WO2021103970A1 - 一种微流控芯片的使用方法及其装置 - Google Patents

一种微流控芯片的使用方法及其装置 Download PDF

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Publication number
WO2021103970A1
WO2021103970A1 PCT/CN2020/126616 CN2020126616W WO2021103970A1 WO 2021103970 A1 WO2021103970 A1 WO 2021103970A1 CN 2020126616 W CN2020126616 W CN 2020126616W WO 2021103970 A1 WO2021103970 A1 WO 2021103970A1
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channel
capture
microfluidic chip
gas
microns
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PCT/CN2020/126616
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English (en)
French (fr)
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杨朝勇
张明霞
邹远
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杭州微著生物科技有限公司
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Priority to EP20894828.1A priority Critical patent/EP4066942A4/en
Priority to US17/779,625 priority patent/US20220410155A1/en
Priority to JP2022530682A priority patent/JP2023503157A/ja
Publication of WO2021103970A1 publication Critical patent/WO2021103970A1/zh

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    • B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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Definitions

  • the invention relates to the fields of microfluidic technology and molecular biology, and in particular to a method for using a microfluidic chip.
  • Cells are the basic unit of biology and life activities. The analysis and processing of the average signal of a large number of cells by traditional methods makes the averaging of the signals obscure the understanding of the heterogeneity of the various life systems of the human body and the cells that make up these systems. With the development of high-throughput sequencing technology, single cell/particle sequencing technology has become one of the most important means of single cell analysis, which greatly improves the efficiency and accuracy of single cell/particle analysis.
  • the microfluidic chip integrates basic operation units such as sample preparation, reaction, separation, detection, cell culture, sorting, lysis and other related fields in biology and chemistry on the microchip, forming a network of microchannels, with controllable fluid running through
  • the whole system miniaturizes and integrates conventional chemical or biological experiments. Due to the low consumption of reaction reagents and short action time based on the microfluidic chip platform, it is especially suitable for single cell/particle analysis research.
  • the purpose of the present invention is to provide a method for using a microfluidic chip.
  • the method of the invention is easy to operate, low in cost, and strong in versatility, can realize rapid exchange of fluids, achieve high-efficiency separation and capture of single particles, and the obtained particles have high purity, and the method can avoid chip clogging and is convenient for recycling.
  • a method for using a microfluidic chip includes injecting gas into the microfluidic chip to replace the liquid injected into the microfluidic chip and form a microfluidic liquid in the microfluidic chip. Reaction chamber.
  • the liquid injected into the microfluidic chip contains particles. In some embodiments of the present invention, the liquid injected into the microfluidic chip does not contain particles.
  • the micro-reaction cavity contains particles; in other embodiments of the present invention, the micro-reaction cavity does not contain particles.
  • the particles of the present invention are well known in the art and need to be captured, analyzed, and reacted.
  • the particle size of the particles is in the range of 5 micrometers to 1000 micrometers, for example, 5 micrometers to 200 micrometers.
  • Microparticles well known in the art include but are not limited to cells, cell clusters, microorganisms, microbial clusters, bacteriophages, exosomes, micelles and artificial microspheres.
  • the artificial microspheres include but are not limited to polyethylene glycol and polyacrylamide.
  • the surface of the artificial microspheres contains Substances that achieve the desired detection purpose, such as but not limited to compounds such as aptamers, and biological macromolecules such as nucleic acids, proteins, and peptides.
  • the artificial microspheres are microspheres modified with nucleic acid sequences for RNA capture.
  • the artificial microspheres are microspheres modified with nucleic acid sequences for gene capture.
  • the artificial microspheres are microspheres modified with molecules such as nucleic acid aptamers or antibodies.
  • the artificial microspheres are microspheres modified with two or more of the above-mentioned molecules.
  • the micro reaction chamber contains a single microsphere; in some embodiments, the micro reaction chamber contains a single cell; in some embodiments, the micro reaction chamber contains no particles.
  • the gas may be any gas commonly used in the art, including but not limited to air, nitrogen, oxygen, helium, hydrogen, carbon dioxide, neon, argon, xenon, and the like. These gases can be used alone or in combination. Those skilled in the art can choose the gas to be used according to actual biochemical reaction compatibility requirements.
  • the gas and the liquid injected into the microfluidic chip enter the microfluidic chip through the same inlet. In some embodiments, the gas and the liquid injected into the microfluidic chip enter the microfluidic chip through different inlets. In some embodiments, the gas enters the microfluidic chip through multiple different inlets.
  • the liquid that has been injected into the microfluidic chip is replaced by the gas and flows out from another opening.
  • the gas of the present invention can be transported to the microfluidic chip through a gas cylinder through a gas pipeline.
  • the same gas cylinder may be used to send gas to the same gas phase inlet or different gas phase inlets; in other embodiments, different gas cylinders may be used to send gas to the same gas phase inlet or different gas phase inlets.
  • Those skilled in the art are aware of conventional methods of using gas cylinders and connecting pipes.
  • the gas is fed into the microfluidic chip at a flow rate of 0.02L/min-1.0L/min; in some embodiments, the gas is fed into the microfluidic chip at a flow rate of 0.05L/min-0.7L /min of gas flow into the microfluidic chip; in some embodiments, the gas is 0.06L/min, 0.07L/min, 0.08L/min, 0.09L/min, 0.1L/min The flow passes into the microfluidic chip.
  • the gas intake time can be adjusted according to actual needs, so that the gas can completely or partially replace the liquid injected into the microfluidic chip.
  • the gas intake time is 10min-90min; in some embodiments, the gas intake time is 10min-40min; in some embodiments, the gas intake time is 15min -25min.
  • microfluidic chip described in the present invention can be routinely used in the field, such as those exemplified in the review literature (Shembekar, Chaipan et al. 2016)/textbook ("Micro-Nanofluidic Chip Laboratory", written by Lin Bingcheng)
  • a variety of microfluidic chips can be applied to the method of using the microfluidic chip provided by the present invention (that is, the air-sealing method).
  • the method of the present invention is particularly suitable for a chip similar to the microfluidic chip described in the patent application CN107012067A, and its oil phase inlet can be replaced with a gas phase inlet for use.
  • the microfluidic chip includes three parts: a capture layer, a control layer, and a slide.
  • the capturing layer includes two parallel particle capturing flow channels and a connecting channel connecting them.
  • the particle capture flow channel contains multiple single particle capture units, each of which includes a flow channel, a liquid storage chamber, and a capture channel.
  • the diameter of the capture channel is smaller than the diameter of the particle to be captured, which can capture a single particle; the flow channel of the previous capture unit
  • the left end is connected to the right end of the flow channel of the latter capture unit.
  • the liquid storage chamber is located between the two ends of the flow channel and is provided with three channels.
  • the first channel has a diameter larger than the single particle to be captured and leads to the fluid inlet end of the flow channel
  • the second channel is a capture channel with a diameter smaller than the single particle to be captured, leading to the liquid outlet end of the flow channel
  • the third channel is a connecting channel, with a diameter smaller than the single particle to be captured, leading to the storage of another parallel capture unit Liquid chamber
  • the connecting channel connects the liquid storage chambers of two matched particle capture units.
  • Each trapping channel is also provided with a particle inlet at the front, a gas phase inlet at the end, and a particle outlet at the end.
  • the size (length, width, height) of the two particle capture flow channels and the shape and size of the capture unit may be the same or different.
  • the control layer includes a barrier channel, which is located directly below or above the connecting channel of the capture layer, and is separated by a diaphragm.
  • the blocking channel is provided with an entrance.
  • the capture layer and the control layer are located above the slide.
  • the microfluidic chip includes a drive pump unit for changing the volume of the liquid storage chamber of the particle capture unit, including a connected drive pump control network channel and a drive pump deformation chamber, the drive The pump control network channel is also provided with a drive pump inlet.
  • the driving pump deformation chamber is located at the top or bottom of the liquid storage chamber of the single particle capture unit of the capture layer, and the driving pump deformation chamber and the liquid storage chamber are separated by a diaphragm.
  • the size of the channel is determined by the size of the particular particle used and analyzed.
  • the width of the capture channel can be 5-500 microns, for example: including but not limited to 5-100 microns, 100-300 microns, 300-500 microns, 5-50 microns, 50-200 microns, 200-400 microns, 10 microns, 20 microns, 50 microns, 80 microns, 100 microns, 200 microns, 300 microns or 400 microns;
  • the depth can be 5-500 microns, for example: including but not limited to 5-100 microns, 100-300 microns, 300- 500 microns, 5-50 microns, 50-200 microns, 200-400 microns, 10 microns, 20 microns, 50 microns, 80 microns, 100 microns, 200 microns, 300 microns or 400 microns;
  • the width of the connection channel can be 3- 100 microns, for example: including but not limited to 3-30 microns,
  • the microparticles have a diameter of 5-200 microns, and can be any microspheres or cells with a micrometer size.
  • the microfluidic chip can be used to pair microspheres and microspheres, or cell and cell pairs, or microspheres and cells.
  • the diameter of the microspheres may be 5-200 micrometers, for example: including but not limited to 5 micrometers, 10 micrometers, 15 micrometers, 20 micrometers, 30 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers. Micrometer, 120 micrometer, 150 micrometer or 180 micrometer.
  • the diameter of the cells may be 5-100 microns, for example, including but not limited to 5 microns, 10 microns, 15 microns, 20 microns, 30 microns, 70 microns, 80 microns, or 90 microns.
  • the material of the capture layer and the carrier used in the microfluidic chip of the present invention can be silicon wafer, glass, polymethyl methacrylate, polyethylene, polypropylene, polyvinyl chloride, polyester, etc.; control The material of the layer can be polymethyl methacrylate, polyethylene, polypropylene, polyvinyl chloride, polyester and the like.
  • the materials of the capture layer and the control layer used may be polydimethylsiloxane PDMS, and the carrier material may be glass.
  • the capture layer and the control layer are reversibly bonded together through thermal calibration, and then bonded to the carrier through plasma.
  • the capture layer and the control layer are surface-activated by plasma and bonded together, and then bonded with the carrier by plasma.
  • the flow rate of microparticles is 0.005mL/h-10mL/h, for example: including but not limited to 0.005mL/h-0.05mL/h, 0.05mL/h-0.1mL/h, 0.1mL/h h-0.5mL/h, 0.5mL/h-2mL/h, 2mL/h-6mL/h, 6mL/h-10mL/h, 0.05mL/h-0.5mL/h, 0.5mL/h-3mL/h , 3mL/h-8mL/h, 0.01mL/h, 0.05mL/h, 0.1mL/h, 0.2mL/h, 0.5mL/h, 0.8mL/h, 1mL/h, 2mL/h, 4mL/h , 6mL/h or 8mL/h.
  • the barrier pump is deformed by passing fluid to play the role of blocking the connecting channel of the capture layer.
  • the fluid filled with the barrier pump can be an aqueous solution, oil or air, which can be injected by a syringe pump.
  • the pressure of the barrier pump is controlled, that is, the degree of barrier to the connecting channel of the capture layer is controlled.
  • the basic workflow of the microfluidic chip is:
  • Step A Increase the pressure of the barrier pump, the barrier channel deforms, and the diaphragm squeezes the uppermost layer of the connecting channel until the connecting channel between the two sets of parallel particle capture channels is completely blocked, and the particles containing particles are introduced from the inlet of a set of particle capture channels
  • the particles enter the liquid storage chamber first. Since the width (pipe diameter) of the capture channel is smaller than the particle diameter, The particles are stuck in front of the capture channel and block the capture channel at the same time. The flow rate of the fluid flowing through the liquid storage chamber approaches zero. At this time, subsequent particles cannot enter the liquid storage chamber again, and can only enter through the flow channel. The next capture unit, in this way, achieves the capture of single particles. Repeat this process in the subsequent particle capture unit to achieve high-throughput single particle capture.
  • Step B Regarding the particle trapping channel that achieves single particle trapping, gas is injected into the gas phase inlet.
  • the gas enters the trap unit, since the width of the trapping channel is much smaller than the width of the flow path, the capillary resistance is relatively large, so the gas phase enters the trapping channel. Do not enter the liquid storage chamber. At this time, the liquid in the liquid storage chamber is retained to form a gas-in-air micro-reaction chamber.
  • the gas flows through the entire chip all the capture units in the flow channel are isolated into a single liquid of the air-in-air liquid. To achieve the separation of a single gas-packed liquid micro-reaction chamber.
  • the workflow may further include step A-1:
  • Step A-1 Pass the liquid required for the next operation into the sample inlet of the particle capture flow channel for realizing single particle capture, and wash away the liquid containing residual particles in the flow channel.
  • step A-1 and/or step B can be repeated multiple times according to actual needs. Each time step A-1 and/or step B is performed, the liquid and/or gas used may be the same or different.
  • step B a method similar to steps A, A-1, and B is used to implement steps C, C-1, and D, respectively, to make another set of particle capture flow that has not been captured by single particles. Channel to achieve high-throughput single particle capture.
  • step C-1 and/or step D can be repeated multiple times according to actual needs. Each time step C-1 and/or step D is performed, the liquid and/or gas used may be the same or different.
  • the workflow further includes:
  • Step E Reduce or close the barrier pump.
  • the connecting channel between the two sets of parallel particle capture channels is opened, and the air-in-air liquid micro-reaction chamber for matching between the two sets of particle capture channels is in a communicable state. Due to the diffusion effect, the gas-in-air liquid micro-reaction chambers in the two liquid storage chambers can realize mutual material exchange, and after a period of sufficient reaction, independent reactions between high-flux paired particles can be realized.
  • step E drive pumps respectively arranged at the bottom of the liquid storage chambers of the two sets of particle capturing flow channel capturing units can be used to alternately change the liquid storage chamber volumes of the two capturing units at intervals, respectively, Promote the rapid exchange of substances in the liquid storage chambers of the two capture units.
  • any step or several steps in the above steps AD can be repeated, for example, step B and step A-1 are repeated, or steps A, B, optional A-1 and C are repeated, Or repeat step B, optional A-1 and step C, etc., through repeated liquid replacement and the generation of air-encapsulated solution micro-reaction chamber, the addition, replacement and reaction chamber of a variety of different reaction particles and reagents can be realized The formation of multi-function operations.
  • step F the last step of the workflow is step F:
  • Step F Separate the capture layer from the control layer, and the particles captured in the liquid storage chamber or after the reaction are exposed on the surface of the chip, and eluted with a buffer solution for subsequent analysis and research.
  • the invention also provides a microfluidic chip containing a micro-reaction cavity in the form of a gas-in-air liquid.
  • the micro-reaction chamber and microfluidic chip in the form of air-in-air liquid are as defined above.
  • the present invention also provides a method for acquiring detection data, including:
  • test sample is loaded on the microfluidic chip, and gas is injected into the microfluidic chip to form a micro-reaction chamber of air-enclosed liquid, and the detection data is obtained by measurement.
  • the method for obtaining detection data further includes comparing the detected data with corresponding data (such as a standard curve) stored in a database to obtain quantitative data for the detection sample.
  • corresponding data such as a standard curve
  • a computer program product embodied in a computer-readable medium. When executed on a computer, the execution steps include:
  • the present invention also provides a kit, including:
  • a computer program product embodied in a computer-readable medium.
  • the method of the present invention can stably realize the generation of a gas-in-air liquid micro-reaction chamber, and solves the problem that the oil-water interface is difficult to demulsify and cannot form effective water-in-oil droplets when the microfluidic chip uses the "oil seal".
  • the gas used in the method of the present invention is cheap and easy to obtain, has no batch difference, and the system is stable, so it solves the problem that the oil phase containing the surfactant in the "oil seal" is expensive, and the large difference between batches leads to unstable results.
  • the method of the present invention can easily realize the removal of solution reagents in the channel of the microfluidic chip, avoid the residue of microemulsion droplets in the channel of the microfluidic chip with the traditional "oil seal" method, and can replace independent droplets with new reagents It makes it possible to add multiple reagents in sequence, greatly expands the types of tests that can be carried out on the microfluidic chip and depends on single particles, and improves the efficiency of experiments, detection and analysis.
  • the method of the present invention can realize stable droplet mixing and reagent replacement, and solves the problem that the oil film isolates the paired droplets in the traditional "oil seal" method, and the paired droplets are difficult to fuse.
  • the effective matching ratio of droplets is low, and particles (such as cells) cannot be lysed.
  • Figure 1 is a top view of the overall structure of the microfluidic chip, in which 1, 2 are sample inlets, 3 are barrier pump inlets, 4 and 5 are gas inlets, and 6, 7 are sample outlets.
  • Figure 2 is 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 a blocking channel, and 13 is a connecting channel.
  • Fig. 3 is a top view of the particle capture unit, where 10 and 11 are particle capturing channels, 12 are blocking channels, 13 are connecting channels, 14 and 15 are liquid storage chambers, and 16, 17 are particle capturing channels, respectively.
  • Fig. 4 is a cross-sectional view of the particle capture unit, in which 13 is a connecting channel, 14 and 15 are liquid storage chambers, 12 is a barrier channel, and 18 is a spacer film.
  • Figure 5 is a top view of the overall structure of the pressure-driven microfluidic chip, in which 19 and 20 are respectively the inlets of the drive pump.
  • Figure 6 is a schematic diagram of the enlarged structure of a pressure-driven microfluidic chip, in which 21 and 22 are respectively driving pump control network channels.
  • Fig. 7 is a top view of a pressure-driven microfluidic chip particle/cell paired capture unit, wherein 23 and 24 are the particle capture unit driving deformation chambers, respectively.
  • Figure 8 is a cross-sectional view of a pressure-driven microfluidic chip particle/cell paired capture unit, where 25 and 26 are spacer films.
  • Fig. 9 shows the micro-reaction chamber in the form of air-in-air shown by the microscope after ventilating the microfluidic chip using the method of the present invention.
  • Fig. 10 shows the micro-reaction chamber in the form of particle capture and water-in-air (containing particles) displayed by the microscope after the microfluidic chip is ventilated by the method of the present invention.
  • Figure 11 is a diagram of the mixing process of the paired micro-reaction chambers shown in the microscope fluorescence field after the microfluidic chip is ventilated by the method of the present invention.
  • Fig. 12 is a schematic diagram and a microscope view of ventilating the microfluidic chip to replace the solution in the chip using the method of the present invention.
  • Figure 13 is a graph showing the statistical results of the recovery rate of the microspheres after the microspheres are captured/reacted by using the method of the present invention (gas phase displacement) to ventilate the chip compared with the prior art method (oil phase displacement).
  • Fig. 14 is a top-view microscope view of the chip before and after the use of the method of the present invention to ventilate the chip, capture, react, and recover the particles.
  • Fig. 15 is a microscope view of a chip using an oil phase to form a water-in-oil micro-reaction chamber in the prior art.
  • Fig. 16 is a fluorescence imaging view of a chip microscope that uses an oil phase to form a water-in-oil micro-reaction chamber in the prior art.
  • Fig. 17 is an in-chip microscopic view of a water-in-oil micro-reaction chamber (containing particles) formed by using an oil phase in the prior art.
  • the use method of the microfluidic chip based on the present invention realizes high-throughput paired capture of single particles.
  • the chip used is shown in Figures 1-4.
  • the chip is processed by standard soft lithography technology and includes a capture layer, a control layer, and a slide. .
  • the capture layer is composed of a capture flow channel 10, a capture flow channel 11, and a connecting channel 13, and the control layer is composed of a barrier channel 12.
  • the capture flow channel contains a plurality of particle capture units 8 connected in series end to end, and each unit is composed of a flow channel 101, a liquid storage chamber (14), and a capture channel (16); see Figure 3, the flow channel 101 includes a U-shaped tube ( In other embodiments, it can also be arc or other zigzag shapes), the left arm end of the U-shaped tube of the previous unit is connected to the right arm end of the U-shaped tube of the next unit, and the connecting part between the U-shaped tubes is Straight tube, the liquid storage chamber 14 is located between the two U-shaped arms, and is provided with three channels.
  • the first channel has a diameter larger than the single particle to be captured, leading to the right arm of the U-shaped tube, and the second channel (capture channel 16)
  • the diameter smaller than the single particle to be captured leads to the left arm of the U-shaped tube;
  • the third channel is a connecting channel 13 whose diameter is smaller than the single particle to be captured and leads to the liquid storage chamber 15 of another particle capture unit 9 in parallel.
  • the capture flow channel 11 contains a plurality of particle capture units 9 connected in series end to end.
  • the particle capture unit 9 and the particle capture unit 8 are symmetrically arranged, and each unit is composed of a flow channel 111, a liquid storage chamber 15, and a capture channel 17; see Fig. 3,
  • the flow channel 111 includes a U-shaped tube.
  • the left arm end of the U-shaped tube of the previous unit is connected to the right arm end of the U-shaped tube of the next unit.
  • the connecting part between the U-shaped tubes is a straight tube.
  • the liquid storage chamber 15 is located in the U-shaped tube. Between the two arms of the shape, there are three channels.
  • the first channel has a diameter larger than the single particle to be captured, leading to the right arm of the U-shaped tube, and the second channel (capture channel 17) has a diameter smaller than the single particle to be captured, leading to The left arm of the U-shaped tube; the third channel is the connecting channel 13, with a diameter smaller than the single particle to be captured, leading to the liquid storage chamber 14 of another particle capturing unit 8 in parallel.
  • the capture flow channel 10 includes a sample inlet 1, a gas inlet 4 and an outlet 7, and the capture flow channel 11 includes a sample inlet 2, a gas inlet 5 and an outlet 6.
  • the control layer is provided with a barrier channel 12; the barrier channel 12 is located below the connecting channel 13, perpendicular to the connecting channel 13 (in other embodiments, it may be a certain angle), and is separated by a diaphragm 18; the barrier channel 12 is connected to one
  • the barrier pump is used to change the pressure blocking of the barrier channel 12 through the barrier pump or the connecting channel (see FIG. 4); the barrier channel 12 is provided with an inlet 3.
  • the width of the flow channel is 60 microns
  • the diameter of the liquid storage chamber is 100 microns
  • the width of the particle capture channel is 6 microns or 15 microns
  • the channel depth is 46 microns
  • the selected particles are A549 cells with a cell diameter of 10-20 microns.
  • the selected particles are polystyrene microspheres with a diameter of 40 microns.
  • the selected polystyrene microspheres contain mRNA capture sequence T30, and T30 can hybridize with mRNA with a polyA tail.
  • the barrier channel and the driving pump deformation chamber in the control layer have a width of 30 micrometers and a height of 30 micrometers.
  • all said inlets are cylindrical holes with a diameter of 1.00 mm.
  • the materials of the capture layer and the control layer are both polydimethylsiloxane PDMS.
  • the material of the slide is glass.
  • both the barrier channel and the driving pump deformation chamber are filled with aqueous solution, and the pressure of the barrier channel and the driving pump deformation chamber is controlled by controlling the driving pressure of the syringe pump.
  • the selected cell flow rate is 0.04 mL/h.
  • the selected flow rate of the microspheres is 0.2 mL/h.
  • the gas flow rate selected is 0.04 L/min.
  • the specific working process of the present invention is:
  • Step A Turn on the barrier pump, the pressure in the barrier channel 12 increases, and the diaphragm 18 deforms upwards. At this time, the connecting channel 13 is blocked.
  • a syringe pump is used to pass the cell A549 suspension into the sample inlet 1.
  • the cells first enter the liquid storage chamber 14. Since the capture channel is smaller than the cells, the cells are stuck in front of the capture channel 16 while blocking the capture channel. At this time, the flow rate of the liquid flowing through the liquid storage chamber 14 approaches zero. The cells of can not enter the particle liquid storage chamber again, and can only enter the next capture unit through the flow channel 101, thus achieving single cell capture. Repeat this process in the subsequent particle capture unit to achieve high-throughput single cell capture.
  • Step A-1 Keep the connection channel 13 closed, and continue to pass the PBS solution into the sample inlet 1 to flush the excess cells in the flow channel 101 out of the chip.
  • Step B Unplug the sample inlet 1 tube, block the outlet 7, and inject air into the gas inlet 4 with a syringe pump system.
  • the air enters the particle capture unit 8 since the capture channel 16 is much smaller than the flow channel 101, the capillary resistance is relatively large Therefore, air enters the flow channel 101 but does not enter the capture channel 16.
  • the solution in the particle storage chamber is retained to form a water-in-air micro-reaction chamber.
  • the micro-reaction chamber contains a single cell, thereby isolating a single cell in In the liquid storage chamber. When air circulates through the entire chip, all single cells are isolated in a single air-in-water microreaction chamber.
  • Step C Inject the coding microsphere (MACOSKO-2011-10 (V+), Barcoded Oligo dT primer ON Beads) suspension with a syringe pump at a flow rate of 0.2 mL/h into the sample inlet 2.
  • a single microsphere enters the microsphere capture unit At 9 o'clock, the microspheres first enter the liquid storage chamber 15. Since the diameter of the capture channel 17 is smaller than that of the microspheres, the microspheres are stuck in front of the capture channel 17 and block the capture channel 17 at the same time.
  • Step C-1 Keep the connection channel 13 closed, pass the cell lysate (0.2% Triton X-100) into the sample inlet 2, flush the excess microspheres in the channel out of the chip, and replace the solution in the capture channel with cells Lysis solution.
  • Step D Unplug the tube of the particle inlet 2 and block the outlet 6, and use the syringe pump system to pass air into the gas inlet 5.
  • the air enters the particle capture unit 9 since the capture channel 17 is much smaller than the flow channel 111, the capillary resistance is relatively large Therefore, the air enters the flow channel 111 but does not enter the capture channel 17.
  • the solution in the particle liquid storage chamber is retained, forming a gas-encapsulated liquid micro-reaction chamber.
  • the micro-reaction chamber contains a single microsphere, thereby removing a single microsphere. Isolated in the liquid storage chamber. When air circulates through the entire chip, all single microspheres are isolated in a single air-in-air microreaction chamber, and the droplets contain cell lysate.
  • Step E The blocking channel 12 is closed, and the connecting channel 13 is opened at this time.
  • the paired droplets of the wrapped cells and the microspheres are connected, and the cell lysate in the microsphere storage chamber 15 can enter the cell storage chamber 14 through diffusion to perform cell lysis. After the cell lysis, it contains the cell contents. It can also enter the microsphere storage chamber 15 through diffusion, where the mRNA with polyA tail in the cell content can hybridize with the T30 on the microsphere. After a period of time, the material between the two paired chambers is sufficient. Exchange can achieve complete cell lysis and single-cell mRNA extraction.
  • paired single microspheres/single cells are separated by FC40, there is no physical contact between cells and microspheres, so there is no cross-contamination, so it can achieve high Throughput a single microsphere captures the mRNA of a single cell.
  • Step F Peel off the capture layer, expose the microspheres or cells captured in the chamber to the surface of the chip, wash them off with a buffer solution, and continue the subsequent analysis and research on the extracted single-cell mRNA.
  • Example 1 the material exchange between two paired droplets is realized by passive diffusion, and the exchange speed is relatively slow. In order to speed up the material exchange between the two paired droplets, it is added on the basis of Example 1.
  • the driving pump unit is used to control the mixing between the two droplets and the distribution of the droplets between the two paired cavities.
  • the chip further includes a drive pump control network channel 21, a drive pump control network channel 22, a drive pump deformation chamber 23, and a drive pump deformation chamber 24.
  • Drive pump control network channels 21, 22 are provided with drive pump inlets 19, 20.
  • the driving pump control network channels 21 and 22 are respectively connected to the driving pump deformation chambers 23 and 24.
  • the driving pump deformation chambers 23 and 24 are respectively located at the bottoms of the liquid storage chambers 14, 15 and pass through the diaphragms 25, 26. Separate from the liquid storage chambers 14, 15.
  • step E the blocking channel 12 is closed, and the connecting channel 13 is opened at this time.
  • the paired droplets of the wrapped cells and the microspheres are connected, increasing the pressure of the driving pump control network channel 22.
  • the driving pump deformation chamber 24 becomes larger, the diaphragm 26 is raised, and the particle liquid storage chamber 15 is reduced in volume.
  • the cell lysate is pumped to the particle liquid storage chamber 14 while reducing the pressure of the driving pump control network channel 21.
  • the driving pump deformation chamber 23 is reduced, the diaphragm 25 is recessed, and the particle liquid storage chamber 14 becomes larger.
  • the solution in the particle liquid storage chamber 15 is also pumped to the particle liquid storage chamber 14; when the pressure of the driving pump control network channel 22 is reduced, the driving pump deformation chamber 24 is reduced at this time, the diaphragm 26 is recessed, and the particle liquid storage chamber The volume of the chamber 15 becomes larger, the solution is pumped to the particle liquid storage chamber 14, and the pressure of the driving pump control network channel 21 is increased at the same time.
  • the driving pump deformation chamber 23 becomes larger, the diaphragm 25 is raised, and the particle liquid storage chamber
  • the volume of the chamber 14 becomes smaller, and the solution in the particle liquid storage chamber 14 is also pumped to the particle liquid storage chamber 15; this cycle is repeated several times, and the material in the particle liquid storage chamber 14 and the particle liquid chamber 15 can be quickly exchanged.
  • the cell lysate in the particle storage chamber 15 can lyse the paired cells.
  • the mRNA with the polyA tail released can enter the particle trap cavity and hybridize with the T30 on the microsphere.
  • Single microspheres/single cells are separated by FC40. There is no physical contact between cells and microspheres, so there is no cross-contamination. Therefore, high-throughput single microspheres can capture mRNA of a single cell.
  • the resulting micrographs are shown in Figure 9-11.
  • Example 1-2 only a single replacement of the solution or reagent in the flow channel was performed.
  • gas phase replacement method of the present invention multiple replacements of the fluid in the flow channel and the generation of multiple independent micro-reaction chambers can also be realized, which can capture The microspheres and/or cells in different solutions act separately or at the same time, or undergo chemical reactions.
  • step E perform the following steps:
  • Step E-1 Increase the pressure of the drive pump control network channel 22.
  • the drive pump deformation chamber 24 becomes larger, the diaphragm 26 is raised, the particle liquid storage chamber 15 is reduced in volume, and the solution in the matching unit is all discharged to Cells are captured inside the cavity.
  • inject 1 ⁇ PBS into the two sample inlets 1 and 2 at a flow rate of 0.2mL/h to thoroughly clean the microspheres, and to prevent free RNA from affecting the microspheres during the cleaning process. Contamination of the ball.
  • Step E-2 Close the barrier channel 12, at this time the connecting channel 13 is opened, and the two sample inlets 1 and 2 are respectively injected with RT mix (1 ⁇ Maxima RT buffer, 1mM dNTPs (Clontech, catalog number) at a flow rate of 0.2mL/h : 639125), 1U/ ⁇ L RNase inhibitor (Lucigen, Item No. 30281-2), 2.5 ⁇ M Template_Switch_Oligo, and 10 U/ ⁇ L Maxima H-RT (ThermoScientific, Item No. EP0751)), replace the solution in the capture channel Is RT mix, and then repeat steps similar to step D to form water-in-air droplets in the liquid storage chamber.
  • RT mix 1 ⁇ Maxima RT buffer, 1mM dNTPs (Clontech, catalog number) at a flow rate of 0.2mL/h : 639125
  • 1U/ ⁇ L RNase inhibitor Lucigen, Item No. 30281-2
  • the process is as follows: open the blocking channel 12, and the connecting channel 13 is blocked at this time, unplug the sample inlet 1, 2 tubes, Block the outlets 6, 7, and inject air into the gas inlets 4 and 5 with a syringe pump.
  • the particle capture chamber 14 and the particle capture chamber 15 respectively form a gas-encapsulated liquid micro-reaction chamber.
  • the micro-reaction chamber contains a single microsphere. , So as to isolate a single microsphere in the liquid storage chamber. When air circulates through the entire chip, all single microspheres are isolated in a single air-in-air microreaction chamber, which contains RT mix, so that all microspheres are in the RT mix solution.
  • the microfluidic chip was placed at 42°C for 1.5 hours to perform mRNA reverse transcription, and the mRNA information of a single cell carried on each microsphere was converted into cDNA information.
  • Exonuclease I mix solution (2 ⁇ L of Exonuclease I (NEW ENGLAND BioLabs Inc., Catalog No.: M0293), 4 ⁇ L of 10 ⁇ Exonuclease I buffer was injected into the two sample inlets 1 and 2 of the chip at a flow rate of 0.2mL/h.
  • Solution (NEW ENGLAND BioLabs Inc., catalog number: M0293), 34 ⁇ L ddH 2 O), incubate the chip at 37°C for 45 min, and then inject 10 ⁇ L TE/TE into the two sample inlets 1 and 2 of the chip at a flow rate of 0.2mL/h.

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Abstract

一种微流控芯片的使用方法，包括向微流控芯片注入气体，以替换已注入微流控芯片的液体，并在微流控芯片中形成气包液体形式的微反应腔。还提供了一种获取检测数据的方法、一种体现在计算机可读介质中的计算机程序产品及一种试剂盒。该使用方法易于操作、成本低、通用性强，可实现流体的快速交换，达成单微粒的高效分离、捕获，所得微粒纯度高，且该使用方法可避免芯片堵塞，便于回收利用。

Description

一种微流控芯片的使用方法及其装置 技术领域
本发明涉及微流控技术及分子生物学领域，具体涉及一种微流控芯片的使用方法。
背景技术
细胞是生物及生命活动的基本单位，传统方法对大量细胞平均信号的分析处理使得信号的平均化模糊了对人体各生命系统及组成这些系统的细胞间异质性的认识。随着高通量测序技术的发展，单细胞/颗粒测序技术已经成为单细胞分析最重要手段之一，其极大地提高了单细胞/颗粒分析的效率与准确性。
微流控芯片将生物和化学等相关领域中的样品制备、反应、分离、检测及细胞培养、分选、裂解等基本操作单元集成至微小芯片上，由微通道形成网络，以可控流体贯穿整个系统，使常规化学或生物实验小型化和集成化。由于基于微流控芯片平台的反应试剂消耗量少、作用时间短，因此特别适用于单细胞/颗粒分析研究。
现有微流控芯片分析平台往往需要在芯片通道内加入各种不同试剂以实现多种生化反应的正常进行，在加入不同试剂后，为实现高通量的单细胞分析通常使用油相(即油封法)对连续的反应腔进行隔断从而防止相邻反应单元间的信号干扰，然而，由于油水不相容的天然属性，油相的使用往往会导致油水液滴生成不稳定、难以进行后续细胞融合、裂解、芯片通道内油相残留、堵塞，使得芯片难于再次利用、油水液滴包裹及破乳效果差、成本高、回收效率低等等，难以在芯片上同时实现高通量及多反应的集成。
发明内容
为解决现有技术中存在的上述问题，本发明的目的在于提供一种微流控芯片的使用方法。本发明所述方法易于操作、成本低、通用性强，可实现流体的快速交换，达成单微粒的高效分离、捕获，所得微粒纯度高，且该方法可避免芯片堵塞，便于回收利用。
本发明通过以下技术方案实现上述目的：
一种微流控芯片的使用方法，包括向所述微流控芯片通入气体，以替换已注入所述微流控芯片的液体并在所述微流控芯片中形成气包液体形式的微反应腔。
本发明的一些实施方案中，所述已注入所述微流控芯片的液体中含有微粒。本发明的一些实施方案中，所述已注入所述微流控芯片中的液体中不含有微粒。
本发明的一些实施方案中，所述微反应腔中含有微粒；本发明的另一些实施方案中， 所述微反应腔中不含微粒。
本发明所述微粒，是本领域熟知的，需要进行捕获、分析、反应的颗粒。所述微粒的粒径大小处于5微米～1000微米的范围内，例如5微米-200微米。本领域熟知的微粒包括但不限于细胞、细胞团簇、微生物、微生物团簇、噬菌体、外泌体、胶束和人造微球，其人造微球包括但不限于聚乙二醇、聚丙烯酰胺、聚甲基丙烯酸、聚甲基丙烯酸酯、聚乙烯醇、聚乙烯、聚苯乙烯、聚酯(如：PLGA和PLA)、二氧化硅和石墨烯等微球，所述人造微球表面含有实现预期检测目的的物质，如：但不限于核酸适体(aptamer)等化合物，以及核酸、蛋白质和多肽等生物大分子。一些实施方案中，所述人造微球是修饰有用于RNA捕获的核酸序列的微球，另一些实施方案中，所述人造微球是修饰有用于基因捕获的核酸序列的微球，另一些实施方案中，所述人造微球是修饰有核酸适体或抗体等分子的微球，另一些实施方案中，所述人造微球是修饰有上述两种或者几种分子的微球。
在一些实施方案中，所述微反应腔含有单个微球；在一些实施方案中，所述微反应腔含有单个细胞；在一些实施方案中，所述微反应腔不含微粒。
在一些实施方案中，所述气体可以是本领域常规使用的任何气体，包括但不限于空气、氮气、氧气、氦气、氢气、二氧化碳、氖气、氩气、氙气等。这些气体可单独或混合使用。本领域技术人员可根据实际生化反应兼容性需求自行选择所使用气体。
在一些实施方案中，所述气体与已注入所述微流控芯片的液体通过相同的入口进入所述微流控芯片中。在一些实施方案中，所述气体与已注入所述微流控芯片的液体通过不同的入口进入所述微流控芯片中。在一些实施方案中，所述气体通过多个不同入口进入所述微流控芯片。
本发明的实施方案中，从所述微流控芯片的一个开口通入气体时，已注入所述微流控芯片的液体被所述气体替换，从另一开口流出。
本发明的气体可通过气瓶经气体管道输送至微流控芯片。在一些实施方案中，可使用同一气瓶向同一气相入口或不同气相入口送气；在另一些实施方案中，可使用不同气瓶向同一气相入口或不同气相入口送气。本领域技术人员知晓使用气瓶和连通管道的常规方法。
在一些实施方案中，将所述气体以0.02L/min-1.0L/min的气流量通入所述微流控芯片；在一些实施方案中，将所述气体以0.05L/min-0.7L/min的气流量通入所述微流控芯片；一些实施方案中，将所述气体以0.06L/min、0.07L/min、0.08L/min、0.09L/min、0.1L/min的气流量通入所述微流控芯片。
本发明中，可根据实际需要调整气体的进气时间，以使气体全部或部分替换已注入所述微流控芯片的液体为宜。在一些实施方案中，所述气体的进气时间为10min-90min；一些实施方案中，所述气体的进气时间为10min-40min；在一些实施方案中，所述气体的进 气时间为15min-25min。
本发明中所述微流控芯片可以是本领域常规使用的，例如综述性文献(Shembekar,Chaipan et al.2016)/教科书(《微纳流控芯片实验室》，林炳承著)中所示例的多种微流控芯片，均可适用本发明提供的微流控芯片使用方法(即，气封方法)。进一步地，本发明方法特别适用于与专利申请CN107012067A中所述微流控芯片相似的芯片，可将其油相入口替换为气相入口使用。
在一些实施方案中，所述微流控芯片包括捕获层、控制层和载片三部分。捕获层包括两个并列的微粒捕获流道及将其连接的连接通道。微粒捕获流道中含有多个单微粒捕获单元，每个单元包括流道、储液腔室、捕获通道，捕获通道的直径小于待捕获微粒的直径，可以捕获单个微粒；前一捕获单元流道的左端连接后一捕获单元流道的右端，所述储液腔室位于流道两端之间，且设有三条通道，第一通道直径大于待捕获的单微粒，通往流道的流体进口端，第二通道为捕获通道，直径小于待捕获的单微粒，通往流道的液体出口端；第三通道为连接通道，直径小于待捕获的单微粒，通往并行的另一捕获单元的储液腔室；所述连接通道连接两个配对的微粒捕获单元的储液腔室。
每个捕获流道还分别设有位于前部的微粒入口、位于末端的气相入口和位于末端的微粒出口。
所述两个微粒捕获流道尺寸(长、宽、高)及捕获单元形状大小可以是相同的，也可以是不同的。
控制层包括阻隔通道，位于捕获层连接通道正下方或上方，其间由隔膜分隔。所述阻隔通道设有一个入口。
捕获层、控制层位于载片上方。
在一些实施方案中，所述还微流控芯片包括用于改变微粒捕获单元的储液腔室体积的驱动泵单元，包括相连通的驱动泵控制网络通道和驱动泵形变腔室，所述驱动泵控制网络通道还设有驱动泵入口。所述驱动泵形变腔室位于捕获层单微粒捕获单元储液腔室顶部或底部，驱动泵形变腔室和储液腔室间通过隔膜分隔。
在一些实施方案中，通道的尺寸由具体使用和分析的微粒的大小决定。一般的，捕获流道宽度可以是5-500微米，例如：包括但不限于5-100微米、100-300微米、300-500微米、5-50微米、50-200微米、200-400微米、10微米、20微米、50微米、80微米、100微米、200微米、300微米或者400微米；深度可以是5-500微米，例如：包括但不限于5-100微米、100-300微米、300-500微米、5-50微米、50-200微米、200-400微米、10微米、20微米、50微米、80微米、100微米、200微米、300微米或者400微米；连接通道的宽度可以是3-100微米，例如：包括但不限于3-30微米、30-50微米、50-100微米、10-40微米、 40-80微米、5微米、10微米、20微米、50微米、60微米、70微米、80微米或者90微米；连接通道深度可以是3-100微米，例如：包括但不限于3-30微米、30-50微米、50-100微米、10-40微米、40-80微米、5微米、10微米、20微米、50微米、60微米、70微米、80微米或者90微米。
在一些实施方案中，所述微粒的直径为5-200微米，可以是微米尺寸的任意微球或细胞。可以使用所述微流控芯片进行微球与微球的配对，或细胞与细胞的配对，或微球与细胞的配对。
在一些实施方案中，所述微球的直径可以是5-200微米，例如：包括但不限于5微米、10微米、15微米、20微米、30微米、70微米、80微米、90微米、100微米、120微米、150微米或者180微米。
在一些实施方案中，所述的细胞的直径可以是5-100微米，例如：包括但不限于5微米、10微米、15微米、20微米、30微米、70微米、80微米或者90微米。
在一些实施方案中，本发明的微流控芯片所用捕获层和载片的材料可以是硅片、玻璃、聚甲基丙烯酸甲酯、聚乙烯、聚丙烯、聚氯乙烯和聚酯等；控制层的材料可以是聚甲基丙烯酸甲酯、聚乙烯、聚丙烯、聚氯乙烯和聚酯等。
在一些实施方案中，所用捕获层和控制层的材料可以为聚二甲基硅氧烷PDMS，载片材料可以为玻璃。
在一些实施方案中，捕获层和控制层通过热校准可逆键合在一起，之后再与载片通过等离子体键合。
在一些实施方案中，捕获层和控制层通过等离子体表面活化后键合在一起，之后再与载片通过等离子体键合。
在一些实施方案中，微粒通入的流速为0.005mL/h-10mL/h，例如：包括但不限于0.005mL/h-0.05mL/h、0.05mL/h-0.1mL/h、0.1mL/h-0.5mL/h、0.5mL/h-2mL/h、2mL/h-6mL/h、6mL/h-10mL/h、0.05mL/h-0.5mL/h、0.5mL/h-3mL/h、3mL/h-8mL/h、0.01mL/h、0.05mL/h、0.1mL/h、0.2mL/h、0.5mL/h、0.8mL/h、1mL/h、2mL/h、4mL/h、6mL/h或者8mL/h。
在一些实施方案中，阻隔泵通过通入流体发生形变发挥阻隔捕获层连接通道的作用。其中阻隔泵充满的流体可以为水溶液、油或者空气，其可由注射泵注入。通过控制注射泵的压力，控制阻隔泵的压力，即控制对捕获层连接通道的阻隔程度。
在一些实施方案中，所述微流控芯片基本工作流程为：
步骤A：增加阻隔泵压力，阻隔通道形变，隔膜挤压连接通道最上层，直至两套并 联的微粒捕获流道间的连接通道被完全阻断，从一套微粒捕获流道入口通入含有微粒的液体，当单微粒进入捕获单元时，因为流体从流道经过的路径大于从储液腔室经过的路径，微粒先进入储液腔室，由于捕获通道的宽度(管径)小于微粒直径，所述微粒被卡在捕获通道前，同时堵住捕获通道，流经储液腔室的流体流速趋近于零，此时后续的微粒无法再次进入该储液腔室，只能通过流道进入下一个捕获单元，这样就实现了单微粒的捕获。在后续的微粒捕获单元中重复该过程，即可实现高通量单微粒的捕获。
步骤B：针对上述实现单微粒捕获的微粒捕获流道，在气相入口注入气体，当气体进入捕获单元时，由于捕获通道宽度远小于流路宽度，其毛细阻力较大，因此气相进入捕获通道而不进入储液腔室，此时储液腔室的液体被保留，形成气包液体的微反应腔，当气体流通整个芯片时，流道中所有的捕获单元均被隔离成气包液体的单个液滴，实现了单个气包液体微反应腔的分隔。
可选地，在一些实施方案中，步骤B前，所述工作流程还可包含步骤A-1：
步骤A-1：在所述实现单微粒捕获的微粒捕获流道的样品入口通入进行下一步操作所需液体，洗去流道中含有残余微粒的液体。
在一些实施方案中，可根据实际需求，多次重复步骤A-1和/或步骤B。每次进行步骤A-1和/或步骤B时，所使用液体和/或气体可以是相同的，也可以是不同的。
在一些实施方案中，在步骤B后，采用与步骤A、A-1、B相类似的方法，分别实施步骤C、C-1、D，使另一套未进行单微粒捕获的微粒捕获流道实现高通量单微粒捕获。在一些实施方案中，可根据实际需求，多次重复步骤C-1和/或步骤D。每次进行步骤C-1和/或步骤D时，所使用液体和/或气体可以是相同的，也可以是不同的。之后，该工作流程进一步包括：
步骤E：减小或关闭阻隔泵，此时两套并行的微粒捕获流道间的连接通道被打开，两套微粒捕获流道间的用于配对的气包液体微反应腔处于可联通状态，由于扩散作用，两个储液腔室里的气包液体微反应腔可以实现相互物质交换，经过一段时间充分反应后，实现高通量配对微粒之间的独立反应。
在一些实施方案中，在步骤E中，可使用分别设置于两套微粒捕获流道捕获单元储液腔室底部的驱动泵，用于分别间隔交替改变两个捕获单元的储液腔室体积，促进实现两个捕获单元储液腔室中物质的快速交换。
在一些实施方案中，步骤E后，可重复进行上述步骤A-D中的任一步或几步，例如重复步骤B和步骤A-1，或重复步骤A、B、任选的A-1和C，或重复步骤B、任选的A-1和步骤C等等，通过反复多次的液体替换与气包溶液微反应腔的生成，可实现多种不同反应微粒及试剂的加入、替换及反应腔的形成等多功能操作。
在一些实施方案中，所述工作流程的最后一步为步骤F：
步骤F：将捕获层与控制层剥离开，储液腔室中被捕获的，或经反应后的微粒被暴露在芯片的表面，用缓冲溶液将其洗脱，进行后续的分析研究。
本发明还提供一种含有气包液体形式微反应腔的微流控芯片。其中所述气包液体形式微反应腔及微流控芯片如上文所定义。
本发明还提供一种获取检测数据的方法，包括：
将检测样本加载于微流控芯片，并向微流控芯片注入气体，形成气包液体的微反应腔，经测定获得检测数据。
获取检测数据的方法，还包括将检测的数据与数据库中储存的对应数据(比如：标准曲线)进行比较，而得到对检测样本进行定量的数据。
一种体现在计算机可读介质中的计算机程序产品，当在计算机上执行时，执行步骤包括：
控制并将气体注入微流控芯片。
另一种体现在计算机可读介质中的计算机程序产品，当在计算机上执行时，执行步骤包括：向加载了检测样本的微流控芯片注入气体，待形成气包液体微反应腔后，再测定并获得样本的检测数据。
本发明还提供一种试剂盒，包括：
微流控芯片；和
体现在计算机可读介质中的计算机程序产品。
本发明有益效果：
1.本发明方法可以稳定地实现气包液微反应腔的生成，解决了微流控芯片使用“油封”时油水界面难以破乳，不能形成有效的油包水液滴的问题。
2.本发明方法所使用气体廉价易得，无批次差异，体系稳定，因此解决了“油封”中含有表面活性剂的油相昂贵，且批次间差异大导致结果不稳定的问题。
3.本发明方法可以方便的实现微流控芯片通道内溶液试剂的去除，避免了传统“油封”方式微乳液滴在微流控芯片通道中的残留，并可以对独立液滴进行新试剂替换，使依次添加多种试剂成为可能，大大扩展了能于微流控芯片上进行的、依赖于单微粒的试验种类，提高了实验、检测及分析效率。
4.传统“油封”方法中需使用破乳剂对“油包水”形式的捕获微粒进行破乳，该方法往往 效果差、成本高；本发明方法可以有效实现“背景扣除”及捕获微粒100％的无损回收，解决了传统“油封”方法中因油相包覆微粒导致的污染，以及萃取回收微球方式导致的微球高损失率。
5.在可进行捕获微粒配对的微流控芯片中，本发明方法可以实现稳定的液滴混合和试剂替换，解决了传统“油封”方法中油膜将配对液滴隔离，配对液滴融合困难导致的有效配对液滴比例低，以及微粒(例如细胞)无法裂解等问题。
附图说明
图1是微流控芯片整体结构俯视图，其中1、2是样品入口，3是阻隔泵入口，4、5是气体入口，6、7是样品出口。
图2是微流控芯片放大结构示意图，其中8、9是微粒捕获单元，10、11是微粒捕获流道，12是阻隔通道，13是连接通道。
图3是微粒捕获单元俯视图，其中10、11分别是微粒捕获流道，12是阻隔通道，13是连接通道，14、15分别是储液腔室，16、17分别是微粒捕获通道。
图4是微粒捕获单元截面图，其中13是连接通道，14、15分别是储液腔室，12是阻隔通道，18是间隔薄膜。
图5是压力驱动微流控芯片整体结构俯视图，其中19、20分别是驱动泵入口。
图6是压力驱动微流控芯片放大结构示意图，其中21、22分别是驱动泵控制网络通道。
图7是压力驱动微流控芯片颗粒/细胞配对捕获单元俯视图，其中23、24分别是微粒捕获单元驱动形变腔室。
图8压力驱动微流控芯片颗粒/细胞配对捕获单元截面图，其中25和26是间隔薄膜。
图9是使用本发明方法对微流控芯片通气后显微镜所显示气包水形式的微反应腔情况。
图10是使用本发明方法对微流控芯片通气后显微镜所显示微粒捕获及气包水(含微粒)形式的微反应腔情况。
图11是使用本发明方法对微流控芯片通气后显微镜荧光场下所示配对微反应腔的混合过程图。
图12是使用本发明方法对微流控芯片通气以对芯片内溶液进行替换的示意图及显微镜图。
图13是与现有技术方法(油相置换)相比，使用本发明(气相置换)方法对芯片通气， 对微球进行捕获/反应后，微球的回收率统计结果图。
图14是使用本发明方法对芯片通气，对微粒进行捕获、反应、回收前后，芯片的俯视显微镜图。
图15是现有技术中使用油相形成油包水微反应腔的芯片显微镜视图。
图16是现有技术中使用油相形成油包水微反应腔的芯片显微镜荧光成像视图。
图17是现有技术中使用油相形成油包水微反应腔(含微粒)的芯片内显微镜视图。
具体实施方式
实施例1
基于本发明的微流控芯片使用方法，实现高通量配对捕获单微粒，所使用芯片如图1-4所示，该芯片由标准软光刻技术加工，包括捕获层、控制层、载片。捕获层由捕获流道10、捕获流道11、连接通道13组成，控制层由阻隔通道12组成。其中捕获流道含有多个首尾串联的微粒捕获单元8，每个单元由流道101、储液腔室(14)、捕获通道(16)组成；参见图3，流道101包括U形管(在其它实施例中，也可以是弧形或其它曲折形状)，前一单元的U形管的左臂端连接后一单元的U形管的右臂端，U形管之间的连接部分为直管，储液腔室14位于U形的两臂之间，且设有三条通道，第一通道直径大于待捕获的单微粒，通往U形管右臂，第二通道(捕获通道16)直径小于待捕获的单微粒，通往U型管的左臂；第三通道为连接通道13，直径小于待捕获的单微粒，通往并行的另一微粒捕获单元9的储液腔室15。
捕获流道11含有多个首尾串联的微粒捕获单元9，微粒捕获单元9和微粒捕获单元8对称设置，每个单元由流道111、储液腔室15、捕获通道17组成；参见图3，流道111包括U形管，前一单元的U形管的左臂端连接后一单元U形管的右臂端，U形管之间的连接部分为直管，储液腔室15位于U形的两臂之间，且设有三条通道，第一通道直径大于待捕获的单微粒，通往U形管右臂，第二通道(捕获通道17)直径小于待捕获的单微粒，通往U型管的左臂；第三通道为连接通道13，直径小于待捕获的单微粒，通往并行的另一微粒捕获单元8的储液腔室14。
捕获流道10含有一个样品入口1和气体入口4以及一个出口7，捕获流道11含有一个样品入口2和气体入口5以及一个出口6。
控制层设有阻隔通道12；阻隔通道12位于连接通道13下方，垂直于连接通道13(在其它实施例中，也可以为一定的夹角)，并由隔膜18隔开；阻隔通道12连接一阻隔泵，通过阻隔泵改变阻隔通道12的压力阻断或连通连接通道(参见图4)；阻隔通道12设有一 个入口3。
作为本发明的优选实施方式，所述流道宽度为60微米，储液腔室直径为100微米，微粒捕获通道宽度为6微米或15微米，通道深度为46微米，微粒捕获单元为720个。
作为本发明的优选实施方式，所选用的微粒为A549细胞，细胞直径为10-20微米。
作为本发明的优选实施方式，所选用的微粒为聚苯乙烯微球，直径为40微米。
作为本发明的优选实施方式，所选用的聚苯乙烯微球上含有mRNA捕获序列T30，T30可以与带有polyA尾巴的mRNA杂交。
作为本发明的优选实施方式，控制层中阻隔通道和驱动泵形变腔室宽度为30微米，高度为30微米。
作为本发明的优选实施方式，所述的所有入口均为圆柱形孔，直径为1.00mm。
作为本发明的优选实施方式，所述的捕获层和控制层的材料均为聚二甲基硅氧烷PDMS。
作为本发明的优选实施方式，所述的载片的材料为玻璃。
作为本发明的优选实施方式，阻隔通道和驱动泵形变腔室都充满水溶液，通过控制注射泵驱动压力，控制阻隔通道和驱动泵形变腔室压力。
作为本发明的优选实施方式，所选用的细胞流速为0.04mL/h。
作为本发明的优选实施方式，所选用的微球流速为0.2mL/h。
作为本发明的优选实施方式，所选用的气相流速为0.04L/min。
作为本发明的优选实施方式，本发明的具体工作过程为：
步骤A：打开阻隔泵，阻隔通道12压力增大，隔膜18向上变形，此时连接通道13被阻断，在样品入口1用注射泵通入细胞A549悬浮液，当单个细胞进入微粒捕获单元8时，首先细胞进入储液腔室14，由于捕获通道小于细胞，细胞被卡在捕获通道16前，同时堵住捕获通道，此时流经储液腔室14的液体流速趋近于零，后续的细胞无法再次进入该微粒储液腔室，只能通过流道101进入下一个捕获单元，这样就实现了单细胞的捕获。在后续的微粒捕获单元中重复该过程，即可实现高通量单细胞的捕获。
步骤A-1：保持连接通道13关闭状态，在样品入口1继续通入PBS溶液，将流道101中的多余细胞冲洗出芯片。
步骤B：拔去样品入口1管子，堵住出口7，在气体入口4用注射泵系统通入空气，空气进入微粒捕获单元8时，由于捕获通道16远小于流道101，其毛细阻力较大，因此空气进入流道101而不进入捕获通道16，此时微粒储液腔室的溶液被保留，形成一个气包水的 微反应腔，微反应腔中含有单个细胞，从而将单个细胞隔离在储液腔室中。当空气流通完整个芯片时，所有的单细胞均被隔离在单个气包水微反应腔中。
步骤C：在样品入口2用注射泵以0.2mL/h的流速注入编码微球(MACOSKO-2011-10(V+)，Barcoded Oligo dT primer ON Beads)悬浮液，当单个微球进入微球捕获单元9时，首先微球进入储液腔室15，由于捕获通道直径17小于微球，微球被卡在捕获通道17前，同时堵住捕获通道17，此时流经储液腔室15的液体流速趋近于零，后续的微球无法再次进入该微球储液腔室，只能通过流道111进入下一个捕获单元，这样就实现了单微球的捕获。在后续的微球捕获单元中重复该过程，即可实现高通量单微球的捕获。
步骤C-1：保持连接通道13关闭状态，在样品入口2通入细胞裂解液(0.2％Triton X-100)，将通道中的多余微球冲洗出芯片，并且将捕获流道内溶液替换为细胞裂解液。
步骤D：拔去微粒入口2管子，堵住出口6，在气体入口5用注射泵系统通入空气，空气进入微粒捕获单元9时，由于捕获通道17远小于流道111，其毛细阻力较大，因此空气进入流道111而不进入捕获通道17，此时微粒储液腔室的溶液被保留，形成一个气包液体的微反应腔，微反应腔中含有单个微球，从而将单个微球隔离在储液腔室中。当空气流通完整个芯片时，所有的单微球均被隔离在单个气包水微反应腔中，液滴中含有细胞裂解液。
步骤E：关闭阻隔通道12，此时连接通道13被打开。此时包裹细胞和微球的配对液滴联通，微球储液腔室15中的细胞裂解液可以通过扩散作用进入细胞储液腔室14进行细胞裂解，细胞裂解后其含有的细胞内含物也可以通过扩散作用进入微球储液腔室15，其中细胞内含物中的带有polyA尾巴的mRNA可以与微球上的T30杂交，经过一段时间后两个配对腔室之间的物质充分交换，可以实现细胞的完全裂解和单细胞mRNA的提取，由于配对单微球/单细胞之间被FC40隔离，细胞之间、微球之间没有物理接触，故没有交叉污染，因此可以实现高通量单个微球捕获单个细胞的mRNA。
步骤F：将捕获层揭下，在腔室中捕获的微球或者细胞被暴露在芯片的表面，用缓冲溶液将其洗脱下来，对提取出的单细胞mRNA继续进行后续的分析研究。
实施例2
实施例1中，两个配对液滴之间的物质交换是通过被动的扩散实现的，其交换速度较慢，为了加快两个配对液滴之间的物质交换，在实施例1的基础上增加驱动泵单元用于控制两个液滴之间的混合，控制液滴在两个配对腔体之间的分配。
下面结合附图和具体实施方式以微球和细胞的配对捕获为例对本发明做进一步详细说明。
如图5-8所示，在实施例1基础上，所述芯片进一步包含驱动泵控制网络通道21、驱 动泵控制网络通道22、驱动泵形变腔室23和驱动泵形变腔室24，所述驱动泵控制网络通道21、22设置有驱动泵入口19、20。所述驱动泵控制网络通道21、22分别与驱动泵形变腔室23、24相连通，所述驱动泵形变腔室23、24分别位于储液腔室14、15底部，并通过隔膜25、26与所述储液腔室14、15相分隔。
作为本发明的优选实施方式，具体工作过程为：
在实施例1基础上，步骤E中，关闭阻隔通道12，此时连接通道13被打开。此时包裹细胞和微球的配对液滴联通，增加驱动泵控制网络通道22的压力，此时驱动泵形变腔室24变大，隔膜26凸起，微粒储液腔室15体积减小，其中的细胞裂解液泵向微粒储液腔室14，同时减小驱动泵控制网络通道21的压力，此时驱动泵形变腔室23减小，隔膜25凹陷，微粒储液腔室14体积变大，微粒储液腔室15的溶液也被泵向微粒储液腔室14；当减小驱动泵控制网络通道22的压力，此时驱动泵形变腔室24减小，隔膜26凹陷，微粒储液腔室15体积变大，其中的溶液泵向微粒储液腔室14，同时增大驱动泵控制网络通道21的压力，此时驱动泵形变腔室23变大，隔膜25凸起，微粒储液腔室14体积变小，微粒储液腔室14的溶液也被泵向微粒储液腔室15；如此循环几次，可以实现微粒储液腔室14和微粒储液腔室15的物质快速交换。微粒储液腔室15中的细胞裂解液可以对于之配对的细胞进行裂解，细胞裂解后，其释放的带有的polyA尾巴的mRNA可以进入微粒捕获腔体与微球上的T30杂交，由于配对单微球/单细胞之间被FC40隔离，细胞之间、微球之间没有物理接触，故没有交叉污染，因此可以实现高通量单个微球捕获单个细胞的mRNA。所得结果显微镜照片如图9-11所示。
实施例3
实施例1-2中，仅进行了流道中溶液或试剂的单次替换，使用本发明气相置换方法，还可实现流道内流体的多次替换以及多次独立微反应腔的生成，可使捕获的微球和/或细胞在不同溶液中分别或同时发生作用，或进行化学反应。
在实施例2基础上，在步骤E后，进行如下步骤：
步骤E-1：增加驱动泵控制网络通道22的压力，此时驱动泵形变腔室24变大，隔膜26凸起，微粒储液腔室15体积减小，配对单元内的溶液全部被排至细胞捕获腔体内。打开阻隔通道12，此时连接通道13被关闭，在两个样品入口1和2分别以0.2mL/h的流速注入1×PBS对微球进行彻底的清洗，同时防止清洗过程中游离RNA对微球的污染。
步骤E-2：关闭阻隔通道12，此时连接通道13被打开，在两个样品入口1和2分别以0.2mL/h的流速注入RT mix(1×Maxima RT buffer,1mM dNTPs(Clontech,货号：639125)、1U/μL RNA酶抑制剂(Lucigen,货号：30281-2)、2.5μM Template_Switch_Oligo，以及10 U/μL Maxima H-RT(ThermoScientific,货号：EP0751))，将捕获流道中的溶液替换为RT mix，然后重复类似步骤D步骤，在储液腔室中形成气包水液滴，过程如下：打开阻隔通道12，此时连接通道13被阻断，拔去样品入口1、2管子，堵住出口6、7，在气体入口4、5用注射泵通入空气，在微粒捕获腔室14和微粒捕获腔室15分别形成气包液体的微反应腔，微反应腔中含有单个微球，从而将单个微球隔离在储液腔室中。当空气流通完整个芯片时，所有的单微球均被隔离在单个气包水微反应腔中，微反应腔中含有RT mix，使得所有微球处于RT mix溶液中。将该微流控芯片置于42℃反应1.5h进行mRNA反转录，每个微球上携带的单个细胞内mRNA信息被转换成cDNA信息。
步骤E-3：反转录结束后，在芯片的两个样品入口1、2分别依次注入TE/SDS(10mM Tris-HCl、1mM EDTA、0.1％SDS、pH＝8.0)、TE/TW(10mM Tris-HCl、1mM EDTA、0.01％吐温、pH＝8.0)、TE(10mM Tris-HCl、1mM EDTA、pH＝8.0)缓冲液进行微球的清洗，溶液流速为0.2mL/h，每种溶液注入量均为20μL。随后在芯片的两个样品入口1、2分别以0.2mL/h流速注入20μL Exonuclease I mix溶液(2μL外切酶I(NEW ENGLAND BioLabs Inc.，货号：M0293)、4μL 10×外切酶I缓冲溶液(NEW ENGLAND BioLabs Inc.，货号：M0293)、34μL ddH 2O)，将芯片置于37℃孵育45min，随后以0.2mL/h流速在芯片的两个样品入口1、2分别注入10μL TE/SDS、10μL TE/TW、10μL ddH 2O。整个流程结束后，在微粒气相入口6、7向芯片内注入氮气并结合芯片内流体的微扰动将芯片内的所有微球进行无损的回收，用于后续扩增建库和测序等一系列反应。所得结果显微镜照片如图12-14所示。
对比例1
参考专利申请CN107012067A实施例2所示方法对细胞和微球进行分别捕获和配对，所得结果如图15-17所示。使用现有技术油相置换方法，不能形成稳定的液滴，配对液滴混合亦不稳定、溶剂替换后往往存在乳剂残留，且微球回收效率低。

Claims (17)

  1. 一种微流控芯片的使用方法，包括向所述微流控芯片通入气体，以替换已注入所述微流控芯片的液体，并在所述微流控芯片中形成气包液体形式的微反应腔。
  2. 根据权利要求1所述的方法，其中所述已注入所述微流控芯片的液体中含有或不含有微粒；所述微反应腔中含有或不含有微粒。
  3. 根据权利要求2所述的方法，其中所述微粒包括细胞、细胞团簇、微生物、微生物团簇、噬菌体、外泌体、胶束和人造微球；
    优选地，所述人造微球包括聚乙二醇、聚丙烯酰胺、聚甲基丙烯酸、聚甲基丙烯酸酯、聚乙烯醇、聚乙烯、聚苯乙烯、聚酯、二氧化硅和石墨烯微球；
    优选地，所述人造微球表面含有实现预期检测目的的物质，包括核酸、核酸适体、蛋白质和多肽；
    优选地，所述人造微球是修饰有用于RNA捕获的核酸序列、修饰有用于基因捕获的核酸序列或修饰有核酸适体或抗体等一种或几种类型分子的微球。
  4. 根据前述任一项权利要求所述的方法，其中所述气体包括以下一种气体或者多种气体的组合：空气、氮气、氧气、氦气、氢气、二氧化碳、氖气、氩气、氙气。
  5. 根据前述任一项权利要求所述的方法，其中所述气体与已注入所述微流控芯片的液体通过相同或不同的入口进入所述微流控芯片中。
  6. 根据前述任一项权利要求所述的方法，其中将所述气体以0.02L/min-1.00L/min或0.05L/min-0.70L/min的气流量通入所述微流控芯片；
    优选地，将所述气体以0.04L/min的气流量通入所述微流控芯片。
  7. 根据前述任一项权利要求所述的方法，其中所述气体的进气时间为10min-90min、10min-40min或15min-25min。
  8. 根据前述任一项权利要求所述的方法，其中所述微流控芯片包括捕获层、控制层、载片；所述捕获层包括两个并行的捕获流道(10，11)及将其连接的连接通道(13)，其中所述捕获流道(10，11)分别由多个捕获单元(8，9)首尾串接组成，每个所述捕获单元包括流道(101，111)、储液腔室(14，15)、捕获通道(16，17)，所述流道(101，111)包括U形管，前一捕获单元U形管的左臂端连接后一捕获单元U形管的右臂端，所述储液腔室位于U形管的两臂之间，且设有三条通道，第一通道直径大于待捕获的单微粒，通往U形管的流体进口端，第二通道为捕获通道，直径小于待捕获的单微粒，通往U型管的液体出口端；第三通道为连接通道(13)，直径小于待捕获的单微粒，通往并行的另一捕获单元的储液腔室；所述连接通道(13)连接两个捕获单元(8，9)的储液腔室(14，15)；
    所述捕获流道(10，11)分别设有样品入口(1，2)、气体入口(4，5)以及样品出口(6，7)；
    所述控制层包括阻隔通道(12)；所述阻隔通道(12)层位于所述连接通道(13)下方或上方，与连接通道(13)相交并由隔膜(18)分隔；所述阻隔通道(12)设有入口(3)。
  9. 根据权利要求8所述的方法，其中所述两个微粒捕获流道尺寸及捕获单元形状大小是相同的或是不同的。
  10. 根据权利要求8或9所述的方法，其中所述的流道的宽度为5-500微米，深度为5-500微米；连接通道的宽度为3-100微米，深度为3-100微米。
  11. 根据权利要求8-10中任一项所述的方法，其中所述微粒的直径在5-200微米。
  12. 根据权利要求8-11中任一项所述的方法，其中所述微粒通入的流速为0.005mL/h-10mL/h。
  13. 根据权利要求8-12中任一项所述的方法，其中所述微流控芯片还包括用于改变所述储液腔室(14，15)体积的驱动泵单元，包括相连通的驱动泵控制网络通道(21，22)和驱动泵形变腔室(23，24)，所述驱动泵控制网络通道(21，22)还设有驱动泵入口(19，20)，所述驱动泵形变腔室(23，24)分别位于储液腔室(14，15)顶部或底部，之间被隔膜(25，26)分隔。
  14. 一种获取检测数据的方法，包括：
    将检测样本加载于微流控芯片，并向所述微流控芯片注入气体，形成气包液体的微反应腔，经测定获得检测数据。
    获取检测数据的方法，还包括将检测的数据与数据库中储存的对应数据(比如：标准曲线)进行比较，而得到对检测样本进行定量的数据。
  15. 一种体现在计算机可读介质中的计算机程序产品，当在计算机上执行时，执行步骤包括：
    控制并将气体注入微流控芯片。
  16. 一种体现在计算机可读介质中的计算机程序产品，当在计算机上执行时，执行步骤包括：
    向加载了检测样本的微流控芯片注入气体，待形成气包液体微反应腔后，再测定并获得样本的检测数据。
  17. 一种试剂盒，包括：
    微流控芯片；和
    体现在计算机可读介质中的计算机程序产品。
PCT/CN2020/126616 2019-11-25 2020-11-05 一种微流控芯片的使用方法及其装置 WO2021103970A1 (zh)

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