the micropores, and then drying to obtain the processed micropore array chip 1. The cleaning agent can be selected from trichloroethylene, acetone, ethanol or isopropanol.
The invention mainly provides an ultrahigh-flux chip for performing ultrahigh-flux real-time fluorescence quantitative analysis on single-cell nucleic acid, and the chip for performing fluorescence quantitative analysis mainly comprises the following steps:
1) manufacturing the chip of the invention;
2) adding a sample to be detected into the micropore array chip 1, capturing single cells through micropores, and capturing target nucleic acid molecules through modified DNA probes in the micropores;
3) PCR amplification detection is carried out, and real-time fluorescence quantitative analysis of the single-cell nucleic acid is realized through fluorescence quantitative analysis.
The chip with the million-level micropores realizes the million-level ultrahigh-flux single cell capture, thereby providing chip support for the method and the system for realizing ultrahigh-flux real-time fluorescence quantitative analysis of the single cell nucleic acid.
In this embodiment, the microfluidic packaging structure 2 includes a bottom substrate 20 and an upper cover plate 21, an upper chip groove 22 is formed in the middle of the lower surface of the upper cover plate 21, and a sample inlet channel groove 23 and a sample outlet channel groove 24 communicated with the upper chip groove 22 are symmetrically formed on two sides of the upper chip groove 22; a lower chip groove 26 is arranged in the middle of the upper surface of the bottom substrate 20, and an auxiliary flow channel groove 25 with the same structure as the sample outlet flow channel groove 24 is arranged on one side of the lower chip groove 26;
after the upper cover plate 21 is attached to the bottom substrate 20, the positions of the upper chip slot 22 and the lower chip slot 26 are opposite to form a chip mounting slot for accommodating the micro-pore array chip 1, a sample injection channel is formed between the sample injection channel slot 23 and the upper surface of the bottom substrate 20, and the positions of the sample outlet channel slot 24 and the auxiliary channel slot 25 are opposite to form a sample outlet channel.
In the invention, the micro-fluidic packaging structure 2 is used for packaging the micro-pore array chip 1, and the rapid sample introduction and the uniform distribution of samples can be realized through the design of the flow channel.
Wherein, the upper cover plate 21 is provided with a sample inlet 27, a total liquid outlet 28, a buffer liquid inlet 29 and a buffer liquid outlet 30; the sample inlet channel groove 23 and the sample outlet channel groove 24 are arranged on both sides of the lower surface of the upper cover plate 21 in the width direction, the sample inlet 27 and the total liquid outlet 28 are arranged on both sides of the upper cover plate 21 in the width direction, and the buffer inlet 29 and the buffer outlet 30 are arranged on both sides of the upper cover plate 21 in the length direction.
The sample inlet channel groove 23, the sample outlet channel groove 24 and the secondary channel groove 25 have the same structure, are all tree-shaped branched structure channels, and are provided with a tree root node port 31 and a plurality of sub-node ports 32;
a root node port 31 of the sample runner groove 23 is communicated with the sample inlet 27, and a sub-node port 32 thereof is communicated to one side of the upper chip groove 22 close to the sample inlet 27;
a sub-node port 32 of the sample runner groove 24 is communicated with one side of the upper chip groove 22 close to the main liquid outlet 28, and a root node port 31 thereof is communicated with the main liquid outlet 28;
a sub-node port 32 of the secondary runner groove 25 is communicated with one side of the lower chip groove 26 close to the main liquid outlet 28, and a root node port 31 thereof is communicated with the main liquid outlet 28;
both ends of the lower chip groove 26 in the longitudinal direction are respectively communicated with a buffer inlet 29 and a buffer outlet 30.
The structure of the integrated flow channel within the microfluidic package structure 2 is shown in fig. 5.
The sample enters from the sample inlet 27 under the action of the driving pump, is uniformly and rapidly distributed on the upper surface of the micropore array chip 1 through the sample injection runner groove 23, and is sucked into the micropores through the capillary force of the micropores, so that the capture of the micropores to single cells is realized, and the redundant sample is discharged from the sample outlet runner groove 24 on the other side of the micropore array chip 1 through the total liquid outlet 28.
The buffer solution enters the lower chip groove 26 through the buffer solution inlet 29, reaches the lower surface of the micro well array chip 1, exchanges substances with the micro wells from the bottoms of the micro wells, and the excess buffer solution can be discharged from the buffer solution outlet 30.
When advancing the appearance, because the reason of capillary force, the sample that micropore array chip 1 upper surface flowed into the micropore need overcome the capillary action and just can flow out from the micropore bottom and flow into the below region, the giving appearance velocity of flow of driving pump is not enough to make the sample can overcome the capillary action of micropore this moment, so the sample can not flow from the micropore lower part, guarantee that the cell can fix in the micropore, the layering of micropore array chip 1 upper and lower region has also been realized simultaneously, can realize upper and lower two-layer independent control, can let in different reagents. When the micropores of the micropore array chip 1 need to be washed and cleaned, the main liquid outlet 28 is connected with a vacuum pump, cleaning liquid enters from the sample inlet 27 and is distributed on the upper surface of the micropore array chip 1 through the sample flow channel groove 23, the suction effect of the vacuum pump enables the cleaning liquid to overcome the capillary force effect of the micropores, and the liquid in the micropores can flow out from the bottoms of the micropores and enter the auxiliary flow channel groove 25 and then is discharged from the main liquid outlet 28.
The flow channel with the tree-shaped branched structure is adopted in the invention, the uniform distribution of samples can be realized, the whole micropore array area 3 is fully distributed, each micropore can be subjected to sample injection, the flow of each tail end branch can be ensured to be basically consistent, and the stable propulsion of the liquid level is realized.
In a further embodiment, the number of the sub-node ports 32 of the flow channel of the tree-like branched structure is the same as the number of the micro-well array regions 3 on the micro-well array chip 1, and is 10. The branch node port 32 of the flow passage with the tree-shaped branched structure is provided with V-shaped guide grooves 33, the number and the positions of the V-shaped guide grooves 33 correspond to the micropore array regions 3 on the micropore array chip 1 one by one, the number of the V-shaped guide grooves 33 is 10, and each V-shaped guide groove 33 corresponds to the side part of one micropore array region 3; the pointed end of the V-shaped guide groove 33 communicates with the sub-node port 32, and the other end communicates with the side of the micro-pore array region 3. Through the arrangement of the V-shaped diversion grooves 33, the liquid can flow rapidly and uniformly and can be distributed and collected conveniently. For the sample channel groove 23, the V-shaped flow guide groove 33 of the sub-node port 32 facilitates to guide the entering sample into the upper part of the micropore array region 3 quickly and uniformly. For the sample channel groove 24 and the secondary channel groove 25, the V-shaped diversion groove 33 of the sub-node port 32 facilitates the liquid in the chip mounting groove to be quickly collected into the sub-node port 32 for efficient discharge.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. The chip is characterized by comprising a micropore array chip and a microfluidic packaging structure, wherein the micropore array chip is arranged in the microfluidic packaging structure;
the micropore array chip is provided with at least one micropore array area on a substrate, the micropore array area is provided with a plurality of micropores, the micropores have the size and the shape which can only accommodate single cells in one micropore, and at least one DNA probe is modified on the inner wall of each micropore.
2. The chip for real-time fluorescence quantitative analysis of ultra-high throughput single-cell nucleic acid molecules according to claim 1, wherein the inner surface of the microwell is treated with hydrophilic treatment, and the other surfaces of the microwell array chip except the inner surface of the microwell are treated with hydrophobic treatment.
3. The chip of claim 2, wherein the microwell array region comprises 10 microwells arranged in a row on the substrate, and the number of microwells included in a single microwell array region is not less than 106And (4) respectively.
4. The chip for real-time fluorescence quantitative analysis of ultra-high throughput single-cell nucleic acid molecules according to claim 3, wherein the microwells are through holes and have regular polygonal structures.
5. The chip for real-time fluorescence quantitative analysis of ultra-high throughput single-cell nucleic acid molecules according to claim 4, wherein the shape of the microwells is regular hexagons, and the diameter of the circumcircles is 1-100 μm.
6. The chip for real-time fluorescence quantitative analysis of ultra-high flux single-cell nucleic acid molecules according to any one of claims 1 to 5, wherein the microfluidic packaging structure comprises a bottom substrate and an upper cover plate, an upper chip groove is formed in the middle of the lower surface of the upper cover plate, and a sample inlet channel groove and a sample outlet channel groove communicated with the upper chip groove are symmetrically formed on two sides of the upper chip groove; a lower chip groove is formed in the middle of the upper surface of the bottom substrate, and an auxiliary runner groove with the same structure as the sample outlet runner groove is formed in one side of the lower chip groove;
the upper cover plate is connected with the bottom substrate in a laminating manner, the upper chip groove and the lower chip groove are aligned to form a chip mounting groove for accommodating the micro-pore array chip, the sample injection channel groove is aligned to form a sample injection channel between the upper surface of the bottom substrate, and the sample injection channel groove and the secondary channel groove are aligned to form a sample injection channel.
7. The chip for real-time fluorescence quantitative analysis of ultra-high flux single-cell nucleic acid molecules according to claim 6, wherein the upper cover plate is provided with a sample inlet, a total liquid outlet, a buffer inlet and a buffer outlet; advance kind runner groove and go out kind runner groove and set up along width direction the lower surface both sides of upper apron, introduction port, total liquid outlet set up along width direction the both sides of upper apron, buffer solution entry and buffer solution export set up along length direction the both sides of upper apron.
8. The chip of claim 7, wherein the sample inlet channel, the sample outlet channel and the secondary channel have the same structure, are all tree-like branched channels, and have a tree root node port and a plurality of sub-node ports;
a root node port of the sample injection runner groove is communicated with the sample injection port, and a sub-node port of the sample injection runner groove is communicated to one side of the upper chip groove close to the sample injection port;
a sub-node port of the sample outlet runner groove is communicated with one side of the upper chip groove close to the main liquid outlet, and a root node port of the sample outlet runner groove is communicated with the main liquid outlet;
a sub-node port of the secondary runner groove is communicated with one side of the lower chip groove close to the main liquid outlet, and a root node port of the secondary runner groove is communicated with the main liquid outlet;
and two ends of the lower chip groove along the length direction are respectively communicated with the buffer solution inlet and the buffer solution outlet.
9. The chip of claim 8, wherein the number of the sub-node ports of the flow channel with the tree-like branched structure is the same as the number of the micro-pore array regions on the micro-pore array chip.
10. The ultra-high flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip of claim 9, wherein the sub-node ports of the flow channel with the tree-like branched structure are provided with V-shaped flow guide grooves, and the number and positions of the V-shaped flow guide grooves correspond to the micropore array regions on the micropore array chip one by one; the sharp-end of the V-shaped diversion trench is communicated with the sub-node port, and the other end of the V-shaped diversion trench is communicated with the side part of the micropore array area.
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| CN201910912693.4A CN110628567B (en) | 2019-09-25 | 2019-09-25 | Ultrahigh-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip |
| PCT/CN2019/112903 WO2021056651A1 (en) | 2019-09-25 | 2019-10-24 | Ultra-high-flux single-cell nucleic acid molecule real-time fluorescence quantitative analysis method, chip and system |
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| CN110241017B (en) * | 2019-05-07 | 2022-09-20 | 中国科学院苏州生物医学工程技术研究所 | Digital biological detection chip and packaging clamp |
| CN116162538A (en) * | 2022-12-16 | 2023-05-26 | 中国科学院苏州生物医学工程技术研究所 | Microfluidic chip and kit for simultaneously detecting protein and RNA |
| CN116162538B (en) * | 2022-12-16 | 2024-01-26 | 中国科学院苏州生物医学工程技术研究所 | Microfluidic chip and kit for simultaneously detecting protein and RNA |
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