CN107828651B - Micro-fluidic chip for preparing single-cell micro-droplet sample - Google Patents
Micro-fluidic chip for preparing single-cell micro-droplet sample Download PDFInfo
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Abstract
The invention relates to the technical field of microfluidic chips, in particular to a microfluidic chip for preparing a single-cell micro-droplet sample, which comprises a chip body, a single-cell micro-droplet sample preparation unit and a blank droplet removal unit, wherein the single-cell micro-droplet sample preparation unit and the blank droplet removal unit are distributed on the chip body and are connected through a micro-channel of the microfluidic chip; the invention realizes the preparation of the single-cell micro-droplet sample with high flux and high ratio by the single-cell micro-droplet sample preparation unit and the blank droplet removing unit; by controlling the size of the blank liquid drop removing unit microstructure, different cell samples can be screened; therefore, 95% of micro-droplets wrap cells in the preparation of the cell micro-droplet sample, the utilization rate of biological reagents is improved, the screening efficiency of experimental results is improved, and the workload and the cost of the biological reagents are reduced for the subsequent micro-droplet-based cell culture, single cell in-situ fluorescence detection, single cell PCR and the like.
Description
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a microfluidic chip for preparing a unicellular micro-droplet sample.
Background
Biological sample processing, including cells, bacteria, proteins, DNA, etc., often plays a crucial role in biological experiments, especially in biological and medical experiments involving single cell sequencing, single cell screening, digital PCR, etc., at the forefront. In addition, a circulating tumor cell is involved in the processes of early diagnosis, clinical treatment, detection after healing and drug screening of cancer. Circulating tumor cells are released from cancer cells in a primary focus and enter a peripheral blood circulation system, and the circulating tumor cells have cell information of protocarcinoma, so that the circulating tumor cells have important significance in cancer research by being used as markers. However, circulating tumor cells are very rare in peripheral blood and are one in a hundred million parts of normal blood cells such as red blood cells or white blood cells. Therefore, it is a great challenge to isolate and purify, and even extract, circulating tumor cells. At present, the capture and separation of circulating tumor cells need to be realized by antigen and antibody, but the separation efficiency is low, the cell purity is low, and the subsequent cell and molecular level research is influenced. How to reduce the interference of normal cells is very critical in the pretreatment of samples. At present, the pretreatment of cell samples is realized by gradient centrifugation on one hand and micro-droplet technology of microfluidic chips on the other hand. Since the size of the cell is generally in the micron level, the microfluidic chip micro-droplet technology is widely applied to the cell sample treatment. The cells in the micro-droplets can avoid interfering with other cells. However, there are limitations to the use of microfluidic chips for preparing cell samples. In the prepared sample, the blank liquid drop accounts for a large amount, the liquid drops wrapping one cell, two cells and three or more cells are distributed in a positive mode, but the micro liquid drops containing the cells account for less than 30 percent of the total amount. Since the size of the micro-droplets is very small, in the order of picoliters or nanoliters, the number of micro-droplets is tens of millions after a biological sample is prepared. Therefore, how to remove blank liquid drops is very important for single cell sample preparation, the number of cell processing is reduced, and the time cost is saved. Therefore, the method realizes the removal of blank liquid drops and improves the proportion of single-cell liquid drops by the channel design of the microfluidic chip, and has important application fields.
Disclosure of Invention
In order to solve the problems existing in the existing microfluidic chip, the invention provides a microfluidic chip for preparing a single-cell micro-droplet sample, and solves the technical problems of large workload, low efficiency, long time consumption and the like in subsequent biological analysis caused by large proportion of blank droplets in the existing microfluidic chip cell micro-droplet sample treatment.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a micro-fluidic chip for preparing a single-cell micro-droplet sample comprises a chip body, a single-cell micro-droplet sample preparation unit and a blank droplet removal unit, wherein the single-cell micro-droplet sample preparation unit and the blank droplet removal unit are distributed on the chip body; the blank droplet removing unit comprises a second oil phase sample feeding unit connected with an external oil phase, a blank droplet removing double-height microstructure, a single-cell micro-droplet collecting unit used for being connected with an external sample collector and a blank micro-droplet collecting unit used for being connected with the external blank micro-droplet collector, the second oil phase sample feeding unit is connected with the second oil phase to enable micro-droplets to flow through the blank droplet separating microstructure, the blank droplet removing double-height microstructure has certain strength through height difference and cell nucleus to realize the separation of the blank micro-droplets and the micro-droplets wrapped with cells, and the single-cell micro-droplet sample preparing unit and the blank droplet removing unit are connected through a micro-channel of a micro-fluidic chip.
Further, the micro-droplet sample preparation unit includes: t-shaped channel, cross-shaped channel, Y-shaped channel and coaxial channel.
Further, the blank droplet removal unit includes a blank micro-droplet separation channel and a separation channel support structure.
Preferably, the height of the blank micro-droplet separation channel is 1-15 micrometers, the width of the blank micro-droplet separation channel is 1-200 micrometers, and the length of the blank micro-droplet separation channel is 10-1000 micrometers.
Preferably, the height of the separation channel supporting structure is 1-15 micrometers, the width is 1-200 micrometers, and the length is 1-100 micrometers.
Furthermore, the material of the microfluidic chip is polydimethylsiloxane, glass, polydimethylsiloxane, polycarbonate, polymethyl methacrylate and polyethylene glycol terephthalate.
Preferably, the microfluidic chip is prepared by bonding polydimethylsilane, polycarbonate, polymethyl methacrylate and polyethylene terephthalate containing the microchannel with a glass substrate by oxygen plasma.
Preferably, the microfluidic chip is prepared by bonding polydimethylsiloxane, polycarbonate, polymethyl methacrylate and polyethylene terephthalate containing the microchannel with a polydimethylsiloxane substrate through oxygen plasma.
Furthermore, the cell sample injection unit, the oil phase sample injection unit and the second oil phase sample injection unit are injected into the microfluidic chip through an injection pump.
Preferably, the cell sample injection unit, the oil phase injection unit and the second oil phase injection unit are positive pressure generated by air pressure to force the sample into the microfluidic chip.
Furthermore, except the blank micro-droplet separation channel, the height of the rest micro-channels of the micro-fluidic chip is 10-200 microns, and the width of the rest micro-channels is 10-500 microns.
Further, the mask layer containing the blank micro-droplet separation channel and the mask layers of other micro-channels are realized by double-layer photoetching.
Compared with the prior art, the invention has the beneficial effects that: according to the micro-fluidic chip for preparing the single-cell micro-droplet sample, the preparation of the single-cell micro-droplet sample with high throughput and high ratio is realized through the single-cell micro-droplet sample preparation unit and the blank droplet removal unit; furthermore, by controlling the size of the blank liquid drop removing unit microstructure, different cell samples can be screened; therefore, 95% of micro-droplets wrap cells in the preparation of the cell micro-droplet sample, the utilization rate of biological reagents is improved, the screening efficiency of experimental results is improved, and the workload and the cost of the biological reagents are reduced for the subsequent micro-droplet-based cell culture, single cell in-situ fluorescence detection, single cell PCR and the like.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic structural diagram of a microfluidic chip according to the present invention;
FIG. 2 is a schematic diagram of a microfluidic chip of the present invention;
FIG. 3 is a schematic diagram of a blank droplet removal unit microstructure according to the present invention;
FIG. 4 is a side view of a blank droplet removal unit microstructure of the present invention;
FIG. 5 is a first photolithography mask layer of the microfluidic chip according to the present invention;
FIG. 6 is a second photolithography mask layer for a microfluidic chip according to the present invention;
FIG. 7 is a diagram showing the alignment effect of the first microfluidic chip lithography mask layer and the microfluidic chip lithography mask layer for single-cell micro-droplet sample preparation according to the present invention.
Fig. 8 is a scanning electron microscope image of a blank micro-droplet separation channel according to the present invention.
In the figure: 0. the system comprises a micro-fluidic chip, 1, a single-cell micro-droplet sample preparation unit, 11, an oil phase sample introduction unit, 12, a cell sample introduction unit, 13, a micro-droplet sample preparation unit, 2, a blank droplet removal unit, 21, a second oil phase sample introduction unit, 22, a blank micro-droplet collection unit, 23, a single-cell micro-droplet collection unit, 24, a blank droplet removal double-height microstructure, 241, a blank micro-droplet separation channel, 242, a separation channel support structure, 33, a micro-droplet preparation channel, 34, a blank micro-droplet collection channel, 4, a micro-fluidic chip photoetching mask layer I, 41-43, a photoetching alignment pattern I, 5, a micro-fluidic chip photoetching mask layer II, 51-53 and a photoetching alignment pattern II.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 1-8, the microfluidic chip for preparing a single-cell micro-droplet sample according to the present invention comprises a chip body, and a single-cell micro-droplet sample preparation unit 1 and a blank droplet removal unit 2 distributed on the chip body, wherein the single-cell micro-droplet sample preparation unit 1 comprises a cell sample injection unit 12 connected to an external cell sample, an oil phase injection unit 11 connected to an external oil phase, and a micro-droplet sample preparation unit 13, and the micro-droplet sample preparation unit 13 is connected to the cell sample injection unit 12 and the oil phase injection unit 11 in a T-shaped or cross-shaped structure and realizes the preparation of micro-droplets through a fluid shear force; the blank droplet removing unit 2 comprises a second oil phase sample introduction unit 21 connected with an external oil phase, a blank droplet removing double-height microstructure 24, a single-cell micro-droplet collecting unit 23 connected with an external sample collector and a blank micro-droplet collecting unit 22 connected with the external blank micro-droplet collector, the second oil phase sample introduction unit 21 is connected with the second oil phase to enable micro-droplets to flow through a blank droplet separating microstructure, the blank droplet removing double-height microstructure 24 realizes the separation of the blank micro-droplets and the micro-droplets wrapped with cells through the characteristic that the height difference and cell nuclei have certain strength, and the single-cell micro-droplet sample preparation unit 1 and the blank droplet removing unit 2 are connected through a micro-channel of a micro-fluidic chip.
Further, the micro droplet sample preparation unit 1 includes: t-shaped channel, cross-shaped channel, Y-shaped channel and coaxial channel.
Further, the blank droplet removing unit 2 includes a blank micro-droplet separation channel 241 and a separation channel support structure 242.
Preferably, the height of the blank micro-droplet separation channel 241 is 1 to 15 micrometers, the width is 1 to 200 micrometers, and the length is 10 to 1000 micrometers.
Preferably, the height of the separation channel support structure 242 is 1-15 micrometers, the width is 1-200 micrometers, and the length is 1-100 micrometers.
Furthermore, the material of the microfluidic chip is polydimethylsiloxane, glass, polydimethylsiloxane, polycarbonate, polymethyl methacrylate and polyethylene glycol terephthalate.
Preferably, the microfluidic chip is prepared by bonding polydimethylsilane, polycarbonate, polymethyl methacrylate and polyethylene terephthalate containing the microchannel with a glass substrate by oxygen plasma.
Preferably, the microfluidic chip is prepared by bonding polydimethylsiloxane, polycarbonate, polymethyl methacrylate and polyethylene terephthalate containing the microchannel with a polydimethylsiloxane substrate through oxygen plasma.
Further, the cell sample injection unit 12, the oil phase sample injection unit 11 and the second oil phase sample injection unit 21 are injected into the microfluidic chip by an injection pump.
Preferably, the cell sample injection unit 12, the oil phase injection unit 11 and the second oil phase injection unit 21 are positive pressure generated by air pressure to force the sample into the microfluidic chip.
Furthermore, except the blank micro-droplet separation channel, the height of the rest micro-channels of the micro-fluidic chip is 10-200 microns, and the width of the rest micro-channels is 10-500 microns.
Further, the mask layer containing the blank micro-droplet separation channel 241 and the mask layers of the other micro-channels are realized by double-layer lithography.
According to the micro-fluidic chip for preparing the single-cell micro-droplet sample, the preparation of the single-cell micro-droplet sample with high throughput and high ratio is realized through the single-cell micro-droplet sample preparation unit and the blank droplet removal unit; furthermore, by controlling the size of the blank liquid drop removing unit microstructure, different cell samples can be screened; therefore, 95% of micro-droplets wrap cells in the preparation of the cell micro-droplet sample, the utilization rate of biological reagents is improved, the screening efficiency of experimental results is improved, and the workload and the cost of the biological reagents are reduced for the subsequent micro-droplet-based cell culture, single cell in-situ fluorescence detection, single cell PCR and the like.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A micro-fluidic chip for preparing a single-cell micro-droplet sample is characterized by comprising a chip body, a single-cell micro-droplet sample preparation unit and a blank droplet removal unit, wherein the single-cell micro-droplet sample preparation unit and the blank droplet removal unit are distributed on the chip body; the blank droplet removing unit comprises a second oil phase sample introduction unit connected with an external oil phase, a blank droplet removing double-height microstructure, a single-cell micro-droplet collecting unit connected with an external sample collector and a blank micro-droplet collecting unit connected with the external blank micro-droplet collector, the second oil phase sample introduction unit is connected with the second oil phase to enable micro-droplets to flow through the blank droplet separating microstructure, the blank droplet removing double-height microstructure realizes the separation of the blank micro-droplets and the micro-droplets wrapped with cells through the height difference and the characteristic of certain strength of cell nuclei, and the single-cell micro-droplet sample preparation unit and the blank droplet removing unit are connected through a micro-channel of the micro-fluidic chip;
the cell sample introduction unit, the oil phase sample introduction unit and the second oil phase sample introduction unit are used for forcing a sample to enter the microfluidic chip by positive pressure generated by air pressure;
except for the blank micro-droplet separation channel, the height of the rest micro-channels of the micro-fluidic chip is 10-200 micrometers, and the width of the rest micro-channels is 10-500 micrometers.
2. The microfluidic chip for single-cell micro-droplet sample preparation according to claim 1, wherein: the micro-droplet sample preparation unit includes: t-shaped channel, cross-shaped channel, Y-shaped channel and coaxial channel.
3. The microfluidic chip for single-cell micro-droplet sample preparation according to claim 1, wherein: the blank droplet removing unit comprises a blank micro droplet separation channel and a separation channel supporting structure, the height of the blank micro droplet separation channel is 1-15 micrometers, the width of the blank micro droplet separation channel is 1-200 micrometers, the length of the blank micro droplet separation channel is 10-1000 micrometers, the height of the separation channel supporting structure is 1-15 micrometers, the width of the separation channel supporting structure is 1-200 micrometers, and the length of the separation channel supporting structure is 1-100 micrometers.
4. The microfluidic chip for single-cell micro-droplet sample preparation according to claim 1, wherein: the material of the micro-fluidic chip is polydimethylsiloxane, glass, polydimethylsiloxane, polycarbonate, polymethyl methacrylate and polyethylene glycol terephthalate.
5. The microfluidic chip for single-cell micro-droplet sample preparation according to claim 4, wherein: the micro-fluidic chip is prepared by bonding polydimethylsilane, polycarbonate, polymethyl methacrylate and polyethylene glycol terephthalate containing a micro-channel with a glass substrate through oxygen plasma.
6. The microfluidic chip for single-cell micro-droplet sample preparation according to claim 4, wherein: the microfluidic chip is prepared by bonding polydimethylsiloxane, polycarbonate, polymethyl methacrylate, polyethylene glycol terephthalate and a polydimethylsiloxane substrate containing a microchannel through oxygen plasma.
7. The microfluidic chip for single-cell micro-droplet sample preparation according to claim 1, wherein: the cell sample injection unit, the oil phase sample injection unit and the second oil phase sample injection unit are injected into the microfluidic chip through an injection pump.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710891682.3A CN107828651B (en) | 2017-09-27 | 2017-09-27 | Micro-fluidic chip for preparing single-cell micro-droplet sample |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710891682.3A CN107828651B (en) | 2017-09-27 | 2017-09-27 | Micro-fluidic chip for preparing single-cell micro-droplet sample |
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| Publication Number | Publication Date |
|---|---|
| CN107828651A CN107828651A (en) | 2018-03-23 |
| CN107828651B true CN107828651B (en) | 2021-02-19 |
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| CN210427605U (en) * | 2018-06-07 | 2020-04-28 | 洛阳华清天木生物科技有限公司 | Micro-droplet processing device |
| CN109112063A (en) * | 2018-08-20 | 2019-01-01 | 北京旌微医学工程研究院有限公司 | A kind of detection of nucleic acids micro-fluidic chip and preparation method thereof |
| US20220041967A1 (en) * | 2018-09-20 | 2022-02-10 | Thunderbio Innovation Ltd | Real-time monitoring of single cell or events |
| CN110923127A (en) * | 2018-09-20 | 2020-03-27 | 北京怡天佳瑞科技有限公司 | Micro-fluidic chip |
| CN111041077A (en) * | 2018-10-15 | 2020-04-21 | 深圳华大生命科学研究院 | Sample loading method, sequencing method and sequencing system for high-throughput sequencing |
| CN112403538A (en) * | 2019-08-23 | 2021-02-26 | 无锡源清天木生物科技有限公司 | Device and method for generating and fusing liquid drops |
| CN110967513B (en) * | 2019-12-18 | 2024-04-05 | 京东方科技集团股份有限公司 | Sample primary screening chip, sample detection method and screening chip device |
| CN112574937A (en) * | 2020-12-31 | 2021-03-30 | 江南大学 | Ultramicro injection method based on micro-fluidic chip and application thereof in high-throughput screening |
| CN115400809B (en) * | 2021-05-28 | 2024-05-31 | 深圳华大生命科学研究院 | Method for recycling content in water-in-oil droplets and droplet generation device |
| CN115521882A (en) * | 2021-06-24 | 2022-12-27 | 清华大学 | Micro-upgrading single-cell droplet generation and culture method and device |
| CN114317585B (en) * | 2021-12-31 | 2023-07-04 | 南京工业大学 | Liquid drop microfluidic assisted method for screening double rhamnolipid high-yield strain |
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