IMPROVED MICROFLUIDIC DEVICES, SYSTEMS AND METHODS

Abstract

Microfluidic devices with microfluidic cassettes allow for on-cassette processing of nucleic acids including amplification by polymerase chain reaction. In systems and methods of using the microfluidic device, the microfluidic cassette is pressurised prior to amplification of nucleic acid in said sample in an amplification/PCR section of the device.

Claims

1. A microfluidic device comprising; a microfluidic fluidic channel having a polymerase chain reaction (PCR) section, said PCR section configured to allow the temperature changes required for polymerase chain reaction (PCR) to occur therein, a fluidically isolatable portion of the device comprising at least the portion of the fluidic channel having a PCR section, at least one closure means actuatable to fluidically isolate the fluidically isolatable portion from the external environment, and at least one means to increase the pressure in said isolatable portion when it is isolated.

2. A microfluidic device according to claim 1 wherein the at least one means to increase the pressure is adapted to increase pressure prior to amplification of a sample by thermocycling within the PCR section.

3. A microfluidic device according to any of the previous claims wherein the at least one means to increase the pressure is a displacement pump.

4. A microfluidic device according to any of the previous claims wherein isolatable portion is the entire fluidic channel present on the device.

5. A microfluidic device according to any of the previous claims wherein the device comprises at least one air-tight valve or sealing means actuatable to fluidically isolate the fluidically isolatable portion.

6. A microfluidic device according to any of the previous claims wherein the means to increase pressure is a positive displacement pump configured to both increase pressure in the fluidically isolatable portion and to move fluid within the fluidically isolatable portion.

7. A microfluidic device according to any of the previous claims wherein the means to increase pressure is actuatable to a first position to apply a first pressure to the isolatable portion, and a second position to apply a second pressure to the isolatable portion.

8. A microfluidic device according to any of the previous claims wherein the means to increase pressure is a bellows pump.

9. A microfluidic device according to any of the previous claims wherein the means to increase pressure is associable with an external pressure source or actuator

10. A microfluidic device according to any of the previous claims wherein there are a plurality of means to increase the pressure.

11. A microfluidic device as in claim 10 wherein the plurality of means to increase the pressure comprise a plurality of positive displacement pumps.

12. A microfluidic device according to any of claim 10 or 11 wherein the plurality of means to increase the pressure are simultaneously or concurrently actuatable to increase the pressure in the isolatable portion.

13. A microfluidic device according to any of the previous claims wherein the means for increasing pressure in the isolatable portion induces a pressure higher than atmospheric pressure and, preferably 1.1 bar or higher and more preferably a pressure of 1.6 bar or higher.

14. A diagnostic system comprising the microfluidic device of any of the previous claims and a host instrument able to receive said microfluidic device.

15. A diagnostic system as in claim 14 wherein the host instrument comprises an interface enabled to convey pressure from said host instrument to said means for increasing pressure on said microfluidic device.

16. A diagnostic system as in any of claims 14 to 15 wherein the host instrument comprises a microprocessor which controls the interactions between the host instrument and the microfluidic device.

17. A method of amplifying nucleic acid from a sample comprising, providing the device or system of any of claims 1 to 16, fluidically isolating at least a portion of the device comprising at least the PCR section, increasing the pressure within the isolated portion, and, after the pressure has been increased within the isolated portion, carrying out polymerase chain reaction (PCR) amplification steps to amplify nucleic acid within the sample.

18. A method of amplifying nucleic acid as in claim 17 wherein the sample is inserted into the device prior to the step of fluidically isolating at least a portion of the device comprising at least the PCR section.

19. A method of amplifying nucleic acid as in claim 17 or 18 wherein wherein one or more fluid-tight valves are closed to fluidically isolate the entire device or only a portion of the microfluidic channel containing the PCR section is fluidically isolated.

20. A method of amplifying nucleic acid as in any of claims 17 to 19 wherein the step of increasing the pressure comprises actuating a plurality of means to increase the pressure.

21. A method of amplifying nucleic acid as in any of claims 17 to 20 wherein the step of increasing the pressure comprises compressing a plurality of positive displacement pumps.

22. A method of amplifying nucleic acid as in any of claims 17 to 21 wherein the step of increasing the pressure comprises compressing a plurality of positive displacement pumps simultaneously or concurrently to increase the pressure in the isolatable portion.

23. A method of amplifying nucleic acid as in any of claims 17 to 22 wherein the step of increasing the pressure comprises compressing a plurality of positive displacement pumps to a first position, wherein the positive displacement pumps may still be further compressed to at least a second position.

24. A method of amplifying nucleic acid as in any of claims 17 to 23 wherein the step of increasing the pressure increases the pressure in the isolated portion to at least 1.6 bar.

25. A method of amplifying nucleic acid as in any of claims 17 to 23 wherein the step of increasing the pressure increases the absolute pressure in the isolated portion to between 1.2 bar and 2 bar.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which like parts are provided with corresponding reference numerals and in which:

[0086] FIG. 1a provides a perspective view of a microfluidics cassette according to an aspect of the present invention, the view showing an outer surface; FIG. 1b provides an exploded perspective view of a similar microfluidics cassette shown from the other side, with various internal features visible.

[0087] FIG. 2a, 2b, 2c is a simplified diagram showing the use of two bellows pumps to pressurise a sealed area of a cassette, with the first applying pressure and the second acting as a reservoir; and

[0088] FIG. 3a, 3b, 3c is a simplified diagram showing the use of two bellows pumps that are actuated simultaneously pressurise a sealed area of a cassette. FIG. 3B shows the pressurization step while FIG. 3C shows the reciprocation mode to move the sample while keeping the pressure in a sellable portion of the cassette.

DETAILED DESCRIPTION

[0089] According to a first aspect of the present invention, and as generally depicted in FIG. 1, there is provided a microfluidic cassette 1, more generally termed a ‘device’, with a micro-channel 2, where the micro-channel 2 allows for continuous flow-through of fluid as required. As shown most clearly in FIG. 1b, which shows the internal workings of a similarly configured cassette, the micro-channel 2 is formed inside the microfluidic cassette 1, in the desired length and shape to allow the passage of a sample, typically a biological sample in liquid format, and/or reagents, some of which may be incorporated on-cassette during the flow-through, along a fluid flow path and through various zones or areas which allow different activities to occur including amplification of DNA from the sample by polymerase chain reaction (PCR). A PCR section of the channel 7 is a portion of the channel that has been configured to allow thermocycling of a sample passing therethrough to occur. The PCR section 7 of the channel (and cassette) is specifically adapted to allow a liquid sample travelling therethrough to be heated and cooled in a cyclical manner to temperatures that allow for denaturing of DNA, annealing of DNA and simplification of DNA. The PCR section 7 of the channel is heatable either by an integral heat source within the channel or the wall of the device, or more typically by being brought into the proximity of an external heat source which applies heat to microfluidic device (such as a microfluidic cassette) in a manner that allows heat to transfer into the PCR section of the channel.

[0090] Various valves and fluidically intercommunicating offshoots such as additional channels, reservoirs or chambers can be used to allow mixing, washing, removal and other actions to occur according to the needs of a diagnostic or biochemical assay to be performed therein in an automated or semi-automated manner. The assay is generally carried out by allowing a sample to interact and/or react with one or more reagents in one or more steps; typically in one or more channels or chambers of the device, for times and at temperatures effective in forming a detectable product that indicates the presence or absence of an analyte in the sample. The channel 2 is formed in a first surface of a first substrate 3, typically a substantially planar, substantially rigid substrate which in this embodiment is polypropylene. The first substrate 3 is overlaid with a second substrate 4, which in this embodiment is a polypropylene film. By bonding the first substrate material 3 to the film 4, for example using laser welding, a substantially closed channel 2 is provided (inlets and outlets can be included as required). It will be understood that if the first substrate 3 is a planar element with an upper and lower surface, the majority of the microchannel 2 can formed in the upper surface or the lower surface. It is however often desirable that the second substrate, i.e. the film 4, forms the upper wall of the microchannel 2 in use. The use of laser welding ensured that a fluid-tight, and more specifically an air-tight seal is created around the microchannels, such that no air can escape or leak out between the first and second substrates. It will be understood that methods other than laser welding can be used and will typically be selected based on the substrate materials themselves. The seal or weld is sufficiently strong to withstand increased pressure levels within the cassette compare to outside of the cassette. Typically, it is strong enough to withstand internal gauge pressures of at least 2 bar.

[0091] Alternatively, it would be understood that although this embodiment has the second substrate as a film 4, the second substrate can be another material and may itself have grooves or channel formed on its surface that can be aligned with the channels of the first substrate. By bonding the substrates 3, 4 together, a closed channel 2 is provided (again inlets and outlets can be included as required).

[0092] The cartridges are typically consumables; i.e., they are used once and then discarded; and contain all or many of the reagents needed for one or more assays to be performed. Such reagents can be held on the cartridge in reservoirs or similar.

[0093] Where necessary, the first and second substrates 3, 4 can be aligned prior to bonding. The length and cross-sectional shape of the channel 2 can be any appropriate shape to allow for the desired transport and processing of a sample and or reagents. Such microfluidic systems ‘lab-on-a-chip’ type systems being well known in the art.

[0094] The cassette 1 is provided with an inlet 5 for receiving a sample into the microfluidic channel. The inlet 5 is provided with an air-tight seal in the form of an insertable cap 6. The insertable cap 6 is hinged to the surface of the cassette for ease of use. The cap 6 is sized to be received into the aperture of the inlet and is provided with a resiliently deformable collar that acts to form an air-tight seal in the inlet after the sample has been inserted and when the cap 6 is closed.

[0095] In this embodiment, the other opposite end of the microchannel 2 to the inlet 5 is permanently sealed as it is a single use cassette. However, a similar cap to that used on the inlet could be used to close an outlet if present. In an alternative embodiment, valves positioned within the microchannel, away from the inlet, can be used to create a smaller volume air-tight area if required (still containing the PCR amplification zone, or PCR section 7). For example, a first air-tight valve could be provided proximate and upstream of the amplification zone 7 and a second air-tight valve could be provided proximate and upstream of the amplification zone 7. Once the sample is between the two valves these can be closed and the pressure in the microchannel 2, containing the amplification zone 7, which is between the two valves raised as in other embodiments.

[0096] The microchannel 2 is a continuous flow channel and, in this embodiment, it includes an amplification zone or PCR section 7 which includes three portions of channel that are heatable to different temperatures to allow the thermocycling of temperatures. In this embodiment there is a portion of the channel in the PCR section 7 that is heated to a temperature of 90 degrees to 98 degrees C. There is another portion of the channel in the PCR section 7 that is heated to a temperature of around 45 degrees to 70 degrees C. A further portion of the channel in the PCR section 7 is heated to a temperature of around 70 degrees to 75 degrees C. In this embodiment the heating of the channel occurs when an external heater source is brought into close proximity with the cassette 1 such that heat exchange occurs through the wall of the cassette into the relevant portions of the micro-channel. The microchannel in the amplification zone or PCR section 7 is serpentine in shape such that the sample will travel through the different temperature zones as it travels along each section of the serpentine shape. In this embodiment the heaters are provided in a separate host instrument which receives the cassette therein, but in theory the heaters could form part of the cassette, for example being positioned within the channels themselves. There are also temperature sensors positioned within or close to the relevant pats of the channel to allow the temperature in the channel to be read and if necessary controlled. Although in this embodiment a serpentine shaped microchannel 2 is used in the PCR section 7 to ensure sample moving through the channel is cyclically taken through the different temperatures required for a PCR reaction to occur, it would be understood that other layouts can be used to thermocycle a sample through different temperatures in a microfluidic cassette. For example, a shuttle flow system could be used in place of the serpentine shaping. In a shuttle flow variant, the sample is shuttled back and forward between two or more sections of the microchannel (as opposed to continuously moving forward as in the serpentine variant), each section being held at one of the required temperatures for a PCR reaction to occur.

[0097] The microchannel includes a first bellows pump 8A in fluid communication with the microchannel 2 (via bellows inlet 10A). The first bellows pump 8A is a positive displacement pump, comprising a hemispherical, compressible bellow which is resiliently biased to return to its expanded shape. The internal dimensions of the hemispherical bellow can be changed by applying pressure to its external surface. c. In the present embodiment, mechanical actuators found on a host instrument into which the cassette is placed act under the control of a microprocessor to actuate the bellows pumps by compression, partial compression, partial decompression or decompression of the same. The actuators could however, in theory, be incorporated onto the cassette.

[0098] The microchannel also includes a second bellows pump 8B in fluid communication with the microchannel 2 (via bellows inlet 10B). The second bellows pump 8B is also a positive displacement pump, in this example again comprising a hemispherical, compressible bellow which is resiliently biased to return to its expanded shape. The second bellows 8B can be partially compressed or decompressed as well as fully compressed or decompressed.

[0099] The microchannel 2 also includes various other reservoirs, branches and chambers (such as a capture and viewing chamber for example) as are required to carry out a molecular assay. Where present, the host instrument will include elements such as actuators, heaters and optical elements.

[0100] Whilst this example describes bellows pumps with a hemispherical bellow, it would be understood by those skilled in the art that other bellows formations could be used, or indeed alternative types of positive displacement pump could be used. For example, another type of positive displacement pump is a syringe pump in which the displacement of the piston of a syringe inside a syringe cylinder causes fluid to be sucked into or pressed out of the syringe outlet.

[0101] In use, a liquid sample 9 is introduced into the inlet 5, which leads to the micro-fluidic channel 2 and the cap 6 is closed to form an air-tight seal. The cassette 1 is placed into a host instrument (not shown). In a first embodiment, as depicted in a basic form in FIG. 2, at rest, air pressure on either side of the liquid sample 9 is equal (see FIG. 2A). The hemispherical bellow 8A is then actuated by pins present in the host instrument pressing down on its external surface and thus reducing the volume that air can fill between the bellow and the liquid sample. The host instrument pins or actuators can be provided with sensors to detect the surface of the bellows to ensure specific control of the pressure is achieved. As the cassette is now a sealed (air-tight) system the air has nowhere to go, so the same amount of air is in a smaller volume, so the pressure of the air must increase (see FIG. 2B where there is an increase in pressure to the left of the sample which will act to push the sample 9 to the right). As the air pressure behind the liquid sample 9 is higher than the air pressure in front of the liquid sample, the liquid sample is pushed forward until the air pressure before and after balance again (see FIG. 2C—both sides have equal pressure again). In this embodiment the second bellow 8B is acting as a pressure ‘reservoir’ thus dampening some of the movement of the sample as the relative change in pressure is not as large, meaning the sample can still move, but pressure inside the channel can stay under 2 bar (but still increase compared to air pressure outside of the sealed cassette). In this embodiment, the absolute pressure inside the channel is increased to 1.6 bar prior to the sample being amplified in the amplification zone.

[0102] Notably, if only one bellow is used, the pressure would still be applied, but the movement of the sample would be much greater. This excessive movement and pressure can be avoided (i.e. dampened) even with only one bellow by using much longer channels, but this requires a greater footprint on the cassette which is typically undesirable.

[0103] Whilst the above example describes how the second bellow can be used as a reservoir, FIG. 3 is a preferred variation of the present invention which shows that utilising a plurality of positive displacement pumps such as bellows or diaphragm pumps has additional benefits. The preferred use of the present invention is to include at least two bellows pumps (or other positive displacement pumps) to induce an increase in pressure to at least 1.6 bar within the microchannel 2. Again, in use, a liquid sample 9 is introduced into the inlet 5, and into the micro-fluidic channel 2; and the cap 6 is closed to form an air-tight seal. The cassette 1 is placed into a host instrument (not shown). Prior to the cassette being closed by the cap 6, both of bellows pump 8A and bellows pump 8B are fully decompressed. In the two examples above, the volume of the entire system changes whenever bellow 8A is depressed. This requires constantly changing the pressure inside the cassette during the assay itself which makes fluid control within the microchannel more difficult. Therefore, in this preferred embodiment, in order to increase the pressure in the sealed portion of the microchannel 2 which contains the amplification zone or PCR section 7, both first bellow 8A and second bellow 8B are actuated simultaneously to keep the overall volume of the system equal, and so keep the overall pressure of the system equal. The host instrument applies a push force to the external surfaces of both first bellow 8A and second bellow 8B such that they are compressed by a first amount (the bellows are compressed approximately half-way) to increase the pressure inside the cassette to approx. 1.6 bar. This can be seen in FIG. 3B and it can be seen that the sample at this point does not move if bellows pump 8A is positioned on the opposite side of the sample to bellows pump 8B i.e. a bellow is provided at either side of the sample, (in this case either side of the inlet 5 into which the sample was initially inserted) and in fact preferably at either side of the amplification zone. At this stage the bellows pumps 8A and 8B can continue to be used to move the liquid sample 9 within the channel as required by the assay. By including various valves, and actuating them to open and close as required, this can allow sample to be very effectively moved throughout various regions of the micro-channel such that different actions and reactions can be carried out. In order to move the liquid sample 9 to the right, bellows pump 8A can be further compressed by a second amount and Bellow B allowed to re-inflate (the resilient material of the bellows allowing it to revert back to its original hemispherical shape). Using this mechanism of concurrently or simultaneously actuating or compressing the two valves—or using two positive displacement pumps simultaneously to raise the pressure in the portion of the microchannel containing the amplification zone 7 prior to the liquid sample 9 being amplified (which requires the heating and cooling to different temperatures) means the pressure inside always stays at 1.6 bar and the liquid sample 9 can also be moved around using the same positive displacement pumps without the danger of increasing the pressure within the microchannel to a point where the bonds, such as laser bonds, valves or cap will leak.

[0104] It is preferred that the pressure in the portion of the microchannel containing the amplification zone is increased to 1.6 bar prior to thermocycling occurring in the amplification zone to obtain the benefits of reducing bubble formation, as well as ensuring that the system works even at high altitude, whilst not damaging or exceeding the limits of any of the valves, caps or seals present in the cassette. However, the pressure could also be raised to between 1.5 bar and 2 bar and significant beneficial effects would still be seen. In some embodiments even raising the pressure to 1.1 bar or 1.2 bar or higher would still allow the benefits of the system working at higher altitudes and some reduction in bubble formation when compared to a standard system where no pressure increase prior to amplification would typically occur.

[0105] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0106] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0107] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations).

[0108] It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being indicated by the following claims.