CN110643502A - Single-cell microfluidic detection chip and preparation method and application method thereof - Google Patents
Single-cell microfluidic detection chip and preparation method and application method thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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
- B01L3/502707—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 characterised by the manufacture of the container or its components
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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
- B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention belongs to the technical field of molecular biology and microfluidics, and particularly relates to a single-cell microfluidic detection chip and a preparation method and a use method thereof. The detection chip comprises an upper layer chip and a lower layer chip, wherein the middle of the upper layer chip and the lower layer chip is separated by a chitosan film; the upper and lower chips have fluid channel structures corresponding to each other; the channel comprises a main channel, and a plurality of capture chambers are arranged on the main channel; a secondary channel is arranged beside the capture chamber and is connected with the main channel; after the capture chamber captures single cells or other microbeads, subsequent cells and microbeads bypass the capture chamber through the secondary channel and then enter the primary channel; after the capture is finished, the dissolving film enables the corresponding capture chambers of the upper layer and the lower layer to be communicated, thereby realizing the cell lysis and the capture of the substance to be detected. The chip can greatly improve the pairing rate of cells and microbeads and improve the cell utilization efficiency of single cell sequencing; cross contamination between single cell nucleic acids can be reduced; the chip can be used for single cell sequencing and other researches.
Description
Technical Field
The invention belongs to the technical field of molecular biology and microfluidics, and particularly relates to a single-cell microfluidic detection chip and a preparation method and a use method thereof.
Background
Microfluidic technology, also known as lab-on-a-chip, can integrate conventional biochemical reactions into a few square centimeters chip. Due to the micron-sized channels of the microfluidic chip, which are equivalent to the size of the cells, the microfluidic chip has attracted more and more attention in the research of single cells.
The first microfluidic chip used for single cell research was the "Quake" chip, which was integrated with a complex pump-valve system to manipulate single cells. Later, with the development of droplet technology, drop-seq technology appeared, which greatly improves the throughput of single cell sequencing. There are also microfluidic chips that have been sequenced for analysis by single cell capture through microstructures.
In summary, in the field of single cell research, the microfluidic chip technology has become a research platform with great development potential, and by the design of the microfluidic chip, single cell capture, control, analysis and the like can be realized. However, most of the existing high-throughput single-cell sequencing is based on a microfluidic droplet technology, and there is a certain probability that one cell and one microbead containing a barcode capture sequence are simultaneously arranged in one droplet in the process of generating a large number of droplets. Since both single cells and single bead coating are subject to poisson distribution during droplet formation, the cell utilization is extremely low (around 1%).
Disclosure of Invention
The invention aims to provide a single-cell microfluidic detection chip which is convenient to capture, control and analyze single cells and high in efficiency, and a preparation method and a use method thereof.
The single-cell microfluidic detection chip comprises an upper chip and a lower chip, wherein a layer of soluble chitosan film is arranged in the middle to separate the upper chip from the lower chip; wherein:
the upper and lower chips contain fluid channel structures which are corresponding to each other up and down; the upper chip and the lower chip respectively comprise a liquid inlet and a liquid outlet which are communicated with the fluid channel;
the upper layer fluid channel and the lower layer fluid channel of the microfluidic detection chip respectively contain a main channel, and a plurality of capture chambers are arranged on the main channel.
The microfluidic detection chip also comprises a secondary channel beside each capture chamber of the upper layer and the lower layer, wherein the secondary channel surrounds the capture chamber, and both ends of the secondary channel are communicated with the main channel (see figure 1).
Further, the microfluidic detection chip comprises 200-1000 capture chambers on the upper and lower main channels.
Furthermore, the height of the main channel and the sub-channel on the upper layer and the lower layer of the microfluidic detection chip is 15-100 micrometers, and the width of the main channel and the sub-channel is 50-150 micrometers; the capture chamber has an inlet width of 50-150 microns; the outlet width of the capture chamber is 10-50 microns.
The preparation method of the microfluidic detection chip provided by the invention comprises the steps of preparing the upper layer chip template and the lower layer chip template by adopting a soft lithography method, and comprises the following specific steps:
(1) spin-coating SU-8 photoresist with the thickness of 15-50 microns on the surface of a silicon wafer or a glass substrate, and baking for 15-60 minutes at the temperature of 95 ℃;
(2) fixing a mask containing an upper layer channel structure or a lower layer channel structure on the surface of a substrate containing photoresist, and polymerizing SU-8 according to the structure of the mask by ultraviolet exposure;
(3) baking for 10-30 minutes at 95 ℃, naturally cooling, and removing unexposed SU-8 glue by using ethyl lactate to form an upper-layer or lower-layer channel structure template;
(4) hardening the film for 1-2 hours at 180 ℃, and cooling to form upper-layer and lower-layer structure templates of the chip;
(5) the chip template was subjected to a low adhesion treatment using trimethylsilane.
The preparation method of the microfluidic detection chip comprises the following specific steps:
(1) respectively preparing upper and lower structures of a PDMS chip by using the prepared upper and lower structure templates of the chip;
(2) spin coating 1-2wt% of chitosan on the surface of a glass substrate, and placing on a hot plate to form a chitosan film;
(3) dipping the lower chip structure with pre-polymerized PDMS with the thickness of about 5-10 microns, placing the pre-polymerized PDMS on a chitosan film, and then placing the chitosan film in an oven with the temperature of 80 ℃ to cure the PDMS;
(4) injecting 0.5-5% NaOH solution into the lower chip channel, and immersing the substrate containing the chitosan film and the lower chip structure combined with the chitosan film into the 0.5-5% NaOH solution;
(5) after 48-72 hours, separating the PDMS on the lower layer of the chip from the substrate by using the chitosan film, washing away the residual NaOH solution by using deionized water, and drying at room temperature or in an oven;
(6) and aligning the upper chip structure and the lower chip structure after the plasma treatment, and sealing the upper chip structure and the lower chip structure on the other side of the chitosan film to form the required microfluidic detection chip.
The invention provides a method for using a microfluidic detection chip, which comprises the following specific steps:
(1) respectively injecting cell suspension and barcode microbeads to be detected into the chip from upper and lower inlets of the chip, and according to the design of the chip, after each cell or microbead enters a corresponding capturing unit, clamping the cell or microbead at a narrow position behind the capturing unit, so that the flow resistance of the capturing unit is increased, subsequent cells or microbeads enter a side secondary channel with smaller flow resistance along with fluid, then enter the next capturing unit, and the like;
(2) after the cells and the microbeads pass through the chip, introducing the oil phase into the chip from the inlets of the upper chip and the lower chip so as to remove the water phase in the fluid channel, and simultaneously forming independent liquid drops by the liquid remained in the capture unit;
(3) injecting oil phase containing 0.1-1wt% acetic acid from the inlets of the upper and lower chips to dissolve the chitosan film under the action of the acetic acid, so that the liquid drops in the upper and lower capturing units are contacted and fused to form one-to-one correspondence of cells and microbeads;
(4) after the required reaction is finished, reversely introducing corresponding buffer solution from the outlet of the chip to wash out the microbeads; or collecting the micro-beads after the upper layer and the lower layer of the chip are uncovered.
After the microbeads are collected, the subsequent processes of reverse transcription, PCR, library building, sequencing, analysis and the like are carried out according to a required method.
The microfluidic detection chip can be used for single cell sequencing and other researches.
The invention realizes the capture of single cells and single microbeads by utilizing the microstructure of the microfluidic chip; the chip contains a soluble chitosan film which plays a role in separation in the capture process of cells and microbeads, and the combination of mRNA and the microbeads is completed after the cells and the microbeads are dissolved; the vertically aligned structure of the chip can greatly improve the pairing rate of cells and microbeads, reduce the consumption of the microbeads and also greatly improve the cell utilization efficiency of single cell sequencing; the structure of the upper and lower chambers can also reduce the cross contamination between single-cell nucleic acids; in addition, the liquid drops formed in the chip capture structure do not contain a surfactant, and the step of emulsion breaking can be reduced in the subsequent operation of collecting the microbeads.
Drawings
Fig. 1 is a diagram of a chip channel structure.
FIG. 2 is a schematic diagram of a cell or bead capture procedure.
Fig. 3 is a schematic diagram of the use of chitosan membrane dissolution in a chip.
Reference numbers in the figures: 1 is the primary channel, 2 is the capture chamber, and 3 is the secondary channel.
Detailed Description
The invention utilizes the micro-fluidic chip technology to prepare the capture chamber with an array structure, and simultaneously utilizes soluble chitosan to separate an upper chip from a lower chip. After the single cells and the microbeads are captured by the chips, the corresponding capture chambers of the upper and lower chips are communicated by dissolving the chitosan of the separation layer, and the nucleic acid molecules after cell lysis are captured by the microbeads; finally, the microbeads are recovered, and then the processes of transcription, PCR, library building, sequencing, analysis and the like are carried out according to the required method.
Example 1
The preparation of the microfluidic chip template adopts a soft lithography method, and the upper and lower chip structure templates are prepared according to the following steps:
(1) spin-coating SU-8 photoresist (the thickness of cell layer photoresist is 30 microns, and the thickness of microsphere layer photoresist is about 50 microns) on the surface of a silicon wafer substrate, and baking for 30 minutes at 95 ℃;
(2) fixing a mask containing an upper layer or a lower layer channel structure on the surface of a substrate containing photoresist, and polymerizing SU-8 according to the structure of the mask by ultraviolet exposure as shown in figure 2;
(3) baking for 30 minutes at 95 ℃, naturally cooling, and removing unexposed SU-8 glue by using ethyl lactate to form an upper-layer or lower-layer channel structure template;
(4) hardening the film for 2 hours at 180 ℃, and cooling to form upper-layer and lower-layer structure templates of the chip;
(5) the chip template was subjected to a low adhesion treatment using trimethylsilane.
The micro-fluidic chip is prepared according to the following steps:
(1) preparing upper and lower structures of the PDMS chip by using the upper and lower templates respectively;
(2) spin-coating 2wt% of chitosan on the surface of a glass substrate, and placing the glass substrate on a hot plate to form a chitosan film;
(3) dipping the lower chip structure with 10 micron thick pre-polymerized PDMS, placing on the chitosan film, and then placing in an oven at 80 ℃ to solidify the PDMS;
(4) injecting 2% NaOH solution into the lower chip channel, and immersing the glass substrate containing the chitosan film and the lower chip structure combined with the chitosan film into the 2% NaOH solution;
(5) after about 48 hours, the PDMS on the lower layer of the chip is separated from the glass substrate by the chitosan film, and the residual NaOH solution is washed by deionized water and then is dried in room temperature or an oven;
(6) and aligning the structure of the upper chip and the lower chip after plasma treatment, and sealing the upper chip and the lower chip on the other side of the chitosan film to form the chip.
Example 2
The use method of the single cell detection chip comprises the following steps:
(1) respectively injecting cell suspension (100/microliter) and barcode microbeads (the diameter is about 40um and 100/microliter) to be detected into the chip from the inlets of the upper layer (cell layer) and the lower layer (microbead layer) of the chip, and according to the design of the chip, each cell or microbead enters a corresponding capturing unit and then is clamped in a narrow part behind the unit, so that the flow resistance of the unit is increased, and subsequent cells or microbeads enter a side secondary channel with smaller flow resistance along with the fluid and then enter the next capturing unit, as shown in fig. 2, and so on;
(2) after the cells and the microbeads pass through the chip, introducing the oil phase into the chip from the inlets of the upper chip and the lower chip so as to remove the water phase in the fluid channel, and simultaneously forming independent liquid drops by the liquid remained in the capture unit;
(3) then injecting an oil phase containing 0.1-1wt% of acetic acid into the upper layer and the lower layer of the chip, wherein the chitosan film can be dissolved under the action of the acetic acid, so that the liquid drops in the capture units of the upper layer and the lower layer are contacted and fused to form one-to-one correspondence of cells and microbeads, as shown in figure 3;
(4) after the required reaction is finished, a corresponding buffer solution can be reversely introduced from the outlet of the chip to wash out the microbeads; or collecting the micro-beads after the upper layer and the lower layer of the chip are uncovered;
(5) after the microbeads are collected, the procedures of reverse transcription, PCR, library construction, sequencing, analysis and the like can be carried out according to a required method.
Experiments show that the capture structure of the microfluidic chip can greatly improve the capture efficiency of single cells (experimental statistics result: more than 80% of capture areas can capture single cells), and meanwhile, the structures of the upper layer and the lower layer can also improve the pairing efficiency of the single cells and microbeads, so that the cell utilization efficiency of single cell sequencing is improved (experimental statistics result: the cell utilization efficiency is more than 50%).
Claims (6)
1. A single cell micro-fluidic detection chip is characterized by comprising an upper chip and a lower chip, wherein a layer of soluble chitosan film is arranged in the middle to separate the upper chip from the lower chip; wherein:
the upper and lower chips contain fluid channel structures which are corresponding to each other up and down; the upper chip and the lower chip respectively comprise a liquid inlet and a liquid outlet which are communicated with the fluid channel;
the upper layer fluid channel and the lower layer fluid channel of the microfluidic detection chip respectively comprise a main channel, and a plurality of capture chambers are arranged on the main channel; a secondary channel is also arranged beside each capture chamber of the upper layer and the lower layer of the microfluidic detection chip, and two ends of the secondary channel are communicated with the main channel.
2. The single-cell microfluidic detection chip of claim 1, wherein the number of the capture chambers on the upper and lower main channels of the microfluidic detection chip is 200-1000.
3. The single-cell microfluidic detection chip of claim 1, wherein the height of the main channel and the sub-channel on the upper layer and the lower layer of the microfluidic detection chip is 15-100 micrometers, and the width is 50-150 micrometers; the capture chamber has an inlet width of 50-150 microns; the outlet width of the capture chamber is 10-50 microns.
4. A method for preparing a single-cell microfluidic detection chip as claimed in any one of claims 1 to 3, comprising the preparation of upper and lower chip templates by soft lithography, comprising the following steps:
(1) spin-coating SU-8 photoresist with the thickness of 15-50 microns on the surface of a silicon wafer or a glass substrate, and baking for 15-60 minutes at the temperature of 95 ℃;
(2) fixing a mask containing an upper layer channel structure or a lower layer channel structure on the surface of a substrate containing photoresist, and polymerizing SU-8 according to the structure of the mask by ultraviolet exposure;
(3) baking for 10-30 minutes at 95 ℃, naturally cooling, and removing unexposed SU-8 glue by using ethyl lactate to form an upper-layer or lower-layer channel structure template;
(4) hardening the film for 1-2 hours at 180 ℃, and cooling to form upper-layer and lower-layer structure templates of the chip;
(5) the chip template was subjected to a low adhesion treatment using trimethylsilane.
5. The method for preparing the single-cell microfluidic detection chip of claim 4, further comprising the step of,
(1) respectively preparing upper and lower structures of a PDMS chip by using the prepared upper and lower structure templates of the chip;
(2) spin coating 1-2wt% of chitosan on the surface of a glass substrate, and placing on a hot plate to form a chitosan film;
(3) dipping the lower chip structure with 5-10 micron thick prepolymerized PDMS, placing on the chitosan film, and then placing in a 60-90 ℃ oven to cure PDMS;
(4) injecting 0.5-5% NaOH solution into the lower chip channel, and immersing the substrate containing the chitosan film and the lower chip structure combined with the chitosan film into the 0.5-5% NaOH solution;
(5) after 48-72 hours, separating the PDMS on the lower layer of the chip from the substrate by using the chitosan film, washing away the residual NaOH solution by using deionized water, and drying at room temperature or in an oven;
(6) and aligning the upper chip structure and the lower chip structure after the plasma treatment, and sealing the upper chip structure and the lower chip structure on the other side of the chitosan film to form the required microfluidic detection chip.
6. A method for using the single-cell microfluidic detection chip as claimed in any one of claims 1 to 3, which comprises the following steps:
(1) respectively injecting cell suspension and barcode microbeads to be detected into the chip from upper and lower inlets of the chip, and according to the design of the chip, each cell or microbead enters a corresponding capturing unit and then is clamped at a narrow position behind the unit, so that the flow resistance of the unit is increased, subsequent cells or microbeads enter a side sub-channel with smaller flow resistance along with fluid and then enter the next capturing unit, and the like;
(2) after the cells and the microbeads pass through the chip, introducing the oil phase into the chip from the inlets of the upper and lower chips to remove the water phase in the fluid channel, and simultaneously enabling the liquid remained in the capturing unit to form independent liquid drops;
(3) injecting an oil phase containing 0.1-1wt% of acetic acid from inlets of the upper and lower chips, dissolving the chitosan film under the action of the acetic acid, and enabling liquid drops in the upper and lower capturing units to contact and fuse to form one-to-one correspondence of cells and microbeads;
(4) after the required reaction is finished, a corresponding buffer solution is reversely introduced from the outlet of the chip to wash out the microbeads, or the microbeads are collected after the upper layer and the lower layer of the chip are uncovered.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111254046A (en) * | 2020-01-21 | 2020-06-09 | 浙江大学 | Device and method for co-capturing single cell and single microsphere |
| CN113046227A (en) * | 2021-03-30 | 2021-06-29 | 扬州大学 | High-flux lymphoma cell microfluidic detection chip and detection method thereof |
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