Contact-less priming method for loading a solution in a microfluidic device and associated system

Abstract

The present invention relates to a contact-less priming system for loading a solution in a microfluidic device comprising: at least one microfluidic device, a pressure chamber configured to enclose said at least one microfluidic device, a pressurization unit fluidly connected to the pressure chamber and at least one closing member. The present invention also relates to a contact-less priming method for loading a solution in a microfluidic device.

Claims

1. A contact-less priming system for loading a solution in a microfluidic device comprising: at least one microfluidic device comprising at least one first port, at least one closed second port and at least one microchannel, wherein each of said at least one first and second ports are fluidly connected to said at least one microchannel and wherein said at least one first port is suitable for containing at least one solution; a pressure chamber with at least one closable, gas tight aperture, configured to enclose said at least one microfluidic device; and a pressurization unit fluidly connected to the pressure chamber for applying pressure in the pressure chamber and upon the at least one first port.

2. The contact-less priming system according to claim 1, wherein the at least one first port has a capacity ranging from 1 to 1000 microliters.

3. The contact-less priming system according to claim 1, wherein the pressurization unit comprises a pressure source, a pressure monitoring device and a feedback control to pressurize the pressure chamber at a pressure suitable to cause a selected amount of the at least one solution to pass from the at least one first port to the at least one microchannel.

4. The contact-less priming system according to claim 1, further comprising at least one closing member disposed on the at least one first port, configured to close at least partially and/or to open at least partially said at least one first port.

5. The contact-less priming system according to claim 1, wherein the at least one closing member is selected from at least one flow restrictor or at least one check-valve.

6. The contact-less priming system according to claim 1, further comprising at least one filter disposed on the at least one first port inhibiting liquid flow and permeable to gas.

7. The contact-less priming system according to claim 1, wherein the at least one microchannel is filled with a carrier fluid.

8. The contact-less priming system according to claim 1, wherein the at least one microchannel comprises at least one network of microchannels.

9. The contact-less priming system according to claim 8, wherein the at least one network of microchannels comprises at least one microchannel and one fluid partitioning zone.

10. The contact-less priming system according to claim 9, wherein the at least one network of microchannels further comprises at least one region for trapping at least one dispersed phase.

11. A contact-less priming method for loading a solution in a microfluidic device comprising the following steps: providing at least one microfluidic device comprising at least one first port, at least one closed second port and at least one microchannel, wherein each of said at least one first and second ports are fluidly connected to said at least one microchannel and wherein the at least one first port is suitable for containing at least one solution; loading at least one solution in the at least one first port; introducing and enclosing said at least one microfluidic device with the at least one solution in a pressure chamber through at least one closable, gas tight aperture of said pressure chamber under atmospheric pressure; and pressurizing the pressure chamber.

12. The contact-less priming method according to claim 11, wherein at least one closing member is disposed on the at least one first port, configured to close at least partially and/or to open at least partially said at least one first port.

13. The contact-less priming method according to claim 11, wherein the at least one closing member is selected from at least one flow restrictor or at least one check-valve.

14. The contact-less priming method according to claim 11, wherein the at least one microchannel is filled with a carrier fluid.

15. The contact-less priming method according to claim 11, wherein the at least one microchannel comprises at least one network of microchannels, one fluid partitioning zone and at least one region for trapping at least one dispersed phase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a microfluidic device according to the present invention comprising no closing member disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a solution in the first port and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(2) FIG. 2 illustrates a microfluidic device according to the present invention comprising a stopper disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a solution in the first port and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(3) FIG. 3 illustrates a microfluidic device according to the present invention comprising a stopper disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a solution in the first port and in the second port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(4) FIG. 4 illustrates a microfluidic device according to the present invention comprising a stopper disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid in the microfluidic network and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(5) FIG. 5 illustrates a microfluidic device according to the present invention comprising a stopper disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid and a dispersed phase in the first port and the carrier fluid in the second port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(6) FIG. 6 illustrates a microfluidic device according to the present invention comprising a stopper disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid, a dispersed phase and a third solution immiscible with the dispersed phase in the first port and the carrier fluid and the third solution in the second port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(7) FIG. 7 illustrates a microfluidic device according to the present invention comprising a flow restrictor disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a solution in the first port and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(8) FIG. 8 illustrates a microfluidic device according to the present invention comprising a flow restrictor disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid in the microfluidic network and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(9) FIG. 9 illustrates a microfluidic device according to the present invention comprising a flow restrictor disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(10) FIG. 10 illustrates a microfluidic device according to the present invention comprising a flow restrictor disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid, a dispersed phase and a third solution immiscible with the dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(11) FIG. 11 illustrates a microfluidic device according to the present invention comprising an outward check-valve disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a solution in the first port and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(12) FIG. 12 illustrates a microfluidic device according to the present invention comprising an outward check-valve disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid in the microfluidic network and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(13) FIG. 13 illustrates a microfluidic device according to the present invention comprising an outward check-valve disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(14) FIG. 14 illustrates a microfluidic device according to the present invention comprising an outward check-valve disposed on the second port and no closing member disposed on the first port; said microfluidic device comprising a carrier fluid, a dispersed phase and a third solution immiscible with the dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(15) FIG. 15 illustrates a microfluidic device according to the present invention comprising an inward check-valve disposed on the first port and a stopper disposed on the second port; said microfluidic device comprising a solution in the first port and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(16) FIG. 16 illustrates a microfluidic device according to the present invention comprising an inward check-valve disposed on the first port and a stopper disposed on the second port; said microfluidic device comprising a carrier fluid and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(17) FIG. 17 illustrates a microfluidic device according to the present invention comprising an inward check-valve disposed on the first port and a stopper disposed on the second port; said microfluidic device comprising a carrier fluid, a dispersed phase and a third solution immiscible with the dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(18) FIG. 18 illustrates a microfluidic device according to the present invention comprising an inward check-valve disposed on the first port and an outward check-valve disposed on the second port; said microfluidic device comprising a solution in the first port and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(19) FIG. 19 illustrates a microfluidic device according to the present invention comprising an inward check-valve disposed on the first port and an outward check-valve disposed on the second port; said microfluidic device comprising a carrier fluid and a dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(20) FIG. 20 illustrates a microfluidic device according to the present invention comprising an inward check-valve disposed on the first port and an outward check-valve disposed on the second port; said microfluidic device comprising a carrier fluid, a dispersed phase and a third solution immiscible with the dispersed phase in the first port; and said microfluidic device being disposed in a pressure chamber. Said pressure chamber being first under atmospheric pressure (1), then pressurized (2) and finally under atmospheric pressure (3). The pressure within the pressure chamber is represented by a continuous line while the pressure within the microfluidic device is represented by a series of circles.

(21) FIG. 21 illustrates a system according to the present invention. In particular, FIG. 21A illustrates the pressure chamber (1) with a movable top lid (15) in the open position and FIG. 21B illustrates the pressure chamber (1) with the movable top lid (15) in the closed position.

REFERENCES

(22) 1—Pressure chamber

(23) 2—Microfluidic device

(24) 3—First port of the microfluidic device

(25) 4—Second port of the microfluidic device

(26) 5—Microchannel/Network of microchannels of the microfluidic device

(27) 6—Stopper

(28) 7—Inward check-valve

(29) 8—Outward check-valve

(30) 9—Flow restrictor

(31) 11—Solution

(32) 12—Carrier fluid

(33) 13—Dispersed phase

(34) 14—Third solution

(35) 15—Movable top lid

(36) 16—Gasket

(37) 17—Aperture

(38) 18—Clamping mechanism

(39) 19—Bottom plate

EXAMPLES

(40) The present invention is further illustrated by the following examples.

(41) Example 1: Priming multiple microfluidic devices with oil using outward check-valves on the output ports of the microfluidic devices.

(42) Material

(43) The pressure chamber consists in a parallelepiped box 18 cm in length, 15 cm in width and 8 cm in height and of inner volume of 1 liter, assembled from an aluminum bottom plate and a milled PMMA casing. An O-ring is placed between the two pieces to provide a gas-tight seal that is able to resist an over-pressure of up to 1 bar applied inside the pressure chamber. The PMMA casing features a PMMA screw lid, 11 cm in diameter, that allows easy opening and closing of the pressure chamber in order to place microfluidic devices to be primed into the pressure chamber. An O-ring is placed between the casing and the screw lid to provide a gas-tight seal.

(44) The pressurization unit consists in a Fluigent MFCS pressure controller with integrated manometer and loop feedback control, connected to a pressure source of 1 bar. The pressurization unit is itself connected to the pressure chamber via silicone tubing through a fluidic port located in the center of the screw lid. The Fluigent MAES Flow software dynamically controls the overpressure applied to the pressure chamber, in real-time or following set pressure commands.

(45) The microfluidic device has an appearance and dimensions similar to that of a microscope slide. It is 36 mm in width, 77 mm in length and 1.5 mm in thickness. It is made by bonding a slab of cyclic olefin polymer (COP) into which are molded networks of microchannels to a thin sheet of COP.

(46) The microfluidic device features 4 identical networks of microchannels, each connecting one first port to one second port. The first and second ports have the dimensions of a standard male Luer connector, as defined by the ISO 594-1 norm. They have a hollow conical structure extending 9 mm from the top surface of the microfluidic device. The outer wall of the connector features a 6% inward taper to meet the Luer ISO 594-1 standard and the inner volume of the port, from the top of the port to its bottom junction with the network of microchannels has an inner volume of 65 μL.

(47) The network of microchannels is described from the first port to the second port. It first features a microchannel of rectangular cross section, 200 μm in width and 150 μm in height, connecting the bottom of the first port to a comb of 33 injection microchannels of smaller dimensions, roughly 52 μm in width and 25 μm in height. These 33 microchannels all lead to a microchamber, spanning 2 of its 4 lateral sides along one of its corner. The microchamber has a width of 22 mm, a length of 15 mm and a height of 120 μm, except on its rim where the floor and roof of the chamber feature a wedge geometry. The wedged rim of the chamber extends 500 μm from the side of the chamber. The height of the chamber on its outer periphery is 25 μm and increases linearly to 65 μm at the inner edge of the wedged rim, where it then abruptly increases to 120 μm. At the corner of the microchamber opposite to the comb of small microchannels, an outlet channel 200 μm in width and 150 μm in height connects the microchamber to the second port of the microfluidic device.

(48) The junction of the injection microchannels to the wedged rim of the microchamber serves as a fluid partitioning zone, as described in Dangla, R., Kayi, S. C., & Baroud, C. N. (2013). Droplet microfluidics driven by gradients of confinement. Proceedings of the National Academy of Sciences, 110(3), 853-858 and Baroud, C., & Dangla, R. (2011). U.S. patent application Ser. No. 13/637,779, and familiar to the man skilled in the art. The central region of the microchamber of central height larger that the maximum height of its wedged rim serves as a trapping region for the dispersed phase to be introduced in the microfluidic device.

(49) To obtain the best performances from the fluid partitioning zone and the trapping region of the microfluidic device, the network of microchannels undergoes a surface treatment in order to optimize the fluid affinity of its walls to the carrier fluid and dispersed phases to be introduced into the microfluidic device.

(50) The closing member is a standard female Luer lock to male Luer lock check-valve with a silicone diaphragm obtained from Cole Palmer, Nordson Medical or Smart Products. The direction of flow is from the female Luer to the male Luer side of the lock. One of such Luer check-valve is disposed onto each of the 4 second ports of the microfluidic device, thus configured as an outward check-valve.

(51) Methods

(52) The four microfluidic networks of three microfluidic devices are primed with oil simultaneously using the materials disclosed above and the method described below.

(53) 60 μL of oil, such as silicone oil (XIAMETER® PMX-200 SILICONE FLUID 50CS, XIAMETER® PMX-200 SILICONE FLUID 500CS), fluorinated oil (3M Fluorinert FC-40, 3M Fluorinert FC-770, 3M Novec 7100), are pipetted into each of the 4 first ports of each of the 3 microfluidic devices.

(54) The 3 microfluidic devices with oil in the first ports and outward check-valves on the second ports are placed into the pressure chamber at atmospheric pressure. The pressure chamber is closed and sealed by tightening the screw lid.

(55) The pressurization unit then increases the pressure in the pressure chamber to 400 mbar above atmospheric pressure during 6 seconds and maintains the pressure to 400 mbar during 1 minute.

(56) The overpressure in the pressure chamber is directly transmitted to the oil contained in the first ports but is not transmitted to the gas contained in the second ports and the networks of microchannels, which are sealed from the pressure in the pressure chamber by the outward check-valves. As a result, the oil is submitted to a pressure gradient which forces it to flow from the first ports into the networks of microchannels and eventually into the second ports. As it does so, it compresses the gas initially contained into the networks of microchannels and second ports. The flow stops once the pressure of this gas equilibrates with the pressure applied to the pressure chamber.

(57) After 1 minute at 400 mbar, all networks of microchannels are primed with the oil. The pressure is rapidly decreased back to atmospheric pressure. During the pressure decrease, the compressed gas in the second ports exits through the outward check-valves. As a result, there is no pressure difference between the first and second ports, such that there is no back-flow of the oil which remains into the networks of microchannels.

(58) Since all networks of microchannels, second ports and check-valves have identical dimensions, the volume of oil injected is identical for each network of microchannel of each microfluidic device and the priming is reproducible.

(59) The screw lid of the pressure chamber is opened and the primed microfluidic devices are extracted for further treatment.

(60) Example 2: Priming multiple microfluidic devices with oil using stoppers on the output ports of the microfluidic devices.

(61) Materials

(62) The pressure chamber, the pressurization unit and the microfluidic devices are identical to those described in example 1.

(63) The closing member is a standard female Luer or Luer-lock cap from Cole Palmer or Nordson Medical. The female Luer caps are not placed onto the microfluidic device prior to implementing the priming method.

(64) Methods

(65) Similarly to example 1, the four microfluidic networks of three microfluidic devices are primed with oil simultaneously using the materials disclosed above and the method described below.

(66) First, 30 μL of oil is pipetted in each first port and each second port. Female Luer caps are then firmly placed onto each second port to act as stoppers.

(67) The 3 microfluidic devices with oil in all ports and stoppers on the second ports are placed into the pressure chamber at atmospheric pressure. The pressure chamber is closed and sealed by tightening the screw lid.

(68) The pressurization unit then increases the pressure in the pressure chamber to 600 mbar above atmospheric pressure during 6 seconds and maintains the pressure to 600 mbar during 1 minute.

(69) Similarly to example 1, the overpressure in the pressure chamber is directly transmitted to the oil contained in the first ports but is not transmitted to the gas contained in the second ports and the networks of microchannels, which are sealed from the pressure in the pressure chamber by the Luer caps. Oil flows into all networks of microchannels and the flows stop once the pressures equilibrate.

(70) At the end of this first phase, the 20 μL of gas initially contained into the networks of microchannels has been forced to flow into the second ports and is replaced by oil initially placed in the first ports. The gas resides above the 30 μL of heavier oil which partially fills the second port.

(71) The pressure is then decreased to atmospheric pressure over 15 seconds. As the pressure decreases in the pressure chamber, the compressed gas in the second ports pushes the oil to back-flow from the second ports through the networks of microchannels to the first ports. A volume of 20 μL of oil, identical to the volume forced into the network of microchannels during pressurization, flows back to the first ports.

(72) At the end of the procedure, all networks of microchannels are primed with oil, with 30 μL of oil in the first ports and 10 μL in the second ports.

(73) Again, since all networks of microchannels, second ports and check-valves have identical dimensions, the volume of oil injected is identical for each network of microchannel of each microfluidic device and the priming is reproducible.

(74) The screw lid of the pressure chamber is opened and the primed microfluidic devices are extracted for further treatment.

(75) Example 3: Production of arrays of aqueous droplets inside multiple microfluidic primed with oil using outward check-valves on the output ports of the microfluidic devices.

(76) Materials

(77) The pressure chamber, the pressurization unit and the microfluidic devices are identical to those described in example 1.

(78) The microfluidic devices are primed with an oil of choice prior to the production of arrays of droplets, using one of the methods described in the example 1. The oil will serve as a carrier phase and contains a surfactant additive to prevent the coalescence of neighboring drops of the aqueous solution. Consequently, the networks of microchannels of the microfluidic devices are completely filled with the carrier phase prior to the loading of the aqueous dispersed phase. Owing to the simultaneous priming method described in example 1, the levels of oil in the first and second ports of the microfluidic devices are all identical from one network of microchannels to the other.

(79) The closing member is a female Luer check-valve similar to the one described in example 1. Check-valves are disposed on all second ports of all microfluidic devices.

(80) Methods

(81) The four microfluidic networks of three microfluidic devices are loaded with arrays of droplets simultaneously using the materials disclosed above and the method described below.

(82) First, 20 μL of aqueous solution is pipetted in each first port, and the 3 microfluidic devices with aqueous solution in all first ports and check-valves on the second ports are placed into the pressure chamber at atmospheric pressure. The pressure chamber is closed and sealed by tightening the screw lid.

(83) The pressurization unit then slowly increases the pressure in the pressure chamber to 351 mbar above atmospheric pressure during 2 minutes and 54 seconds by steps of +1.5 mbar every second and maintains the pressure to 351 mbar during 3 minutes.

(84) Similarly to example 1, the overpressure in the pressure chamber is directly transmitted to the aqueous mix contained in the first ports but is not transmitted to the gas contained in the second ports which are sealed from the pressure in the pressure chamber by the Luer check-valves. The aqueous mixes slowly flow into all networks of microchannels. The regular increase in pressure provides further control of the injection flow rate, which does not exceed approximately 5 μL/min. Limiting the maximum value of the flow rate is necessary to ensure that the droplets to be produced are monodisperse in volume, as described in Dangla, R., Kayi, S. C., & Baroud, C. N. (2013). Droplet microfluidics driven by gradients of confinement. Proceedings of the National Academy of Sciences, 110(3), 853-858.

(85) The aqueous mix first displaces the oil from the distribution microchannel and then penetrates the comb of injection microchannels. As soon as the aqueous mix reaches the junction of an injection microchannel with the wedged rim of the microchamber, the aqueous mix spontaneously partitions into droplets. The wedge propels the newly formed droplets which are collected inside the microchamber.

(86) At the end of the 3 minutes at 351 mbar, the pressures applied to the first ports and in the second ports are equilibrated and the flow of the aqueous dispersed phase has stopped. At this point, approximately 70% of all 4 microchambers of all 3 microfluidic devices are filled with an array of approximately 25 000 droplets of the aqueous dispersed phase.

(87) Last, the pressure is rapidly decreased to atmospheric pressure over 15 seconds. During the pressure decrease, the compressed gas in the second ports exits through the outward check-valves. As a result, there is no pressure difference between the first and second ports, such that there is no back-flow of the oil or of the aqueous droplets which remains in the microchambers of the microfluidic devices.

(88) The screw lid of the pressure chamber is opened and the microfluidic devices filled with arrays of aqueous droplets are extracted to be used for further treatment such as thermal cycling and digital PCR.

(89) Example 4: Production of arrays of aqueous droplets inside multiple microfluidic primed with oil using stoppers on the second ports of the microfluidic devices.

(90) Materials

(91) The pressure chamber, the pressurization unit and the primed microfluidic devices are identical to those described in example 3.

(92) The closing member is a female Luer cap serving as a stopper, similar to the one described in example 2. Female Luer caps are disposed on all second ports of all microfluidic devices prior to the following method.

(93) Methods

(94) The four microfluidic networks of three microfluidic devices are loaded with arrays of droplets simultaneously using the materials disclosed above and the method described below.

(95) First, 20 μL of aqueous solution is pipetted in each first port, and the 3 microfluidic devices with aqueous solution in all first ports and female Luer caps on the second ports are placed into the pressure chamber at atmospheric pressure. The pressure chamber is closed and sealed by tightening the screw lid.

(96) The pressurization unit then slowly increases the pressure in the pressure chamber to 552 mbar above atmospheric pressure during 3 minutes and 4 seconds by steps of +3 mbar every second and maintains the pressure to 552 mbar during 3 minutes.

(97) Similarly to example 2, the overpressure in the pressure chamber is directly transmitted to the aqueous mix contained in the first ports but is not transmitted to the gas contained in the second ports which are sealed from the pressure in the pressure chamber by the female Luer caps which serve as stoppers.

(98) Similarly to example 3, the aqueous mixes flow into all networks of microchannels and are partitioned into droplet arrays which are collected in the microchamber.

(99) At the end of the 3 minutes at 552 mbar, the pressures applied to the first ports and in the second ports are equilibrated and the flow of the aqueous dispersed phase has stopped. At this point, approximately 70% of all 4 microchambers of all 3 microfluidic devices are filled with an array of approximately 25 000 droplets of the aqueous dispersed phase. The bottoms of the second ports are filled with a volume of oil that is equal to the volume of aqueous solution that has filled the networks of microchannels.

(100) Last, the pressure is slowly decreased to atmospheric pressure over 6 minutes and 8 seconds by steps of −1.5 mbar every second. During the pressure decrease, the compressed gas in the second ports forces a back-flow of the oil contained in the bottom of the second ports through the networks of microchannels. During the back-flow, the microchamber serves as a trapping region for the partitioned aqueous phase. Indeed, the wedged rim of the microchamber expels droplets of the aqueous dispersed phase because of surface tension effects.

(101) As a result, during the pressure decrease, only the carrier phase, i.e. the oil, flows back from the second port to the first port through the networks of microchannels and around the array of droplets contained in the microchambers. The arrays of aqueous droplets remain in the microchambers of the microfluidic devices.

(102) The screw lid of the pressure chamber is opened and the microfluidic devices filled with arrays of aqueous droplets are extracted to be used for further treatment such as thermal cycling and digital PCR.

(103) Example 5: Production of arrays of aqueous droplets at a constant flow rate inside multiple microfluidic primed with oil using check-valves on the second ports of the microfluidic devices.

(104) Materials

(105) The pressure chamber, the pressurization unit, the primed microfluidic devices and the closing members are identical to those described in example 3.

(106) Methods

(107) The four microfluidic networks of three microfluidic devices are loaded with arrays of droplets simultaneously by injecting an aqueous solution at a constant flow rate using the materials disclosed above and the method described below.

(108) The method is identical to the one described in example 3 except for the pressure profile applied to the pressure chamber during the pressure increase phase.

(109) Instead of a linear increase in pressure from atmospheric pressure to an overpressure of 351 mbars, the pressure is increased following an exponential profile. The pressure increases following such a profile from 1 bar to 1.351 bar during 2 minutes and 54 seconds.

(110) While the exposed priming method does not directly allow to control in the injection flow rate of the aqueous dispersed phase, the exponential profile is optimal to minimize the flow rate variations throughout the increase of pressure and the injection of the aqueous dispersed phase.

(111) Example 6: Combined priming with oil and production arrays of aqueous droplets inside multiple microfluidic using stoppers on the output ports of the microfluidic devices.

(112) Materials

(113) The pressure chamber, the pressurization unit, the microfluidic devices and the closing members are identical to those described in example 2.

(114) Methods

(115) First, 55 μL of fluorinated oil, which will serve as a carrier fluid, is pipetted in each second port and a female Luer cap is firmly placed on top of each second port.

(116) Second, 15 μL of the same fluorinated oil is pipetted in each first port and 20 μL of an aqueous solution which serves as a dispersed phase is pipetted on top of the 15 μL of fluorinated oil. Because fluorinated oil is denser than the aqueous solution, the aqueous solution remains on top of the carrier fluid.

(117) The 3 microfluidic devices with the fluorinated oil and the aqueous solution in all first ports and female Luer caps on the second ports are placed into the pressure chamber at atmospheric pressure. The pressure chamber is closed and sealed by tightening the screw lid.

(118) The pressurization unit then slowly increases the pressure in the pressure chamber to 350 mbar above atmospheric pressure during 5 minutes and 50 seconds by steps of +1 mbar every second and maintains the pressure to 350 mbar during 3 minutes.

(119) Similarly to example 2, the overpressure in the pressure chamber is directly transmitted to the aqueous mix contained in the first ports but is not transmitted to the gas contained in the second ports which are sealed from the pressure in the pressure chamber by the female Luer caps which serve as stoppers.

(120) As the pressure increases, the fluorinated oil at the bottom of the first port first starts to flow into the network of microchannels, forcing the air contained in the network of microchannels into the closed second port. Once all the oil initially present in the first port has flown into the network of microchannels, the aqueous solution starts to flow into the network of microchannels.

(121) Similarly to example 3, the aqueous dispersed phase is partitioned into arrays of droplets as it reaches the microchambers.

(122) At the end of the 3 minutes at 350 mbar, the pressures applied to the first ports and in the second ports are equilibrated and the flows of the oil and aqueous dispersed phase have stopped. At this point, approximately 70% of all 4 microchambers of all 3 microfluidic devices are filled with an array of approximately 25 000 droplets of the aqueous dispersed phase. The second ports are filled with the initially pipetted fluorinated oil plus the oil that has flown from the first port to the second port during the increase in pressure.

(123) Similarly to example 4, the pressure is then slowly decreased back to atmospheric pressure during 5 minutes and 50 seconds by steps of −1 mbar every 1 second. Although there is a back-flow of oil during the pressure decrease, the microchamber acts as a trapping region for the droplets of aqueous solution and only the carrier fluid flows back from the second port to the first port.

(124) The screw lid of the pressure chamber is opened and the microfluidic devices filled with arrays of aqueous droplets are extracted to be used for further treatment such as thermal cycling and digital PCR.

(125) Example 7: The combined production arrays of aqueous droplets inside multiple microfluidic devices primed with oil using stoppers on the output ports of the microfluidic devices and temperature treatment of the arrays of aqueous droplets for amplification of a DNA template by polymerase chain reaction (PCR).

(126) Materials

(127) The pressurization unit and the primed microfluidic devices are identical to those described in example 4. The closing members are female Luer caps identical to those described in example 2.

(128) The pressure chamber is identical to the one described in example 1. However, the pressure chamber is combined with a heating element to control the temperature of its bottom aluminum plate. The pressure chamber is placed on top of the heat block of a PeqLab peqStar in situ X thermocycler. Thermal contact between the heat block of the thermocycler and the bottom plate of the pressure chamber is optimized by applying a layer of Artic Silver© 5 thermal paste between the heat block and the bottom plate of the pressure chamber.

(129) The aqueous solution is a PCR reaction mixture containing 10.sup.3 target sequences of pUC18 plasmid, 1 μM forward primer, 1 μM reverse primer and 250 nM of fluorescently-labeled hydrolysis probe. The reaction is assembled using 1 Unit of Taq DNA Polymerase, 200 μM of deoxynucleosides triphosphate and 1× MP buffer (MP biomedicals).

(130) Methods

(131) The four microfluidic networks of three microfluidic devices are primed with oil and loaded with arrays of droplets simultaneously using the materials and the methods described in example 4.

(132) First, 20 μL of fluorinated oil, which will serve as a carrier fluid, is pipetted in each second port and a female Luer cap is firmly placed on top of each second port.

(133) Second, 15 μL of the same fluorinated oil is pipetted in each first port and 20 μL of the aqueous solution described above, which serves as a dispersed phase is pipetted on top of the 15 μL of fluorinated oil. Because fluorinated oil is denser than the aqueous solution, the aqueous solution remains on top of the carrier fluid.

(134) The 3 microfluidic devices with the fluorinated oil and the aqueous solution in all first ports and female Luer caps on the second ports are placed into the pressure chamber at atmospheric pressure onto the bottom plate of the pressure chamber at ambient temperature. The pressure chamber is closed and sealed by tightening the screw lid.

(135) The pressurization unit then slowly increases the pressure in the pressure chamber to 750 mbar above atmospheric pressure during 6 minutes and 15 seconds by steps of +2 mbar every second and the pressure of 750 mbar is maintained in the pressure chamber.

(136) Similarly to example 4, the overpressure in the pressure chamber is directly transmitted to the aqueous mix contained in the first ports but is not transmitted to the gas contained in the second ports which are sealed from the pressure in the pressure chamber by the female Luer caps which serve as stoppers.

(137) As the pressure increases, the fluorinated oil at the bottom of the first port first starts to flow into the network of microchannels. Once all the oil initially present in the first port has flown into the network of microchannels, the aqueous solution starts to flow into the network of microchannels.

(138) Similarly to example 4, the aqueous dispersed phase is partitioned into arrays of droplets as it reaches the microchambers.

(139) At the end of the 6 minutes and 15 of pressure increase, approximately 70% of all 4 microchambers of all 3 microfluidic devices are filled with an array of approximately 25 000 droplets of the aqueous dispersed phase. The second ports are filled with the initially pipetted fluorinated oil plus the oil that has flown from the first port to the second port during the increase in pressure.

(140) Using the peqStar in situ thermocycler and while maintaining the pressure in the pressure chamber at 750 mbar, the temperature of the bottom plate is first increased to 95° C. for 10 minutes. The bottom plate heats the arrays of droplets contained in the microfluidic devices to the same temperature.

(141) Following this initial heating phase, the temperature of the bottom plate then undergoes 40 cycles of cooling at 58° C. for 1 minute and heating at 95° C. for 30 seconds, again while maintaining the pressure in the pressure chamber at 750 mbar. Finally, the temperature of the bottom plate is decreased back to ambient temperature and maintained at this temperature.

(142) This temperature treatment of the bottom plate is transmitted to the arrays of droplets contained in the microchamber of the microfluidic devices. As a result, the droplets that contain at least one copy of the target nucleic acid, called positive droplets, undergo a polymerase chain reaction (PCR) that amplifies the concentration of the target nucleic acid in these positive droplets, detected through an increase in fluorescence intensity of the droplets due to the hydrolysis of the targeted probe. The droplets that do not contain a copy of the target nucleic acid do not undergo PCR amplification, and therefore do not show an increase in fluorescence

(143) Similarly to example 4, the pressure is then slowly decreased back to atmospheric pressure during 6 minutes and 15 seconds by steps of −2 mbar every 1 second. Although there is a back-flow of oil during the pressure decrease, the microchamber acts as a trapping region for the droplets of aqueous solution and only the carrier fluid flows back from the second port to the first port.

(144) The screw lid of the pressure chamber is opened and the microfluidic devices filled with arrays of aqueous droplets which have undergone PCR amplification are extracted to be used for further treatment or analysis.

(145) This example has the advantage of combining the production of droplet arrays in multiple microfluidic devices with PCR amplification.