METHOD FOR FORMING AND RESPECTIVELY EXPORTING DROPLET WRAPPING SINGLE PARTICLE IN MICRO-FLUIDIC CHIP

Abstract

A micro-fluidic chip that can be used for screening a single particle and forming and exporting a droplet wrapping same. The micro-fluidic chip is connected to a liquid sample introduction apparatus, and can constitute a micro-fluidic chip apparatus for forming a droplet for wrapping a single particle. The micro-fluidic chip apparatus can further constitute, with a particle capture apparatus, a micro-fluidic operating system for forming a droplet wrapping a single particle. Further provided is a method for forming and respectively exporting a droplet wrapping a single particle in a micro-fluidic chip.

Claims

1. A microfluidic chip, which comprises a cover layer and a substrate layer; wherein the substrate layer comprises at least one sample channel, and the sample channel comprises a microfluidic channel and a detection cell; and the cover layer comprises a sample injection hole and an oil storage well hole; wherein the oil storage well hole and the substrate layer form an oil storage well; the inlet of the sample channel is connected with the sample injection hole, and an outlet of the sample channel is connected with the oil storage well.

2. The microfluidic chip according to claim 1, wherein the microfluidic channel comprises a straight channel and at least one curved channel.

3. The microfluidic chip according to claim 1, wherein the detection cell and the oil storage well hole are connected through a channel, and the channel is a straight channel.

4. The microfluidic chip according to claim 1, wherein the sample channel further comprises at least one liquid storage well, and the detection cell is located between the liquid storage well and the oil storage well hole, wherein the liquid storage well and the detection cell are connected through a channel, and the liquid storage well and the curved channel are connected through a channel, wherein the channels comprise at least one straight channel or at least one curved channel.

5. Preparation method of the microfluidic chip according to claim 1, comprising steps: (i) etching the substrate layer according to design of the sample channel, (ii) punching the sample injection holes and oil storage well holes on the cover layer, (iii) aligning the cover layer and the substrate layer and bonding the same using low-temperature bonding method, and (iv) hydrophobization of the surfaces.

6. A microfluidic chip device for forming single microparticle-encapsulated liquid droplets, which comprises the microfluidic chip according to claim 1, and a liquid sample injection device, wherein the liquid sample injection device is connected with the sample injection hole, and the liquid sample injection device is selected from a group consisting of a gravity-driven sample injection device, a syringe, a peristaltic pump, a syringe pump, and combinations thereof.

7. The microfluidic device according to claim 6, wherein the gravity-driven sample injection device comprises a height-adjustable sample holder, a sample container, and a catheter, wherein the sample container is connected with the sample injection hole through the catheter, and the sample container is capable of moving up and down on the height-adjustable sample holder.

8. A microfluidic operating system for forming single microparticle-encapsulated droplets, comprising the microfluidic chip according to claim 1, and a microparticle capture device, wherein the microparticle capture device is selected from optical tweezers and magnetic tweezers.

9. A method for forming and exporting single microparticle-encapsulated droplets, wherein the method utilizes the microfluidic chip according to claim 1, comprising: (i) injecting an oil phase into the oil storage well; (ii) injecting the microparticle phase solution into the sample channel of the microfluidic chip through the injection hole; (iii) adjusting the liquid flow in sample channel so that the interface between the microparticle phase and the oil phase in the oil storage well is steadily stationary nearby the outlet of sample channel; (iv) capturing the target microparticles with a microparticle capture device, moving the microfluidic chip, and dragging the target microparticles to the vicinity of the interface between the aqueous phase and oil phase; (v) adjusting liquid flow in sample channel to allow the target microparticles to enter oil storage well and to form liquid droplets encapsulating single target microparticles; and (iv) exporting the droplets encapsulating single target microparticles; wherein method for adjusting liquid flow in sample channels is selected from a group consisting of gravity-driven adjusting, injection pump-driven adjusting, and peristaltic pump-driven adjusting and combinations thereof, and method for exporting the droplets encapsulating single target microparticles is selected from a group consisting of capillary tube exporting, pipette exporting and combinations thereof.

10. Use of the microfluidic chip according to claim 1, for screening of single microparticles, formation of single microparticle-encapsulated droplets, or exportation of single microparticle-encapsulated droplets.

11. A microfluidic operating system for forming single microparticle-encapsulated droplets, comprising the microfluidic chip device according to claim 6, and a microparticle capture device, wherein the microparticle capture device is selected from optical tweezers and magnetic tweezers.

12. A method for forming and exporting single microparticle-encapsulated droplets, wherein the method utilizes the microfluidic device according to claim 6, comprising: (i) injecting an oil phase into the oil storage well; (ii) injecting the microparticle phase solution into the sample channel of the microfluidic chip through the injection hole; (iii) adjusting the liquid flow in sample channel so that the interface between the microparticle phase and the oil phase in the oil storage well is steadily stationary nearby the outlet of sample channel; (iv) capturing the target microparticles with a microparticle capture device, moving the microfluidic chip, and dragging the target microparticles to the vicinity of the interface between the aqueous phase and oil phase; (v) adjusting liquid flow in sample channel to allow the target microparticles to enter oil storage well and to form liquid droplets encapsulating single target microparticles; and (iv) exporting the droplets encapsulating single target microparticles; wherein method for adjusting liquid flow in sample channels is selected from a group consisting of gravity-driven adjusting, injection pump-driven adjusting, and peristaltic pump-driven adjusting and combinations thereof, and method for exporting the droplets encapsulating single target microparticles is selected from a group consisting of capillary tube exporting, pipette exporting and combinations thereof.

13. Use of the microfluidic chip device according to claim 6 for screening of single microparticles, formation of single microparticle-encapsulated droplets, or exportation of single microparticle-encapsulated droplets.

14. A method for forming and exporting single microparticle-encapsulated droplets, wherein the method utilizes the microfluidic operating system according to claim 8, comprising: (i) injecting an oil phase into the oil storage well; (ii) injecting the microparticle phase solution into the sample channel of the microfluidic chip through the injection hole; (iii) adjusting the liquid flow in sample channel so that the interface between the microparticle phase and the oil phase in the oil storage well is steadily stationary nearby the outlet of sample channel; (iv) capturing the target microparticles with a microparticle capture device, moving the microfluidic chip, and dragging the target microparticles to the vicinity of the interface between the aqueous phase and oil phase; (v) adjusting liquid flow in sample channel to allow the target microparticles to enter oil storage well and to form liquid droplets encapsulating single target microparticles; and (iv) exporting the droplets encapsulating single target microparticles; wherein method for adjusting liquid flow in sample channels is selected from a group consisting of gravity-driven adjusting, injection pump-driven adjusting, and peristaltic pump-driven adjusting and combinations thereof, and method for exporting the droplets encapsulating single target microparticles is selected from a group consisting of capillary tube exporting, pipette exporting and combinations thereof.

15. Use of the microfluidic operating system according to claim 8 for screening of single microparticles, formation of single microparticle-encapsulated droplets, or exportation of single microparticle-encapsulated droplets.

16. A method for forming and exporting single microparticle-encapsulated droplets, wherein the method utilizes the microfluidic operating system according to claim 11, comprising: (i) injecting an oil phase into the oil storage well; (ii) injecting the microparticle phase solution into the sample channel of the microfluidic chip through the injection hole; (iii) adjusting the liquid flow in sample channel so that the interface between the microparticle phase and the oil phase in the oil storage well is steadily stationary nearby the outlet of sample channel; (iv) capturing the target microparticles with a microparticle capture device, moving the microfluidic chip, and dragging the target microparticles to the vicinity of the interface between the aqueous phase and oil phase; (v) adjusting liquid flow in sample channel to allow the target microparticles to enter oil storage well and to form liquid droplets encapsulating single target microparticles; and (iv) exporting the droplets encapsulating single target microparticles; wherein method for adjusting liquid flow in sample channels is selected from a group consisting of gravity-driven adjusting, injection pump-driven adjusting, and peristaltic pump-driven adjusting and combinations thereof, and method for exporting the droplets encapsulating single target microparticles is selected from a group consisting of capillary tube exporting, pipette exporting and combinations thereof.

17. Use of the microfluidic operating system according to claim 11 for screening of single microparticles, formation of single microparticle-encapsulated droplets, or exportation of single microparticle-encapsulated droplets.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 shows a schematic diagram of microfluidic chip design.

[0054] FIG. 2 shows a picture of a microfluidic chip.

[0055] FIG. 3 shows a diagram of microfluidic chip device for forming single microparticle-encapsulated droplets. A shows a schematic diagram of a microfluidic chip device composed of a liquid sample injection device and a microfluidic chip. B shows a picture of a microfluidic operating system, which is composed of a microfluidic chip device and a microparticle capture device, and is used to form a single microparticle-encapsulated droplet.

[0056] FIG. 4 shows the process of forming a single microparticle-encapsulated droplet.

[0057] FIG. 5 illustrates the process of sorting individual cells by the microfluidic chip device has low impacts on cell viability. A shows a just wrapped droplet that is then sucked out of the open oil storage well by the capillary tube, the whole process of which does not require external power and is based on the capillary force of the oil phase in the capillary tube. B shows that by placing the capillary tube at the channel outlet, the oil phase will flow into the capillary tube by capillary force, and the single cell encapsulated droplets in the oil phase will then flow into the capillary tube along with the oil phase. CF are video screenshots of this process. G and H are the cell encapsulated droplets under the field of view of the 50 objective lens, taking E. coli cell and yeast cell as model cells respectively, in which it is found that two cells of different sizes can be successfully encapsulated in the droplets and exported.

[0058] FIG. 6 shows the DNA amplification results. A and C show the MDA amplification results, and B and D show the 16S identification results, wherein N is the negative control of the MDA (multiple substitution) amplification. The experiment proved that the droplets can successfully encapsulate and export the cells, and the exported cells can be successfully used for downstream expansion and sequencing analysis.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The microfluidic chip, the microfluidic chip device, the microfluidic operating system and the method for forming and exporting single microparticle-encapsulated droplets, provided by this invention, can be used to separate single microparticles from biological and non-biological sources, for example, the isolation of single particles of eukaryotic cells (such as animal cells, plant cells, fungal cells and so on), prokaryotic cells (such as bacterial cells, and so on), single cell organisms, virus particles, organelles, particles formed by biological macromolecules, drug particles, drug carrier particles, lipid of plastids, polymer particles, and other natural or synthetic particles.

[0060] The present invention will be further described below with reference to the specific examples. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without detailed conditions in the following examples are generally performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Unless stated otherwise, percentages and parts are by weight.

Example 1

[0061] 1. Preparation of the Microfluidic Chip:

[0062] (i) A sample injection hole and a liquid storage well hole were punched in the upper layer of quartz glass by ultrasonic drilling, and the distance between the holes depended on the length of the channel. The upper layer of quartz glass was a smooth flat surface with a thickness of 0.5 to 1 mm and had no etched pattern. (The order of the upper and lower layers of quartz glass was determined by the optical path of the optical tweezers. Since the microscope used in this experiment was an upright microscope, wherein the optical tweezers passed through the chip from top to bottom, it was to be ensured that the upper surface of the chip was a smooth optical surface. Therefore, the upper layer was made of smooth quartz glass, and the quartz glass engraved with channels was on the lower layer.)

[0063] (ii) The surface of the lower quartz glass was etched according to the channel design, and the channel height was about 15-50 microns.

[0064] (iii) The two layers of glass were aligned and bonded with low-temperature bonding method.

[0065] (iv) Surfaces were treated by hydrophobization (using silylation reagent), to form a hydrophobic and lipophilic surface at the location of the oil storage well.

[0066] (v) For single microparticle-encapsulated droplets, the oil phase was mineral oil (containing 2% wt surfactant Span 80). Because the density of this oil phase is less than that of water, the generated droplets would be located at the outlet of the microchannel at the bottom of the oil storage well, which is convenient for observation and exportation.

[0067] The design schematic the microfluidic chip are shown in FIG. 1.

[0068] 2. Preparation of Microfluidic Chip Devices and Systems for Forming Single Microparticle-Encapsulated Droplets

[0069] The liquid sample injection device selected here was a gravity-driven sample injection device. The sample container was suspended on a height-adjustable sample holder, and the sample container was connected with the sample injection hole on the microfluidic chip through a catheter. A schematic diagram of a microfluidic chip device consisting of a liquid sample injection device and a microfluidic chip is shown in FIG. 2A. When the microfluidic chip was placed on the microscope platform, the microfluidic chip device and the microparticle capturing device together constructed a microfluidic operating system for forming single microparticle-encapsulated droplets.

Example 2

[0070] The schematic figure of steps for formation and exportation of target single microparticle-encapsulated droplets is shown in FIG. 3.

[0071] (i) Oil phase (usually mineral oil containing surfactants) was injected into the oil storage well at the outlet of the sample channel.

[0072] (ii) Microparticle phase (cell phase in this example) solution was injected into the chip channel through the injection hole by static pressure injection method, and the height of the sample holder was adjusted to h0, so that the interface between the cell phase and the oil phase at the sample outlet was steadily stationary near the sample outlet.

[0073] (iii) The characteristic map (such as Raman spectrum, 532 nm laser) of single cells was collected in the detection cell, and the desired target cells were captured by optical tweezers (such as 1064 nm laser). The microfluidic chip was moved, so that the target cells were dragged to the vicinity of the interface between the aqueous phase and oil phase.

[0074] (iv) The height of the sample holder was adjusted to h, the cells and part of the water was pushed into the oil phase by gravity to form water-in-oil droplets, and then the height of the sample holder was adjusted back to h0, so that the interface between the cell liquid and the oil phase was stationary again near the outlet of the sample channel.

[0075] (v) The microparticle-encapsulated droplets were contacted by a capillary tube. Since the oil phase has good wetting effect on the capillary tube, the droplets automatically entered the capillary tube with part of the oil, due to capillary force. FIGS. 5A-F show that a single-cell-encapsulated droplet can be successfully exported by a capillary tube.

[0076] The height of the sample holder is determined by the internal fluid resistance of the quartz chip. According to the channel size of the quartz chip used and the height of the microscope platform, a 1-meter-high sample holder was selected for this experiment. The sample holder was designed as a slide rail that has an electrically movable slider for fixing the sample container, as shown in FIG. 3B.

Example 3

[0077] DNA amplification and electrophoresis were performed on the isolated single cells.

[0078] The droplets separated in Example 2 were observed under a 50 objective lens, and it was observed that the single cell was successfully wrapped in the droplet. This method is applicable to cells with different sizes, from about one micrometer to about tens of micrometers, as shown in FIG. 5G and FIG. 5H wherein the of E. coli cells and yeast cells were encapsulated in droplets using this method. Because the density of the mineral oil used in this method is less than that of the cell suspension (water), the droplets will automatically distribute at the bottom of the capillary tube during the droplet transferring process. Droplets can be exported by just contacting the wall of the test tube or centrifuge tube using the capillary tube containing the droplet, which makes the transferring process simpler and more convenient with higher success rate.

[0079] The single cell encapsulated droplets in the capillary tube were centrifuged and introduced into a separate centrifuge tube, and the amplification substrate was added for DNA amplification, as shown in FIG. 6. In this test, E. coli (FIG. 6A and FIG. 6B) and yeast (FIG. 6C and FIG. 6D) were used as model cells, and some empty droplets were used as controls (E1 and E2 are E. coli sample control empty droplets, E3E4E5 are yeast control empty droplets). It was found that no amplification products appeared in the empty droplets, and that the single-cell-encapsulated droplets were successfully expanded, which proves that the cells isolated by this method can be successfully used for downstream analysis such as single-cell sequencing, and that pollution can be successfully avoided in the single cell screening process.

[0080] All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.