METHOD FOR USING MICROFLUIDIC CHIP AND DEVICE THEREOF

Abstract

The present invention relates to a method of using a microfluidic chip comprising introducing a gas into the microfluidic chip to replace the liquid that has been introduced into the microfluidic chip and forming a micro-reaction chamber in the form of a liquid-in-gas in the microfluidic chip. The present invention also relates to a method for obtaining assay data, a computer program product embodied in a computer-readable medium and a kit. The methods described in the present invention are easy to operate, low cost, versatile, enabling rapid exchange of fluids, achieving efficient separation and capture of single particles with high purity. In addition, the methods can avoid clogging the chip and facilitate recycling.

Claims

1. A method of using a microfluidic chip, comprising: introducing a gas into the microfluidic chip to replace the liquid that has been injected into the microfluidic chip, and forming one or more micro-reaction chambers in the form of a liquid-in-gas in the microfluidic chip.

2. The method according to claim 1, wherein the liquid that has been injected into the liquid contains or does not contain particles.

3. The method according to claim 2, wherein the particles include cells, cell clusters, microorganisms, microbial clusters, phages, exosomes, micelles, and artificial microspheres; preferably, the artificial microspheres include polyethylene glycol, polyacrylamide, polymethacrylic acid, polymethacrylate, polyvinyl alcohol, polyethylene, polystyrene, polyester, silica, and graphene microspheres; preferably, the artificial microspheres contain on their surface substances to achieve the intended detection purpose, including nucleic acids, nucleic acid aptamers, proteins, and polypeptides; preferably, the artificial microspheres are microspheres modified with a nucleic acid sequence for RNA capture, modified with a nucleic acid sequence for gene capture, or modified with one or more types of molecules such as nucleic acid aptamers or antibodies.

4. The method according to claim 1, wherein the gas comprises one or a combination of the following gases: air, nitrogen, oxygen, helium, hydrogen, carbon dioxide, neon, argon, and xenon.

5. The method according to claim 1, wherein the gas enters the microfluidic chip through the same or different inlets as the liquid that has been injected into said microfluidic chip.

6. The method according to claim 1, wherein the gas is introduced into the microfluidic chip at a gas flow rate of 0.02 L/min to 1.00 L/min, or 0.05 L/min to 0.70 L/min; preferably, the gas is introduced into the microfluidic chip at a gas flow rate of 0.04 L/min.

7. The method according to claim 1, wherein the time required for introducing the gas is 10 min-90 min, 10 min-40 min, or 15 min-25 min.

8. The method according to claim 1, wherein the microfluidic chip comprises a capture layer, a control layer, and a slide; wherein, the capture layer includes two parallel sets of capture flow channels (10, 11) and a connecting channel (13) connecting them, wherein the capture flow channel (10, 11) comprises a plurality of capture unit (8, 9) in series at the beginning and end, respectively; each capture unit comprises a flow channel (101, 111), a reservoir chamber (14, 15), and a capture channel (16, 17), respectively, and the flow channel (101, 111) comprises a U-shaped tube, with the left arm end of the U-shaped tube of the former capture unit being connected to the right arm end of the U-shaped tube of the latter capture unit; the reservoir chamber locates between the two arms of the U-shaped tube and is provided with three channels, with the first channel leading to the fluid inlet end of the U-shaped tube and being larger in diameter than the single particle to be captured, the second channel being a captured channel, leading to the fluid outlet end of the U-shaped tube and being smaller in diameter than the single particle to be captured, the third channel being the connection channel (13), leading to the reservoir chamber of another capture unit in parallel and smaller in diameter than the single particle to be captured; the connecting channel (13) connects the reservoir chambers (14, 15) of the two capture units (8, 9); the capture flow channels (10, 11) are provided with a sample inlet (1, 2), a gas inlet (4, 5), and a sample outlet (6, 7), respectively; the control layer comprises a blocking channel (12), which is located below or above the connection channel (13), intersecting with the connection channel (13), and is separated from the connection channel (13) by a membrane (18); the blocking channel (12) is provided with an inlet (3).

9. The method according to claim 8, wherein the dimensions of the two sets of particle capture flow channels and the shape and size of the capture units therein are the same or different.

10. The method according to claim 8, wherein the flow channels have a width of 5-500 μm and a depth of 5-500 μm; the connecting channel has a width of 3-100 μm and a depth of 3-100 μm.

11. The method according to claim 8, wherein the particles have a diameter in the range of 5-200 microns.

12. The method according to claim 8, wherein the particles are introduced at a flow rate of 0.005 mL/h-10 mL/h.

13. The method according to claim 8, wherein the microfluidic chip further comprises a driving pump unit for changing the volume of the reservoir chambers (14, 15), respectively, comprising connected driving pump control network channels (21, 22) and driving pump deformation chambers (23, 24); wherein the driving pump control network channels (21, 22) are further provided with driving pump inlets (19 20); the driving pump deformation chambers (23, 24) are located at the top or bottom of the reservoir chambers (14, 15), respectively, separated by a membrane (25, 26).

14. A method for obtaining an assay data, comprising: loading a sample to be assayed into the microfluid chip and introducing a gas into the microfluid chip to form one or more micro-reaction chambers in the form of a liquid-in-gas, and obtaining the assay data by measurement; the method for obtaining the assay data further comprising comparing the assayed data with the corresponding data stored in a database (e.g., a standard curve), and obtaining the data for quantifying the assayed sample.

15. A computer program product embodied in a computer readable medium, when executed on a computer, the execution steps comprising: controlling and introducing a gas into the microfluidic chip.

16. (canceled)

17. A kit, comprising: a microfluidic chip; and a computer program product embodied in a computer readable medium according to claim 15.

Description

DESCRIPTION OF DRAWINGS

[0064] FIG. 1 illustrates a top view of the overall structure of the microfluidic chip, where 1 and 2 are the sample inlets, 3 is the blocking pump inlet, 4 and 5 are the gas inlet, and 6 and 7 are the sample outlet.

[0065] FIG. 2 illustrates a schematic diagram of the enlarged structure of the microfluidic chip, in which 8 and 9 are particle capture units, 10 and 11 are particle capture flow channels, 12 is the blocking channel, and 13 is the connection channel.

[0066] FIG. 3 illustrates a top view of the particle capture unit, where 10 and 11 are the particle capture flow channels, 12 is the blocking channel, 13 is the connection channel, 14 and 15 are the reservoir chambers, and 16 and 17 are the particle capture channels, respectively.

[0067] FIG. 4 illustrates a cross-sectional view of the particle capture unit, where 13 is the connecting channel, 14 and 15 are the reservoir chambers, 12 is the blocking channel, and 18 is the membrane.

[0068] FIG. 5 illustrates a top view of the overall structure of the pressure-driving microfluidic chip, where 19 and 20 are the driving pump inlets.

[0069] FIG. 6 illustrates a schematic diagram of the enlarged structure of the pressure-driving microfluidic chip, in which 21 and 22 are the driving pump control network channels.

[0070] FIG. 7 illustrates a top view of the particle/cell pairing capture unit of the pressure-driving microfluidic chip, where 23 and 24 are the driving pump deformation chambers of the particle capture units.

[0071] FIG. 8 illustrates a cross-sectional view of the particle/cell paired capture units of the pressure-driving microfluidic chip, where 25 and 26 are membranes.

[0072] FIG. 9 illustrates micro-reaction chambers in the form of a water-in-gas as shown by the microscope after the gas is introduced into the microfluidic chip using the method of the present invention.

[0073] FIG. 10 illustrates micro-reaction chambers in the form of water-in-gas (with particles) after the gas is introduced into the microfluidic chip using the method of the present invention.

[0074] FIG. 11 illustrates a diagram of the mixing process of the paired micro-reaction chambers shown in the microscope fluorescence field after the gas is introduced into the microfluidic chip using the method of the present invention.

[0075] FIG. 12 illustrates a schematic and microscopic view of the replacement of the solution inside the chip after the gas is introduced into the microfluidic chip using the method of the present invention.

[0076] FIG. 13 illustrates a graph of the statistical results of the recovery of microspheres after capturing/reacting the microspheres using the method of the present invention (gas phase replacement) by introducing the gas into the chip compared to the prior art method (oil phase replacement).

[0077] FIG. 14 illustrates a top microscopic view of the chip before and after the particles are captured, reacted, and recovered after the gas is introduced into the chip using the method of the present invention.

[0078] FIG. 15 illustrates a microscopic view of the chip wherein water-in-oil micro-reaction chambers are formed when using the prior art oil phase.

[0079] FIG. 16 illustrates a microscopic fluorescence imaging view of the chip wherein water-in-oil micro-reaction chambers are formed when using the prior art oil phase.

[0080] FIG. 17 illustrates an on-chip microscopic view of the chip wherein water-in-oil micro-reaction chambers are formed when using the prior art oil phase.

MODE OF CARRYING OUT THE INVENTION

Example 1

[0081] High throughput paired capture of single particles based on the methods of the present invention of using the microfluidic chips. The chip in used is shown in FIGS. 1-4. The chip which includes a capture layer, a control layer, and a slide is prepared by a standard soft lithography technique. The capture layer consists of a capture flow channel 10, a capture flow channel 11, and a connecting channel 13. The control layer consists of a blocking channel 12. The capture flow channel 10 further conclude a plurality of particle capture unit 8 connected in series at the beginning and end. Each unit consists of a flow channel 101, a reservoir chamber 14, and a capture channel 16. Referring to FIG. 3, the flow channel 101 includes a U-shaped tube (in other embodiments, it may also be an arc or other zigzag shape), with the left arm end of the U-shaped tube of the former unit connecting to the right arm end of the U-shaped tube of the latter unit, and the connecting part between the U-shaped tubes being straight. The reservoir chamber 14 is located between the two arms of the U-shape and is provided with three channels. The first channel leads to the right arm of the U-shaped tube and is larger in diameter than the single particle to be captured. The second channel (capture channel 16) leads to the left arm of the U-shaped tube and is smaller in diameter than the single particle to be captured. The third channel is the connecting channel 13, leading to a reservoir chamber 15 of another parallel particle capture unit 9 and is smaller in diameter than the single particle to be captured.

[0082] The capture flow channel 11 contains a plurality of particle capture unit 9 connected in series at the beginning and end. The particle capture unit 9 and the particle capture unit 8 are set symmetrically. And the particle capture unit 9 consists of a flow channel 111, a reservoir chamber 15, and a capture channel 17. Referring to FIG. 3, the flow channel 111 includes a U-shaped tube, with the left arm end of the U-shaped tube of the former unit connecting to the right arm end of the U-shaped tube of the latter unit and the connection part between the U-shaped tubes being straight. The reservoir chamber 15 is located between the two arms of the U-shaped and is provided with three channels. The first channel leads to the right arm of the U-shaped tube and is larger in diameter than the single particle to be captured. The second channel (capture channel 17) leads to the left arm of the U-shaped tube and is smaller in diameter than the single particle to be captured. The third channel is the connecting channel 13, leading to the reservoir chamber 14 of another parallel particle capture unit 8, and is smaller in diameter than the single particle to be captured.

[0083] The capture flow channel 10 contains a sample inlet 1, a gas inlet 4, and an outlet 7. The capture flow channel 11 contains a sample inlet 2, a gas inlet 5, and an outlet 6.

[0084] The control layer is provided with a blocking channel 12, which is located below and perpendicular to the connection channel 13 (or in other embodiments, it may also be at a certain angle), and separated by a membrane 18.

[0085] The blocking channel 12 is connected to a blocking pump, by which the pressure of the blocking channel 12 is varied to block or connect the connecting channel (see FIG. 4). The blocking channel 12 is provided with an inlet 3.

[0086] As a preferred embodiment of the invention, the width of the flow channels is 60 μm, the diameter of the reservoir chambers is 100 μm; the width of the particle capture channels is 6 μm or 15 μm; the depth of the channels is 46 μm, and the number of particle capture units is 720.

[0087] As a preferred embodiment of the present invention, the particles used are A549 cells with a cell diameter of 10-20 μm.

[0088] As a preferred embodiment of the present invention, the particles used are polystyrene microspheres with a diameter of 40 μm.

[0089] As a preferred embodiment of the present invention, the polystyrene microspheres used contain mRNA capture sequence T30, which can hybridize with mRNA with polyA tails.

[0090] As a preferred embodiment of the present invention, the width of the blocking channel and the driving pump deformation chamber in the control layer is 30 μm and the height is 30 μm, respectively.

[0091] As a preferred embodiment of the present invention, all the inlets are cylindrical holes with a diameter of 1.00 mm.

[0092] As a preferred embodiment of the present invention, the material of the capture and control layers are both polydimethylsiloxane PDMS.

[0093] As a preferred embodiment of the present invention, the material of the slide is glass.

[0094] As a preferred embodiment of the present invention, both the blocking channel and the driving pump deformation chamber are filled with an aqueous solution, and the pressure thereof is controlled by controlling the driving pressure of the syringe pump.

[0095] As a preferred embodiment of the present invention, the cell flow rate used is 0.04 mL/h.

[0096] As a preferred embodiment of the present invention, the microsphere flow rate used is 0.2 mL/h.

[0097] As a preferred embodiment of the present invention, the gas phase flow rate used is 0.04 L/min.

[0098] As a preferred embodiment of the present invention, the specific workflow of the present invention is as follows:

[0099] Step A: The blocking pump is opened to increase the pressure of the blocking channel 12. The membrane 18 deforms upward. At the time the connection channel 13 is blocked. Suspension of cell A549 at the sample inlet 1 is injected with a syringe pump. When a single cell enters the particle capture unit 8, the cell first enters the reservoir chamber 14. Because the capture channel is smaller in size than that of the cell, the cell is stuck in front of the capture channel 16, while blocking the capture channel. At this point, the liquid flow rate through the reservoir chamber 14 tends to zero, and the subsequent cells cannot re-enter the particle reservoir chamber, but can only enter the next capture chamber through the flow channel 101, thus achieving a single-cell capture. High throughput single-cell capture is achieved by repeating this process in subsequent particle chambers.

[0100] Step A-1: Connection channel 13 is kept closed and the PBS solution is introduced at the sample inlet 1 to elute the excess cells from flow channel 101 out of the chip.

[0101] Step B: The tube of the sample inlet 1 is removed, and outlet 7 is plugged, with the air being introduced through the gas inlet 4 with the syringe pump system. When the air enters the particle capture chamber 8, the air enters the flow channel 101 without entering the capture channel 16 because the capture channel 16 is much smaller in size than that of the flow channel 101, with higher capillary resistance. At which time the solution in the particle reservoir chamber is retained, forming a water-in-gas micro-reaction chamber, such that each micro-reaction chamber contains a single cell, thus isolating the single cell in the reservoir chamber. When the airflow completes the chip, all single cells are isolated in single water-in-gas micro-reaction chambers separately.

[0102] Step C: A suspension of encoded microspheres (MACOSKO-2011-10 (V+), Barcoded Oligo dT primer ON Beads) is injected at a flow rate of 0.2 mL/h at the sample inlet 2 using a syringe pump. When a single microsphere enters the microsphere capture unit 9, first the microsphere enters the reservoir chamber 15. Because the capture channel diameter 17 is smaller in size than that of the microsphere, the microsphere is stuck in front of the capture channel 17, while blocking the capture channel 17. At the time, the liquid flow rate through the reservoir chamber 15 tends to zero, and subsequent microspheres cannot enter the microsphere reservoir chamber again, but only enter the next capture unit through flow channel 111, thus achieving a single microsphere capture. High throughput single microsphere capture is achieved by repeating this process in subsequent chambers.

[0103] Step C-1: The connecting channel 13 is kept closed. A cell lysate (0.2% Triton X-100) is introduced at the sample inlet 2, eluting the excess microspheres from the channel out of the chip, and replacing the solution in the capture flow channel with the cell lysate.

[0104] Step D: The tube from the particle inlet 2 is removed and outlet 6 is plugged. Air is injected at the gas inlet 5 with a syringe pump system. Because the capture channel 17 is much smaller in size than that of the flow channel 111, with a higher capillary resistance, when the air enters the particle capture unit 9, it enters the flow channel 111 without entering the capture channel 17, At this point the solution in the particle reservoir chamber is retained, forming a liquid-in-gas micro-reaction chamber, such that each micro-reaction chamber contains a single microsphere, thus isolating the individual microspheres in the reservoir chamber. When the airflow completes the chip, all the single microspheres are isolated in single water-in-gas micro-reaction chambers separately in which the droplets contain cell lysate.

[0105] Step E: The blocking channel 12 is closed, at which point the connecting channel 13 is opened. At the time the paired droplets of the encapsulated cells and microspheres are connected, the cell lysate in the microsphere reservoir chamber 15 can enter the cell reservoir chamber 14 for cell lysis by diffusion. After cell lysis, the inclusions of the cell contained in the cell lysate can also enter the microsphere reservoir chamber 15 by diffusion, where the mRNA with polyA tails in the cell inclusions can hybridize with T30 on the microsphere. After a period, sufficient exchanges of substances between the two paired chambers can be achieved for complete cell lysis and single-cell mRNA extraction. Since the paired single microspheres/single cells are isolated by FC40, there is no physical contact between the cells and microspheres, so there is no cross-contamination, and thus high throughput capture of mRNA from single cells with single microspheres can be achieved.

[0106] Step F: The capture layer is peeled off and the microspheres or cells captured in the chambers are exposed to the surface of the chip, which is eluted off with buffer solution. The extracted single-cell mRNAis subjected to subsequent analytical studies.

Example 2

[0107] In Example 1, the substance exchanges between the two paired droplets are achieved by passive diffusion, and the exchange rate is slow. To accelerate the substance exchanges between the two paired droplets, a driving pump unit is added to the chip exemplified in Example 1 for controlling the mixing between the two droplets and controlling the distribution of the droplets between the two paired chambers.

[0108] The following is a further detailed description of the present invention in conjunction with the accompanying drawings and specific embodiments using paired capture of microspheres and cells as an example.

[0109] As shown in FIGS. 5-8, based on Example 1, the chip further comprises a driving pump control network channel 21, a driving pump control network channel 22, a driving pump deformation chamber 23, and a driving pump deformation chamber 24. The driving pump control network channels 21, 22 are provided with driving pump inlets 19, 20. The driving pump control network channels 21, 22 are respectively connected to the driving pump deformation chambers 23, 24, respectively. The driving pump deformation chambers 23, 24 are located at the bottom of the reservoir chambers 14, 15, respectively, and separated from the reservoir chamber 14, 15 by membranes 25 and 26.

[0110] As a preferred embodiment of the present invention, the specific working process is as follows:

[0111] Based on Example 1, in step E, the blocking channel 12 is closed, at which time the connecting channel 13 is opened. At that time, the paired droplets encapsulating the cells and microspheres are connected. The pressure of the driving pump control network channel 22 is increased. When the driving pump deformation chamber 24 becomes larger, the membrane 26 protrudes upward. The volume of the particle reservoir chamber 15 is reduced and the cell lysate therein is pumped into the particle reservoir chamber 14, while the pressure of the driving pump control network channel 21 is reduced. At the time, the driving pump deformation chamber 23 is reduced, and the membrane 25 is recessed downward. The volume of the particle reservoir chamber 14 becomes larger, and into which the cell lysate in the particle reservoir chamber 15 is pumped into. When the pressure of the driving pump control network channel 22 is reduced, the driving pump deformation chamber 24 becomes smaller and the membrane 26 is recessed downward. The volume of the particle reservoir chamber 15 becomes larger, and the solution therein is pumped into the particle reservoir chamber 14; while the pressure of the driving pump control network channel 21 is increased, and the driving pump deformation chamber 23 becomes larger, the membrane 25 is recessed downward. The solution in the particle reservoir chamber 15 is pumped into the particle reservoir chamber 14. The chamber 23 becomes larger, the membrane 25 protrudes upward. The volume of the particle reservoir chamber 14 becomes smaller, and the solution therein is also pumped into the particle reservoir chamber 15. By repeating this cycle several times, a rapid exchange of substances in the particle reservoir chamber 14 and the particle reservoir chamber 15 can be achieved. The cell lysate in the particle reservoir chamber 15 can lyse the cells paired with it. After cell lysis, the released mRNA with polyA tails can enter the particle capture chamber to hybridize with T30 on the microspheres. Since the paired single microsphere/single cell is isolated by FC40, there is no physical contact between the cell and microsphere. So, there is no cross-contamination, and thus high throughput capture of mRNA from single cells with single microspheres can be achieved. Microscopic photographs of the obtained results are shown in FIGS. 9-11.

Example 3

[0112] In Examples 1-2, only a single replacement of solutions or reagents in the flow channel was performed. Using the gas phase replacement method of the present invention, multiple replacements of fluids in the flow channels and independent micro-reaction chambers can also be achieved by multiple times, allowing the captured microspheres and/or cells to interact separately or simultaneously in different solutions, or to undergo chemical reactions.

[0113] Based on Example 2, after step E, the following steps are carried out:

[0114] Step E-1: The pressure of the driving pump control network channel 22 is increased, at which point the driving pump deformation chamber 24 becomes larger, with the membrane 26 protruding upward. The volume of the particle reservoir chamber 15 is reduced, and the solution in the paired unit is all drained into the cell capture chamber. The blocking channel 12 is opened, at which time the connecting channel 13 is closed, and 1×PBS at a flow rate of 0.2 mL/h is injected at the two sample inlets 1 and 2 respectively for thoroughly washing the microspheres while preventing contamination of the microspheres by free RNA during the washing process.

[0115] Step E-2: The blocking channel 12 is closed, at which point the connecting channel 13 is opened. RT mix (1× Maxima RT buffer, 1 mM dNTPs (Clontech, Ref 639125), 1 U/μL RNAase inhibitor (Lucigen, Ref. 30281-2), 2.5 μM Template_Switch_Oligo, and 10 U/μL Maxima H-RT (ThermoScientific, Ref. EP0751)) are injected at a flow rate of 0.2 mL/h in both sample inlets 1 and 2, to replace the solution in the capture flow channel with the RT mix. Then a step similar to step D is repeated to form water-in-air droplets in the reservoir chamber. The process is as follows: the blocking channel 12 is opened, at which point the connecting channel 13 is blocked. The tubes of sample inlet 1 and 2 are removed and the outlets 6 and 7 are plugged. Air is injected at the gas inlets 4 and 5 with a syringe pump. Liquid-in-gas micro-reaction chambers are formed in the particle capture chamber 14 and the particle capture chamber 15, respectively. Each micro-reaction chamber contains a single microsphere, which is thus isolated in the reservoir chamber. When the airflow completes the chip, all of the single microspheres are isolated separately in the single water-in-air micro-reaction chamber that contains RT mix, such that all microspheres are in RT mix solution. The microfluidic chip is placed at 42° C. for 1.5 h for mRNA reverse transcription, and the mRNA information within the single-cell carried on each microsphere is converted into cDNA information.

[0116] Step E-3: After the reverse transcription, TE/SDS (10 mM Tris-HCl, 1 mM EDTA, 0.1% SDS, pH=8.0), TE/TW (10 mM Tris-HCl, 1 mM EDTA, 0.01% Tween, pH=8.0) and TE (10 mM Tris-HCl, 1 mM EDTA, pH=8.0) buffer are injected sequentially at the two sample inlets 1 and 2 at a flow rate of 0.2 mL/h with 20 μL of each solution to wash the microspheres. Subsequently, 20 μL of Exonuclease I mix solution (2 μL Exonuclease I (NEW ENGLAND BioLabs Inc., item no.

[0117] M0293), 4 μL of 10× Exonuclease I buffer solution (NEW ENGLAND BioLabs Inc., item no. M0293), and 34 μL of ddH.sub.2O are injected at a flow rate of 0.2 mL/h into each of the two sample inlets 1 and 2 of the chip. The chips are incubated at 37° C. for 45 min, followed by the injection of 10 μL TE/SDS, 10 μL TE/TW, and 10 μL ddH.sub.2O at the two sample inlets 1 and 2 of the chips at a flow rate of 0.2 mL/h, respectively. At the end of the whole process, nitrogen is injected into the chip at the particle gas-phase inlets 6 and 7, and combined with the micro-turbulence of the fluid inside the chip, all microspheres inside the chip are recovered nondestructively for a series of reactions such as subsequent amplification library building and sequencing. The microscopic photographs of the results are shown in FIGS. 12-14.

Comparative Example 1

[0118] The cells and microspheres were captured and paired separately referring to the method shown in Example 2 of patent application CN107012067A, and the results obtained are shown in FIGS. 15-17. By using the oil phase replacement method in the prior art, stable droplets could not be formed, the paired droplets are not mixed stably, emulsion residues often existed after solvent replacement, and the recovery efficiency of microspheres was low.