IMPROVEMENTS IN OR RELATING TO A DEVICE AND METHOD FOR DISPENSING A DROPLET

Abstract

A device for dispensing one or more microdroplets is provided. The device comprising a microfluidic chip having an oEWOD structure configured to create an optically-mediated electrowetting (oEWOD) force, the microfluidic chip includes a first region and a second region, wherein said first and second regions are separated by a constriction; wherein the first region is adapted to receive and manipulate one or more microdroplets dispersed in a carrier fluid at first flow rate; and wherein the second region is configured to receive the microdroplet via the constriction from the first region and transfer said microdroplet to an outlet port of the microfluidic chip in a second flow rate; wherein the second region is configured to receive said microdroplet via the constriction from the first region by application of an optically -mediated electrowetting (oEWOD) force; and wherein the second flow rate in the second region is higher than the first flow rate in the first flow region. A method and apparatus for dispensing one or more microdroplets are also provided.

Claims

1-29. (canceled)

30. A device for dispensing one or more microdroplets, the device comprising a microfluidic chip having an optically-mediated electrowetting (oEWOD) structure configured to create an oEWOD force, the microfluidic chip includes a first region and a second region, wherein said first and second regions are separated by a constriction; wherein the first region is adapted to receive and manipulate one or more microdroplets dispersed in a carrier fluid at a first flow rate; and wherein the second region is configured to receive the microdroplet via the constriction from the first region and transfer said microdroplet to an outlet port of the microfluidic chip in a second flow rate; wherein the second region is configured to receive said microdroplet via the constriction from the first region by application of an optically-mediated electrowetting (oEWOD) force; and wherein the device further comprises a valve and/or a pump, the valve and/or the pump is configured to enable the microdroplet to be dispensed through and out of the outlet port of the chip; and a controller configured to control the valve and/or the pump such that the second flow rate in the second region is higher than the first flow rate in the first region.

31. The device according to claim 30, wherein the constriction is a physical barrier.

32. The device according to claim 30, wherein the constriction is a semi-permeable membrane.

33. The device according to claim 30, wherein the microdroplets comprise a biological material, one or more cells or one or more beads.

34. The device according to claim 30, wherein the constriction comprises an opening, wherein the width of the opening is between 20 to 400 microns.

35. The device according to claim 30, wherein the geometry of the second region is a substantially crescent-shaped channel.

36. The device according to claim 30, wherein the second region further comprises a plurality of channels, each channel is configured to receive the microdroplet from the first region and transfer said microdroplet to the outlet port of the microfluidic chip.

37. The device according to claim 36, wherein the controller is configured to control the flow of the or each of the microdroplets simultaneously in each of the channels in the second region.

38. The device according to claim 30, further comprising a detection system for detecting a detection signal from the microdroplet dispensed from the outlet port of the microfluidic chip.

39. The device according to claim 30, further comprising a reader module configured to read and transmit the generated signal from a sensor or a detection module to the controller, upon which the controller is further configured to position the valve into an open position such that the microdroplet is dispensed.

40. The device according to claim 30, further comprising a receptacle, wherein the receptacle is configured to receive a dispensed microdroplet.

41. The device according to claim 40, wherein the receptacle is a multi-well plate, a PCR tube or a microcentrifuge tube.

42. The device according to claim 41, wherein the multi-well plate is mounted onto a multi-axis motion controlled stage, wherein the multi-axis motion controlled stage is configured to move the multi-well plate into a first position such that a target well is positioned under a valve provided to the outlet port of the microfluidic chip.

43. The device according to claim 38, wherein the detection system includes an optical detector.

44. A method for dispensing one or more microdroplets out of a microfluidic chip, the method comprising the steps of: providing a device comprising the microfluidic chip having a first region and a second region separated by a constriction, the device further comprises a pump and/or a valve; transporting the microdroplet from the first region into the second region, wherein the microdroplet is dispersed in a carrier fluid at a first flow rate in the first region; wherein the second region is configured to receive the microdroplet via the constriction means from the first region and transfer said microdroplet to an outlet port of the microfluidic chip at a higher carrier fluid flow rate, wherein the second region is configured to receive said microdroplet via the constriction from the first region by application of an optically-mediated electrowetting (oEWOD) force; and activating the pump and/or the valve using a controller to control the flow of the carrier fluid such that the microdroplet is dispensed through and out of the outlet port, wherein the activation of the pump and/or valve using the controller causes the second flow rate in the second region to be higher than the first flow rate in the first region.

45. The method according to claim 44, further comprising the step of mounting a multi-well plate onto a multi-axis motion controlled stage, wherein the multi-axis motion controlled stage is configured to move the multi-well plate to a target well using the controller such that the target well is positioned under a valve provided to the outlet port of the microfluidic chip.

46. The method according to claim 45, further comprising the step of recording the target well using the controller.

47. The method according to claim 44, further comprising the step of generating a signal using a detection module or a sensor.

48. The method according to claim 47, further comprising the step of detecting the generated signal from the detection module or the sensor and transmitting the generated signal to the controller, upon which the controller is further configured to switch a valve into an open position such that the microdroplet is dispensed.

49. An apparatus for dispensing one or more microdroplets, the apparatus comprising: a microfluidic device according to claim 30; a conduit connected to the outlet port of the microfluidic chip for receiving the microdroplet once it is dispensed from the chip; a sensor located in the vicinity of the conduit configured to generate a signal; a reader module configured to read and transmit the generated signal from the sensor to a controller; wherein the controller is configured to control a valve and/or pump; and wherein in response to the signal generated by the sensor, the controller is configured to switch the valve into a position such that the microdroplet is dispensed from the apparatus, or the controller is configured to switch the valve into a position such that the microdroplet is dispensed onto a receptacle.

Description

[0149] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which:

[0150] FIG. 1 shows a microfluidic device for dispensing one or more microdroplets as disclosed in the present invention;

[0151] FIGS. 2A, 2B, 2C illustrates the chip loading and dispensing sequence as disclosed in the present invention;

[0152] FIGS. 3A and 3B illustrating the dispensing procedure and detection of droplets according to FIGS. 2A to 2C;

[0153] FIGS. 4A, 4B, 4C and 4D showing droplet manipulation within the microfluidic device;

[0154] FIGS. 5A and 5B showing Droplets dispensed in a multi-phase flow; and

[0155] FIG. 6 providing an apparatus or system for dispensing droplets.

[0156] Referring to FIG. 1, there is provided a microfluidic device 10 for dispensing one or more microdroplets comprising an enclosed volume 12. The enclosed volume 12 includes a first region 14 and a second region 16. The first region 14 may be a large area as shown in FIG. 1 where droplets are stored, handled and/or manipulated. The first 14 and second regions 16 are separated by a constriction means 18 such as a wall or barrier. The wall or barrier 18 as shown in FIG. 1 would comprise a gap 20 sufficiently wide enough to allow the droplets to go through and enter the second region 16. Droplets containing cells of interest are selected and then moved through the gap 20 via by the application of an optoelectrowetting (oEWOD) force. The device also has a number of ports 22, 24, 26, 28 which can be independently opened or sealed using connected valves.

[0157] The first region 14 is adapted to receive and manipulate one or more microdroplets dispersed in a carrier fluid at a low carrier fluid flow rate. In some instances, the low carrier fluid flow rate is zero in the first region 14. This ensures that droplets can be easily manipulated and handled. If the flow rate is too high in the first region 14 then it overcomes the oEWOD force holding droplets in place or manipulating droplets.

[0158] The flow rate in the first region can be within the range of 0 to 20 μL/min, or it may exceed 0, 2, 4, 6, 8, 10, 12, 14, 16 or 18 μL/min. In some instances, the flow rate of the first region may be less than 20, 18, 16, 14, 12, 10, 8, 6, 4 or 2 μL/min.

[0159] The second region 16 may have two different flow rates. During the standby mode, where the droplets are moved into the second region, the flow rate in the second region can be between 0 to 20 μL/min, or it may exceed 0, 2, 4, 6, 8, 10, 12, 14, 16 or 18 μL/min. In some instances, the flow rate of the second region in standby mode may be less than 20, 18, 16, 14, 12, 10, 8, 6, 4 or 2 μL/min.

[0160] During the dispensing mode, where droplets are being dispensed out of the chip, the flow rate of the second region is 10-100 μL/min, or it may exceed 10, 20, 30, 40, 50, 60, 70, 80 or 90 μL/min. In some instances, the flow rate of the second region during dispense is less than 100, 90, 80, 70, 60, 50, 40, 30, 20 or 15 μL/min.

[0161] A droplet may contain biological material, cells or beads. A droplet may contain a single or multiple cells. A droplet may contain a single or multiple beads. A droplet can be of any shape or size but preferably, the droplet is of spherical or cylindrical shape. The size of a droplet may be between 20 to 600 μm but it may be more than 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 550, 560 or 580 82 m. In some embodiments, the size of the droplet may be less than 600, 580, 560, 550, 540, 520, 500, 480, 460, 440, 420, 400, 380, 360, 340, 320, 300, 280, 260, 250, 240, 220, 200, 180, 160, 150, 140, 120, 100, 80, 60, 50, 40 or 30 μm. A plurality of droplets can be merged together to form a larger droplet. Alternatively, a large droplet can be divided to form smaller sized droplets as appropriate.

[0162] If the droplet is too small relative to the height of the microfluidic chamber then the droplet is not in contact with both sides of the walls within the microfluidic chamber and hence, the droplet cannot be moved by oEWOD. In contrast, large droplets with respect to the device geometry can be hard and/or slow to move within the microfluidic chamber and often disrupt other operations simply by getting in the way by obstructing other correctly-sized droplets or by merging with correctly-size droplets.

[0163] Referring to FIG. 1, the second region 16 is a channel such as a microchannel connected between an inlet port and an outlet port contained within the second region 16. The microchannel 16 is configured to receive the microdroplet via through the gap 20 within the constriction means 18 from the first region 14 and transfer said microdroplet to an outlet port of the second region 16 of the microfluidic chip 10 at a higher carrier fluid flow rate. The higher carrier fluid flow rate may be generated by attaching a pump source at one of the ports located within the second region 16. The pump source may be a syringe pump or pressure pump, connected to one or more inlet or outlet ports of the microfluidic chip 10. In addition, the valve can be a software controlled valve connected to one or more outlet or inlet ports of the microfluidic chip 10.

[0164] The microchannel can be patterned inside the second region 16 of the microfluidic chip 10 in such a way that the microchannel is connected between an inlet port and an outlet port within the second region 16.

[0165] A pump source is connected to one or more outlet ports such as port 22 and port 28 through a 2-way valve, as shown in FIG. 1. Droplets are loaded through another port, for example port 24 by aspirating from port 22. Droplets are manipulated in the first region 14 and then selected to move into the second region 16 via the application of the oEWOD force. FIG. 1 also illustrates that the droplets are then dispensed from the outlet port e.g. outlet port 26 by pumping into port 28, whilst ports 22 and 24 are shut off via a valve. The pump source is then switched off and the valve at the outlet ports of the microfluidic chip 10 is in a closed position.

[0166] Referring to FIGS. 2A, 2B and 2C, there is shown microdroplets 30 loading onto the microfluidic chip 10 and the dispensing sequence. The microfluidic chip 10 comprises an enclosed volume 12. The enclosed volume 12 includes a first region 14 and a second region 16. As shown in FIG. 2A, the valves 32 are connected to the ports 22, 24, 26, 28 of the microfluidic chip 10 such that each port 22, 24, 26, 28 can be opened or closed. Droplets 30 are loaded into the first region 14 of the chip 10 by passing a stream of carrier fluid containing droplets between two of the ports, whilst the other ports are sealed by the valves 32. The valves 32 are closed to reduce the flow rate inside the first region 14 to zero. Droplets 30 are then stored and/or manipulated in the first region 14.

[0167] Referring to FIG. 2B, there is shown a droplet 30 being selected and moved from the first region 14 through the gap 20 located within the constriction means 18 into the second region 16 by application of the oEWOD force.

[0168] As shown in FIG. 2C, the droplet 30 is then dispensed from the outlet port e.g. outlet port 26 by pumping into port 28 using a syringe pump 35, whilst ports 22 and 24 are shut off via by a valve 32. The droplets 30 can be dispensed into a receptacle such as a multi-well plate 34 as illustrated in FIG. 2C. The voltage may be switched off during the dispense cycle to allow the droplet to be released from the oEWOD force.

[0169] Referring to FIG. 3A, there is provided a microfluidic chip 10 showing the second region 16. The ports 26, 28 of the microfluidic chip are connected a valve 32. Both valves are in an open position as illustrated in FIG. 3A. A carrier fluid is infused from the pump 35, causing the droplet 30 to move through the second region 16 and out of the chip 10 into the conduit 40. The outlet valve 32 is open to a waste channel 36 or container. A sensor 38 is located in the vicinity of the conduit 40 to monitor for the presence of the droplet within the conduit 40.

[0170] The detection module 38 can be an optical sensor such as a photodiode or phototransistor, or an electrical sensor such as a capacitance or impedance sensor, or a combination of several such sensors. The set of sensors can be positioned around the vicinity of the conduit. The sensors would then generate an electrical signal when a droplet passes a detection window. Optionally, inspection cameras can be positioned to image the inside of the tubing either side of the valve such that videos or images from the inspection cameras can be recorded and analysed by a reader module.

[0171] The apparatus further comprises a reader module such as a microcontroller (not shown in the accompanying figures) which is configured to read and transmit the generated signal from the sensor or the detection module to the controller. The reader module such as a microcontroller is configured to read the output signals of the sensors and transmit the status of the sensors to the controller.

[0172] Referring to FIG. 3B, there is illustrated the detection of a droplet using sensors or optical detectors 38, the software controller will position the valve 32 to be open to a second conduit 42 directed into the receptacle 34 so that the droplet is transferred into the receptacle 34.

[0173] Referring to FIG. 4A, there is shown target droplets 43 being manipulated and assayed inside the optofluidic chamber within the first region 14. The droplets 43 can be moved and re-arranged into an array.

[0174] Referring to FIGS. 4B and 4C, there is shown a selected droplet 43 being moved by oEWOD force through the constriction means 18 into the second region 16.

[0175] As illustrated in FIG. 4D, the droplet 43 is dispensed by opening the valves connected to the ports of the second region 16 and infusing carrier fluid to create a high flow rate within the second region 16 to transport the droplet out of the device.

[0176] Referring to FIG. 5A, droplets can be dispensed in a multi-phase flow. Two independent pumps, one with aqueous medium 44 and one with immiscible carrier fluid 46 are connected to the inlet port 48, possibly by a junction component 47. Valves 32 are also provided as shown in FIG. 5A. The valves 32 can open or close independently of each other. In some cases, the valves may open or close in sequence or in tandem or they may open or close simultaneously. A plug of medium 50 is infused into the second region 16 of the chip 10 before and/or after a volume of immiscible carrier fluid. The droplet 30 is moved into the immiscible carrier fluid section and then the mixed phase fluids are infused through the outlet 49 to the dispense receptacle. This reduces the volume of immiscible fluid introduced into the dispense receptacle.

[0177] As shown in FIG. 5B, a larger sized droplet 52 can be created by selecting smaller droplets 54 and merging them together to form a larger droplet 52. The merged droplet 52 may have a diameter size of approximately 50 μm in diameter. In some instances, the smaller droplets 54 can be merged together to form a larger droplet 52 upon a voltage application to the device of between 5 to 10 V, preferably at 10 V. The merged droplet 52 can be pushed out of the outlet port of the microfluidic chip by using a pump. The flow rate within the second region may be between 10 to 100 μL/min.

[0178] In some instances, where droplets are merged into a stream of aqueous (or a plug) soon after leaving the chip, the amount of oil that ultimately has to be ejected from the chip and ultimately in to the well is minimised. Thus, this would avoid filling the receptacle such as a well with oil. Additionally or alternatively, the small droplet 54 can precede the big aqueous plug 52 or merged droplet and can be pumped out of the microfluidic chip via a syringe pump, hence it avoids filling the receptacle such as a well with oil.

[0179] Referring to FIG. 6, there is provided a dispensing apparatus or system 100. The dispensing apparatus or system 100 comprises a device as disclosed in the previous aspects of the invention. The apparatus 100 for dispensing one or more microdroplets comprises a microfluidic chip 102, the microfluidic chip 102 (A) includes a first region and a second region, wherein the first and second regions are separated by a constriction means; where the first region is adapted to receive and manipulate one or more microdroplets dispersed in a carrier fluid at a low carrier fluid flow rate; and where the second region is configured to receive the microdroplet via the constriction means from the first region and transfer said microdroplet to an outlet port of the microfluidic chip at a higher carrier fluid flow rate, whereby the second region is configured to receive said microdroplet via the constriction means from the first region by application of an optically-mediated electrowetting (oEWOD) force.

[0180] The apparatus further comprises a controller configured to control the valve and/or pump connected to the outlet port of the microfluidic chip 102. A valve 103 (B) is connected to an outlet port of the microfluidic device 102 as shown in FIG. 6 and a pump (not shown in the accompanying drawings) is connected to an inlet port of the microfluidic device 102. The outlet port of the microfluidic chip 102 is connected with a conduit 104 such as a tube. The conduit can be transparent.

[0181] The receptacle 108 is a multi-well plate 108. The multi-well plate is mounted on a multi-axis controlled stage 110 with an XYZ configuration. The multi-well plate 108 may be a 96 or a 384-well plate. The stage 110 can be manually or automatically controlled. The receptacle 108 can also be a waste container or reservoir or a PCR tube or a microcentrifuge tube such as an Eppendorf tube. Optionally, each well pre-filled with a volume of cell media. The controller is configured to control the movement of the stage where the multi-well plate is mounted onto during the dispensing procedure. One droplet may be dispensed in each well and/or multiple droplets can be dispensed into one well.

[0182] During the dispensing procedure, the dispense head 106 moves down into a well containing an aqueous buffer. Additionally or alternatively, the well may move towards the dispense head 106. Alternatively, the dispense head 106 may be fixed in position and the well plate may move towards the dispense head 106. The pump connected to an inlet port of the microfluidic chip 102 is activated and pumps for a length of time and at an appropriate speed to pump the required volume of buffer through the microfluidic chip. The precise time and speed depends on the size of the microchannel, interconnecting tubing and interface connections. For example, the pump connected to an inlet port of the microfluidic chip 102 can be activated and pumps for 12 seconds at 50 μL/min. The valve connected to an outlet port of the microfluidic chip 102 is opened such that a certain amount of volume typically about 7 μL to 10 μL to is pushed out of the microfluidic chip into the tubing 104 and dispensed into a waste container. The fixed volume of 7 μL to 10 μL may be adequate to fully purge the microchannel and the interconnecting tubing and interface connections.

[0183] Droplet(s) can then be moved from the microfluidic chip 102 and into the tubing 104 stopping just before the valve. The pump is then deactivated and stops pumping fluids out of the microfluidic device 102 whilst the valve moves into a dispensing position. The pump is reactivated by the controller for a further 4 seconds and the droplet is dispensed into the well 108. The valves are then manually closed or the valve can be automatically closed by the software controlled controller.

[0184] In some examples, the method of dispensing or the sequence of dispensing a droplet can be as follows: the pump source is switched off and the valve is in a closed position as controlled by the controller. The target droplets are manipulated and assayed inside the optofluidic chamber within the microfluidic chip. Optoelectrowetting transport moves a target droplet from the optofluidic chamber into the microchannel within the microfluidic chip. The 3-axis stage moves the multi-well plate such that a target well is positioned under the outlet tubing of the software-controlled valve. The software-controlled pump is activated and starts displacing a fixed volume of a fluid, typically 7 microlitres to adequately purge the microchannel and the interconnecting tubing and interface connections.

[0185] The software-controlled valve is then switched to an open position, such that the fluid is routed into the multi-well plate. The resulting flow of fluids in the microchannel purges the droplet, along with a volume of carrier phase, into the multi-well plate. Optionally, the sensors or cameras are interrogated and the presence of a droplet in the outlet tubing before the multi-well plate is determined. If no droplet is detected the pump source is commanded to dispense extra volume to recover the droplet. The pump is switched off and the valve is closed. The dispense head is withdrawn from the multi-well plate and/or the multi-well plate is withdrawn from the dispense head. Optionally the multi-well plate is moved to place a waste well or alternative waste receptacle below the dispense head and the microfluidic pathway are purged. The steps as described above are repeated until all the target droplets have been recovered from the microfluidic device. The multi-well plate is recovered for further experiments such as DNA sequencing or cell expansion.

[0186] Alternatively, the droplets may be recovered by relying on the pump and valve to meter the correct volume to recover a droplet. By way of example only, 2 to 5 μL metering volume can be provided for 20 cm tube length and a 0.1 mm inner diameter, 0.1 μm dispensing at 20 μl/min. This means that there is not a requirement to provide sensors or camera to detect the droplets within the tubing. Additionally or alternatively, the apparatus as disclosed in the present invention may support multiple dispense pathways and multiple pump sources and valves as appropriate in order to parallelise the recovery process of one or more droplets of interest.

[0187] The device, apparatus and methods of the present invention can be used for many applications such as dispensing of single cells. In some instances, the droplet may contain a plurality of cells. Droplets may contain random number of cells, including single cells. Furthermore, the recovered droplet containing single cells or multiple cells can be assayed which may include, but is not limited to PCR amplifications, DNA sequencing, RNA sequencing and cell expansion. Efficiency of dispensing may be assessed by dyeing droplets with trypan blue and using cameras to film the droplet before and after dispense valve. By way of example only, a dispense is considered successful if a droplet is detected after the dispense valve in <12 seconds from dispense start. In one example only, efficiency of dispensing single cells and doing PCR is around 80% ( 40/50), while overall efficiency of PCR after dispense is 79% ( 66/84).

[0188] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0189] “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0190] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0191] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.