CN113005022B - Synchronous detection chip and method for same single cell secretion protein, cytoplasmic mRNA and nuclear DNA - Google Patents
Synchronous detection chip and method for same single cell secretion protein, cytoplasmic mRNA and nuclear DNA Download PDFInfo
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- CN113005022B CN113005022B CN202010639514.7A CN202010639514A CN113005022B CN 113005022 B CN113005022 B CN 113005022B CN 202010639514 A CN202010639514 A CN 202010639514A CN 113005022 B CN113005022 B CN 113005022B
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- 230000001360 synchronised effect Effects 0.000 title claims abstract description 11
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Abstract
The invention belongs to the technical field of molecular biology, and relates to a detection chip and a detection method for single-cell secreted proteins, cytoplasmic mRNA and nuclear DNA. A synchronous detection chip for the same single cell secretion protein, cytoplasmic mRNA and nuclear DNA, which comprises a valve control layer, a detection layer and a substrate; the valve control layer set up the top of testing layer, the base sets up the below at the testing layer, the testing layer include a plurality of single cell detection unit, total inlet, advance appearance microchannel, play appearance microchannel and total outlet, total inlet is connected with advance appearance microchannel, total outlet is connected with play appearance microchannel, every single cell detection unit is connected with advance appearance microchannel and play appearance microchannel respectively. The beneficial effects of the invention are as follows: each detection unit can acquire and detect multiple information of the same single cell nuclear gDNA, cytoplasmic mRNA and cell secretion protein; each chip can simultaneously perform a plurality of single-cell operations, and the reagent consumption is less, the cost is low, and the accuracy is high.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a detection chip and a detection method for secretory proteins, cytoplasmic mRNA and nuclear DNA of the same single cell.
Background
In recent years, many studies have shown that cells have significant differences, whether in normal tissues or diseased tissues, even though cells of the same class have heterogeneity in gene expression, genetic genes, or secreted proteins. The single cell level of biological information is therefore an important basis for accurate medical treatment. Accurate medicine is the most important direction of modern medical development. The advocated "precision medical treatment" includes precision diagnosis and precision treatment. The accurate diagnosis not only needs comprehensive gene information, but also needs multifaceted biological information, including information on gene phenotype, gene expression, protein substances (antibodies, antigens, saccharides and the like), exosomes and the like. The substances show disease-related variability in the occurrence and development processes of the serious diseases, and the comprehensive analysis of multi-level information has unpredictable significance in clinical accurate diagnosis and treatment. The current development of gene sequencing technology has faster development, can carry out gene sequencing at a single cell level, has various means for detecting protein substances, and has relatively weak exosome detection. Moreover, these multiple levels of information are difficult to obtain at the single cell or small cell level.
The bottleneck in high quality single cell sequencing is the quality of single cell whole genome gene acquisition. In major diseases, such as tumors, the variant tissue cells have strong heterogeneity, and the very small number of certain cells in the tumor tissue actually play an important role in the disease, so that the biological information at the single cell level has very important significance. With the rapid development of sequencing technology, gene sequencing technology itself has become well established. However, sequencing of a small number of cells or single-cell genes, to obtain high quality sequencing results, has a major bottleneck in how to obtain high quality single-cell genes prior to sequencing. In the process of extracting single cell or small amount of cell genes, the conventional operation process causes serious gene pollution and loss. The number of copies of the gene per single cell is limited, and if lost, even with higher-end sequencing techniques, no real genetic information can be obtained.
More importantly, there is currently no tool or method that can detect three levels of information, the same single cell whole genome gDNA/mRNA and multiple secreted proteins, simultaneously.
Disclosure of Invention
The invention aims to provide a detection chip and a method capable of simultaneously carrying out three layers of whole genome gDNA/mRNA and various secreted proteins on the same single cell.
In order to achieve the above object, the present invention firstly adopts a technical scheme that a synchronous detection chip for same single cell secretion protein, cytoplasmic mRNA and nuclear DNA is provided, the chip comprises a valve control layer, a detection layer and a substrate; the valve control layer is arranged above the detection layer, and the substrate is arranged below the detection layer; the detection layer comprises a plurality of single-cell detection units, a total sample inlet, a sample injection micro-channel, a sample outlet micro-channel and a total sample outlet, wherein the total sample inlet is connected with the sample injection micro-channel, the total sample outlet is connected with the sample outlet micro-channel, and each single-cell detection unit is respectively connected with the sample injection micro-channel and the sample outlet micro-channel.
As a preferred mode of the present invention, the single cell detection unit includes a sample inlet, a microchannel, a cDNA amplification chamber, a gDNA amplification chamber, and a secreted protein detection chamber, the microchannel is provided with:
a first branch connected to the cDNA amplification chamber;
The second branch is connected with the gDNA amplification cavity;
a third branch connected with the secreted protein detection cavity; the rear end of the secreted protein detection cavity is connected with the sample outlet micro-channel;
The front ends of the first branch and the third branch are communicated with the front end of the second branch;
And a capture antibody array bar code is arranged at a position corresponding to the secreted protein detection cavity on the substrate.
As a preferred mode of the present invention, the valve control layer includes a plurality of control units, each of which includes: a master control valve;
the first branch is provided with a first inlet valve arranged in front of the cDNA amplification cavity and a first outlet valve arranged behind the first inlet valve;
the second branch is provided with a second inlet valve arranged in front of the gDNA amplification cavity and a second outlet valve arranged behind the gDNA amplification cavity;
the third branch is provided with a third inlet valve arranged in front of the secreted protein detection cavity;
and the master control valve, the second inlet valve and the micro-flow channel between the master control valve and the second inlet valve form a single-cell capturing cavity.
Further preferably, the first branch is communicated with the front end of the second branch through a bypass, and a valve is arranged on the bypass.
Further preferably, at least one intermediate valve is provided between the first inlet valve and the cDNA amplification chamber, and at least one reaction chamber is formed between the first inlet valve and the intermediate valve.
Further preferably, at least one intermediate valve is arranged between the second inlet valve and the gDNA amplification chamber; at least one reaction chamber is formed between the second inlet valve and the intermediate valve.
The detection chip provided by the invention is prepared by the following method: firstly, preparing a mask plate according to a chip design drawing, transferring a pattern onto a silicon wafer through photoetching, pouring silicon gel by taking the silicon wafer as a master plate in an injection molding mode, curing the silicon gel through baking and removing the silicon gel from the silicon wafer, aligning a detection layer and a valve control layer, bonding to form a silicon gel chip, and bonding the silicon gel chip with a substrate provided with a capture antibody array bar code.
The invention also provides a synchronous detection method for the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA by adopting the detection chip, which comprises the following steps:
Injecting the cell suspension into a total sample inlet, capturing single cells in a single cell capturing cavity, culturing, and enabling proteins secreted by the cells in the culturing process to enter a secreted protein detection cavity and be specifically captured by a capture antibody array bar code;
After culturing for a period of time, adding a cell membrane lysate to lyse a cell membrane, flushing released cytoplasm into a first branch, and sequentially carrying out reverse transcription, two-chain generation reaction and cyclization reaction of mRNA in the first branch;
flushing the liquid after cyclization reaction into a cDNA amplification cavity, loading a gene amplification reagent, and carrying out cDNA amplification;
Adding cell nucleus lysate, and releasing gDNA by cell nucleus lysis; carrying out denaturation reaction and neutralization reaction in the second branch in sequence;
flushing the neutralized solution into a gDNA amplification cavity, loading a gene amplification reagent, and carrying out gDNA amplification reaction;
And after the amplified cDNA and gDNA products are collected, the detection chip is removed, a fluorescent-labeled detection antibody is loaded on the substrate, the detection antibody is specifically combined with cell secretion proteins, then the substrate is washed, and a fluorescent signal is detected after the substrate is dried by nitrogen.
In a preferred embodiment of the present invention, the reverse transcription reaction, the two-chain formation reaction, and the cyclization reaction of the mRNA are performed in separate reaction chambers.
Further preferably, the denaturation reaction and the neutralization reaction are performed in different reaction chambers, respectively.
Compared with the prior art, the invention has the beneficial effects that:
1. Each detection unit can be used for acquiring and detecting multiple information of single cell nuclear gDNA, intracellular mRNA and cell secretion proteins;
2. each chip can simultaneously perform a plurality of single-cell operations, and the reagent consumption is low and the cost is low;
3. The chip of the invention is matched with the micro-flow channel through the valve to form different reaction cavities, different reactions are carried out in the different reaction cavities, the liquid volume can be accurately controlled according to the volume of the reaction cavities, the human error is reduced, and the detection accuracy is improved.
Drawings
FIG. 1 is an overall block diagram of a detection chip provided in an embodiment of the present invention;
FIG. 2 is a schematic diagram of one of the detecting units in FIG. 1;
FIG. 3 is a schematic diagram of single cell capture, cell membrane lysis, cytoplasmic release, and nuclear lysis in the detection method provided by the practice of the present invention;
FIG. 4 shows fluorescence signals of single cell secreted protein detection in the detection method provided by the implementation of the present invention;
FIG. 5 shows the result of electrophoresis of amplified cDNA and gDNA in the detection method provided by the practice of the present invention;
FIG. 6 shows the result of PCR electrophoresis of different gene loci of amplified cDNA and gDNA products in the detection method provided by the implementation of the present invention.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The first embodiment provided by the invention is a synchronous detection chip for the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA, and the detection chip of the embodiment comprises a valve control layer, a detection layer and a substrate from top to bottom. The valve control layer comprises a control valve inlet 105 and a plurality of control valves, the control valve inlet 105 is connected with each control valve through a runner, and the valves are pressed down by loading liquid into the control valves to block the liquid passages below, so that the trend of the liquid in the micro-runner of the lower detection layer is controlled.
As shown in FIG. 1, the total of 20 single-cell detection units U1-U20 are arranged on the detection chip, and the detection of secreted proteins, cytoplasmic mRNA and nuclear DNA can be simultaneously carried out on 20 single cells.
The detection layer comprises 20 single-cell detection units, a total sample inlet 101, a sample injection micro-channel 102, a total sample outlet 103 and a sample outlet micro-channel 104. The total sample inlet 101 is connected with a sample injection micro-channel 102, and the total sample outlet 103 is connected with a sample outlet micro-channel 104. The 20 single-cell detection units are respectively connected with the sample injection micro-channel 102 and the sample discharge micro-channel 103. The total sample inlet 101 and the sample microchannel 102 are used as sample channels for loading cell solutions and various reagents into each single cell detection unit. The total sample outlet 103 and the sample outlet micro flow channel 104 are used as liquid discharge channels for discharging liquid in each single cell detection unit.
As shown in fig. 2, each single cell detection unit includes a sample inlet 107 and a micro flow channel 120, wherein the sample inlet 107 is opened on the sample injection micro flow channel 102 and is connected with the micro flow channel 120, so that the cell solution and the reagent loaded through the total sample injection port 101 and the sample injection micro flow channel 102 are distributed into each single cell detection unit through the sample inlet 107.
The micro flow channel 120 has 4 branches, namely, a first branch 108, a second branch 111, a third branch 114 and a fourth branch 117. The first branch 108 is connected to a cDNA amplification chamber 109, and a cDNA collection port 110 is provided behind the cDNA amplification chamber 109 for obtaining cytoplasmic cDNA. The second branch 111 is connected with a gDNA amplification cavity 112, and a gDNA collecting port 113 is arranged behind the gDNA amplification cavity 112 and used for acquiring cell nucleus gDNA. The third branch 114 is connected with a secreted protein detection cavity 115, and a capture protein antibody array bar code 116 is arranged on a substrate corresponding to the secreted protein detection cavity 115 and used for specifically capturing and detecting cell secreted proteins. A liquid outlet 118 is arranged at the rear of the secreted protein detection cavity 115, and the liquid outlet 18 is connected with the sample outlet micro-channel 103 and is used for discharging the waste liquid in the third branch 114 and the secreted protein detection cavity 115.
The fourth branch 117 is a flushing liquid discharge branch, and the rear end thereof is connected to the sample ejection micro flow channel 103 through a liquid discharge port.
As shown in fig. 2, the micro flow channel 120 and the second branch 111 are positioned on a straight line, the first branch 108 is communicated with the second branch 111 through a bypass, and the front ends of the third branch 114 and the fourth branch 117 are communicated with the front end of the second branch 111, so that the liquid entering the micro flow channel 120 can be split at the front ends of the respective branches, and can be switched between different branches as required.
In order to control the liquid direction of each branch of each single cell detection unit, a control unit is arranged above each single cell detection unit, each control unit comprises a plurality of control valves, and the opening and closing of the valves are controlled through the control valve inlets 105 and the flow channels.
As shown in fig. 2, each control unit includes a master valve V0, where the master valve V0 is located at the rear end of the micro flow channel 120 and the front end of the second branch 111.
A first inlet valve V2 is provided at the front end of the first branch 108, and a first outlet valve V13 is provided at the rear of the cDNA amplification chamber 109. Four intermediate valves are provided between the first inlet valve V2 and the cDNA amplification chamber 109, in order along the first leg 108: intermediate valve V6, intermediate valve V8, intermediate valve V10, intermediate valve V11. The region between the first inlet valve V2 and the intermediate valve V8 is a reverse transcription reaction chamber in which the reverse transcription reaction of mRNA is performed. The region between the first inlet valve V2 and the intermediate valve V10 is a two-chain reaction chamber in which the two-chain reaction is performed after the completion of the reverse transcription reaction. The region between the first inlet valve V2 and the intermediate valve V11 is a cyclization reaction chamber in which cyclization reaction is performed after the two-chain formation reaction. Single-cell cDNA amplification is performed in the cDNA amplification chamber 109 after the cyclization reaction.
A second inlet valve V5 is provided at the front end of the second branch 111, and a second outlet valve V14 is provided behind the gDNA amplification chamber 112. Three intermediate valves are provided between the second inlet valve V5 and the gDNA amplification chamber 112, and the following steps are sequentially performed along the second branch 111: intermediate valve V7, intermediate valve V9, intermediate valve V12. The area between the master valve V0 and the second inlet valve V5 is a single cell capturing chamber 119, and after the cell suspension enters the micro flow channel 120, when single cells appear in the area, the inlet valves of the master valve V0, the second inlet valve V5 and other branches are closed immediately, so that the single cells can be captured, and then cultured. The area between the main control valve V0 and the intermediate valve V7 is a cracking reaction cavity, and in the cavity, cell membrane or cell nucleus cracking reaction can be carried out. The region between the main control valve V0 and the intermediate valve V9 is a denaturation reaction chamber in which the double-stranded DNA undergoes denaturation reaction. The area between the main control valve V0 and the intermediate valve V12 is a neutralization reaction cavity, and the neutralization reaction is carried out on the reaction system of the double-stranded DNA denaturation reaction in the cavity. Single cell gDNA amplification is performed in the gDNA amplification chamber 112 after the neutralization reaction.
At the front end of the third branch 114 is a third inlet valve V3, which third inlet valve V3 is located after the main control valve V0 and before the second inlet valve V5.
A fourth inlet valve V1 is provided in the fourth branch 117, which fourth inlet valve V1 is located after the main control valve V0 and before the third inlet valve V3.
A valve V4 is provided in the bypass between the first branch 108 and the second branch 111, which valve is used for switching between the first branch 108 and the second branch 111.
The detection chip provided by the embodiment is prepared by the following method: according to the chip structure design diagrams shown in fig. 1 and 2, mask preparation is firstly carried out, then patterns are transferred onto a silicon wafer through photoetching, then the silicon wafer is used as a master plate to pour silica gel in an injection molding mode, and the silica gel is solidified through baking and is removed from the silicon wafer to prepare the detection layer and the valve control layer.
And aligning the detection layer and the valve control layer, bonding to form a silica gel chip, and bonding the silica gel chip with a substrate printed with the secreted protein specific capture antibody bar code.
The second embodiment provided by the invention is: by adopting the detection chip, the method for detecting the single cell secreted protein, the cytoplasmic mRNA and the nuclear DNA comprises the following specific steps:
1. Single cell capture: all the initial states of the control valves are closed states, namely the valves are pressed down to block the flow channels below. The master valve V0 is opened and the single cell trapping chamber 119, which is closed by the master valve V0 to the second inlet valve V5, is filled with BSA blocking solution.
Cells were suspended in PBS and configured as a cell suspension. The fourth inlet valve V1 is opened, then the cell suspension enters the micro flow channel 120 from the total sample inlet 101 through the sample micro flow channel 102 and the sample inlet 107, when single cells exist in the single cell capturing cavity 119, the total control valve V0 and the fourth inlet valve V1 are immediately closed, and the PBS solution entering the fourth branch 117 enters the sample micro flow channel 104 through the liquid outlet 118 and is discharged from the total sample outlet. The single cells captured are shown in FIG. 3 (a).
2. Cell secretion protein capture: the captured single cells are cultured in the capture chamber, and all other control valves are closed except that the third inlet valve V3 is semi-closed, allowing liquid to pass but not cells. Thus, the protein secreted by the cells during the culturing process diffuses into the secreted protein detection chamber 115 through the third inlet valve V3, is captured and immobilized by the specific antibody array bar code 116 on the substrate, and the excessive liquid enters the sample outlet micro-channel 104 through the liquid outlet 118 and is discharged from the total sample outlet 103.
3. MRNA reverse transcription and amplification: the third inlet valve V3 is completely closed, the total control valve V0 and the second inlet valve V5 are opened, and the cell membrane lysate is loaded into the detection unit from the total sample inlet 101: 0.5-5% Triton,10-80nM NaCl,5-50nM Tris, and 2% DEPC. When the liquid is filled up to the middle valve V7, the master valve V0 is closed, cells are subjected to cell membrane lysis in the lysis reaction chamber, and cytoplasm is released by lysis within 5 minutes as shown in fig. 3 (b) and (c). Then the master control valve V0 and the valve V4 are opened, the cytoplasmic solution is flushed into the reaction cavity between the valve V4 and the intermediate valve V6 by using the buffer solution at a slower speed, and the master control valve V0 and the valve V4 are closed.
The first inlet valve V2 and the intermediate valve V6 are opened, reverse transcription reagents (including reverse transcriptase, dNTPs, multi-T DNA ligation chains and reaction buffer) are loaded from the total injection port 101, the liquid is filled up to the intermediate V8, the first inlet valve V2 is closed, and the reverse transcription reaction of mRNA is performed in the reverse transcription chamber.
After the completion of the reverse transcription reaction, the first inlet valve V2 and the intermediate valve V8 are opened, and the reagents for forming the second strand (including the two-strand buffer, dNTPs, DNA polymerase, DNA LIGASE, RHaseH, etc.) are loaded from the total inlet 101, and when the intermediate valve V10 is filled with the liquid, the first inlet valve V2 is closed, and the two-strand reaction is performed in the two-strand reaction chamber.
After the two-chain formation reaction, the first inlet valve V2 and the intermediate V10 are opened, the cyclized connection reaction reagent is loaded from the total sample inlet 101, and when the liquid is filled up to the intermediate valve V11, the first inlet valve V2 is closed, and the cyclized reaction is performed in the cyclized reaction chamber.
Finally, the first inlet valve V2 and the intermediate valve V11 are opened, the gene amplification reagent is loaded from the total sample inlet 101, and when the solution is filled up to the first outlet valve V13, the first inlet valve V2 is closed, and the amplification reaction is performed in the cDNA amplification chamber 109.
4. Nuclear gDNA amplification: the total control valve V0 and the second inlet valve V5 were opened, the cell nucleus lysate was loaded from the total injection port 101, and when the liquid was filled to the intermediate valve V7, the total control valve V0 was closed, and gDNA was released by cell nucleus lysis within 5 minutes, as shown in fig. 3 (d).
And opening the main control valve V0 and the intermediate valve V7, loading double-stranded DNA denaturing agent from the main injection port 101, and closing the main control valve VO when the liquid is filled into the intermediate valve V9, so that the double-stranded DNA undergoes a denaturation reaction in the denaturation reaction cavity.
And opening the total control valve V0 and the intermediate valve V9, loading the neutralizing agent from the total injection port 101, closing the total control valve V0 when the liquid is filled into the intermediate valve V12, and neutralizing the denaturant in the neutralization reaction cavity by adopting the neutralizing agent.
Then, the total control valve V0 and the intermediate valve V12 are opened, the gene amplification reagent is loaded from the total injection port 101, and when the solution is filled up to the second outlet valve V14, the total control valve V0 is closed, and the nuclear gDNA amplification reaction is performed in the gDNA amplification chamber 112.
5. CDNA and gDNA amplification product collection: after the amplification reaction of cDNA and gDNA is finished, opening a first inlet valve V2 and a first outlet valve V13, loading PBS from a total sample inlet 101 to flush cDNA out, and collecting cDNA at a cDNA collecting port 110; then the first inlet valve V2 is closed, the master valve V0 and the second outlet valve V14 are opened, the gDNA is flushed out by the loading PBS and the amplified gDNA is collected at the gDNA collection port 113. The cDNA and gDNA collected were used for subsequent detection.
6. Single cell secreted protein detection: and after the amplified cDNA and gDNA are collected, removing the silica gel chip, loading a fluorescent-labeled detection antibody on the substrate, specifically combining the detection antibody with the captured cell secretory protein, flushing the substrate, and detecting a fluorescent signal after blowing the substrate with nitrogen, thereby completing the specific detection of the cell secretory protein.
By adopting the detection chip and the detection method provided by the invention to detect the secreted proteins of 6 single cell samples, the result is shown in figure 4, each band in the figure is a fluorescence detection signal of a protein secreted by one cell, and the intensity of the fluorescence signal represents the amount of the detected protein expression.
By adopting the detection chip and the detection method provided by the invention, mRNA and gDNA of 6 single cell samples are detected. The collected mRNA reverse transcription amplification product cDNA and gDNA amplification product are long fragments of DNA, and in order to determine whether there is an amplification product, a small amount of the amplification product is taken for gel electrophoresis, and white bands represent the amplification product, and the brighter the bands, the more the amplification product. The results of 6 single cells are shown in FIG. 5.
The cDNA and gDNA amplification products of mRNA reverse transcription amplification products collected from 2 cells were amplified by Polymerase Chain Reaction (PCR) at different gene sites, the gel electrophoresis results of the amplification products are shown in FIG. 6, M is DNA MARKER, which represents the electrophoresis position of the standard DNA length, and the band represents the amplification products at the corresponding gene sites.
Claims (8)
1. A synchronous detection chip for the same single cell secretion protein, cytoplasmic mRNA and nuclear DNA, which comprises a valve control layer, a detection layer and a substrate; the valve control layer set up the top of testing the layer, the base sets up the below at the testing layer, its characterized in that: the detection layer comprises a plurality of single-cell detection units, a total sample inlet, a sample injection micro-channel, a sample outlet micro-channel and a total sample outlet, wherein the total sample inlet is connected with the sample injection micro-channel, the total sample outlet is connected with the sample outlet micro-channel, and each single-cell detection unit is respectively connected with the sample injection micro-channel and the sample outlet micro-channel; the single cell detection unit include sample entry, microchannel, cDNA amplification chamber, gDNA amplification chamber and secretion protein detection chamber, the microchannel is equipped with:
a first branch connected to the cDNA amplification chamber;
The second branch is connected with the gDNA amplification cavity;
The third branch is connected with the secreted protein detection cavity, and the rear end of the secreted protein detection cavity is connected with the sample outlet micro-channel;
The front ends of the first branch and the third branch are communicated with the front end of the second branch;
the substrate is provided with a capture antibody array bar code at a position corresponding to the secreted protein detection cavity;
The detection chip is prepared by the following method: firstly, preparing a mask plate according to a chip design drawing, transferring a pattern onto a silicon wafer through photoetching, pouring silicon gel by taking the silicon wafer as a master plate in an injection molding mode, curing the silicon gel through baking and removing the silicon gel from the silicon wafer, aligning a detection layer and a valve control layer, bonding to form a silicon gel chip, and bonding the silicon gel chip with a substrate provided with a capture antibody array bar code.
2. The synchronous detection chip for the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA according to claim 1, wherein: the valve control layer comprises a plurality of control units, and each control unit comprises: a master control valve;
the first branch is provided with a first inlet valve arranged in front of the cDNA amplification cavity and a first outlet valve arranged behind the first inlet valve;
the second branch is provided with a second inlet valve arranged in front of the gDNA amplification cavity and a second outlet valve arranged behind the gDNA amplification cavity;
the third branch is provided with a third inlet valve arranged in front of the secreted protein detection cavity;
and the master control valve, the second inlet valve and the micro-flow channel between the master control valve and the second inlet valve form a single-cell capturing cavity.
3. The synchronous detection chip for the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA according to claim 2, wherein: the first branch is communicated with the front end of the second branch through a bypass, and a valve is arranged on the bypass.
4. The synchronous detection chip for the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA according to claim 2, wherein: at least one intermediate valve is arranged between the first inlet valve and the cDNA amplification cavity, and at least one reaction cavity is formed between the first inlet valve and the intermediate valve.
5. The synchronous detection chip for the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA according to claim 2, wherein: at least one intermediate valve is arranged between the second inlet valve and the gDNA amplification cavity; at least one reaction chamber is formed between the second inlet valve and the intermediate valve.
6. A method for simultaneous detection of the same single cell secreted protein, cytoplasmic mRNA and nuclear DNA using the detection chip of any one of claims 1-5, comprising:
Injecting the cell suspension into a total sample inlet, capturing single cells in a single cell capturing cavity, culturing, and enabling proteins secreted by the cells in the culturing process to enter a secreted protein detection cavity and be specifically captured by a capture antibody array bar code;
After culturing for a period of time, adding a cell membrane lysate to lyse a cell membrane, flushing released cytoplasm into a first branch, and sequentially carrying out reverse transcription, two-chain generation reaction and cyclization reaction of mRNA in the first branch;
flushing the liquid after cyclization reaction into a cDNA amplification cavity, loading a gene amplification reagent, and carrying out cDNA amplification;
Adding cell nucleus lysate, and releasing gDNA by cell nucleus lysis; carrying out denaturation reaction and neutralization reaction in the second branch in sequence;
flushing the neutralized solution into a gDNA amplification cavity, loading a gene amplification reagent, and carrying out gDNA amplification reaction;
And after the amplified cDNA and gDNA products are collected, the detection chip is removed, a fluorescent-labeled detection antibody is loaded on the substrate, the detection antibody is specifically combined with cell secretion proteins, then the substrate is washed, and a fluorescent signal is detected after the substrate is dried by nitrogen.
7. The method according to claim 6, wherein the reverse transcription, the two-strand formation reaction and the cyclization reaction of the mRNA are performed in different reaction chambers.
8. The method for simultaneous detection of a single-cell secreted protein, cytoplasmic mRNA and nuclear DNA according to claim 6, wherein the denaturation reaction and the neutralization reaction are performed in different reaction chambers, respectively.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108267581A (en) * | 2012-08-24 | 2018-07-10 | 耶鲁大学 | For the systems, devices and methods of high-throughput multi detection |
| CN212560270U (en) * | 2020-07-06 | 2021-02-19 | 山东大学 | Synchronous detection chip for same single cell secretion protein, mRNA and DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108267581A (en) * | 2012-08-24 | 2018-07-10 | 耶鲁大学 | For the systems, devices and methods of high-throughput multi detection |
| CN212560270U (en) * | 2020-07-06 | 2021-02-19 | 山东大学 | Synchronous detection chip for same single cell secretion protein, mRNA and DNA |
Non-Patent Citations (1)
| Title |
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| Co-detection and sequencing of genes and transcripts from the same single cells facilitated by a microfluidics platform;Lin Han et al;Scientific Reports;第1-9页 * |
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