The present invention relates to a microfluidic device comprising a heating chamber and temperature regulation means for the heating chamber. Also contemplated are microfluidic PCR devices having multiple heating chambers with associated temperature regulation means.

Background to the Invention

Manual processing of several biological samples, including blood, urine and saliva, for molecular analysis requires an extended array of laboratory expertise and equipment to analyse accurately, it is therefore cost-prohibitive in many application, prone to error and risks sample contamination or degradation during time of transport. Time to results and back log of testing can create strain on centralised laboratories reducing the quality of care decentralised clinics can provide for their patients. Cost restrictions also play a part in reducing the implementation of robotic fluid handling systems for point of care or doctor's office analysis. There is a current and escalating need for molecular analysis of biological samples at the point of care, this requirement can only be met by less expensive, user-friendly and automated systems that are less prone to error, contamination and sample degradation then conventional technology.

The Polymerase chain reaction is widely accepted as the gold standard in bacterial identification as it allows real time quantification of DNA down to a single molecule (Tang, Wang, Kong, & Ho, 2016a). The target nucleic acid (NA) is amplified by PCR and detected using fluorescent dyes which fluoresce in the presence of double stranded DNA (dsDNA). There are currently several systems which utilise PCR at the point of care, the GeneXpert platform from Cepheid (USA) provides fully integrated cartridges for detection of bacteria & viruses by multiplex PCR (Czilwik et al., 2015a). This system however is limited by its one reaction chamber due to the maximum multiplexing capability of fluorescent dyes, therefore the maximum target

sequences per test is also limited (Czilwik et al., 2015b). The FilmArray from

BioFire Diagnostics and the Verigene system from Nanosphere (A Luminex company) are two Point of Care Molecular Diagnostic (PoC-MDx) systems which offer a greater range of bacterial and viral identification however the systems complexity, driven by their complex, non-monolithic cartridge integration and employment of microarray technology for nucleic acid detection, commands a far greater price point (Czilwik et al., 2015b). Due to this fact these systems are cost- prohibitive towards key areas of need such as doctor's office analysis. Microfluidic platforms have become increasingly important in many application areas such as biotechnology, diagnostics or medical. Microfluidic systems also lead to a concept of lab-on-a-chip, which is the integration of an entire bio/chemical laboratory onto a polymer chip. To create these microfluidic systems, a driving force moves samples within microfluidic structures towards unit operations. By rotating the microfluidic systems on a compact disc-shaped substrate the fluid is inherently drive radially outward from the centre of rotation. With a suitable design of microchannels, valves, vents, chambers, etc., the functions such as fluid transport, splitting, merging can be realized The centrifugal microfluidic lab-on-a-disk platform offers a solution to the technical complexity of fluid manipulations for molecular analysis of bio-fluids, however accurate temperature control for PCR has remained a challenge due to the inherent rotating reference frame of a spinning disk. Purpose built centrifugal microfluidic PCR platforms have been developed, an example of this is the Hahn-Schickard Labdisk Platform, this platform provides a method of disk rotation, circulating currents of hot and cold air to control the total disk temperature as well as fluorescent detection of amplified DNA strands (Strohmeier et al., 2015). Although a brilliant step forward for lab-on-a-disk PCR the device must heat and cool the entire disk which increases thermal mas and therefore run time, only one temperature can be reached at a given time which limits primer design. Another significant problem of currently implemented centrifugal microfluidic platforms is individually localised temperature regulation and specifically activating a single heating unit whilst keeping several heating units inactivated without the need for complex electronics within the reference frame of the rotating disk.

Methods to solve this problem have been explored using battery backs, wireless power transfer and IR lasers.

Jung et al. presented a system described as "Ultrafast Rotary PCR system for multiple influenza viral RNA detection" which is capable of amplifying RNA targets by reverse-transcription PCR. The device sequentially rotates a Microfluidic chip over specific heating blocks set to individual temperatures which correspond to the steps of denaturing (95 ^° C), annealing (58 ^° C) and extension (72 ^° C), however this requires the chip to remain stationary over the heating block during each

temperature step and limits the number of PCR reactions that can be achieved at once (Jung et al., 2012a; Tang, Wang, Kong, & Ho, 2016b) This concept does not include a method of DNA extraction or purification prior to amplification thereby undermining its practical applications.

What is required is a Lab-on-a-disk platform with the capability to directly heat multiple localised fluid filled micro-chambers to a range of temperatures

simultaneously thereby minimising thermal mass and allowing a greater range of primer annealing temperatures. Chen et al ("Wirelessly Addressable Heater Array for Centrifugal Microfluidics and Escherichia Coli Sterilisation" - 35 ^th Annual International Conference of the IEEE EMBS, Osaka, Japan 3-7 July, 2013) discloses using of wireless induction heating of a chamber in a microfluidics device for the purpose of sterilisation of bacterial cells. In this instance a metal alloy is patterned in a coiled configuration which responds to an externally generated alternating magnetic field, the frequency of the alternating magnetic field is changed to the resonant frequency of an individual patterned coil, each patterned coil activates at a different resonant frequency thereby allowing the activation of one or several coils at a time. A problem associated with this art is the maximum temperature achievable by the process which was 93 ^° C, therefore making it inapplicable in its current form to PCR.

It is an object of the invention to overcome at least one of the above-referenced problems. Summary of the Invention

The present invention is based on the application of wireless induction heating to microfluidic devices, especially disk-type microfluidic devices, configured for manipulating liquid samples. The Applicant has discovered that heating chambers of microfluidic devices can employ induction heating to generate heating gradients within the chamber and thereby generate convection currents within the chamber, which allow a liquid sample to be cycled through a range of temperatures. This is achieved by disposing two spaced-apart heating elements in the chamber, each configured to heat to a different temperature in the presence of an RF magnetic field. Application of a suitable RF magnetic field (or magnetic fields) causes the heating elements to heat to different temperatures creating a convection current within the chamber which cycles the fluid through different temperatures. One specific application of the invention is a PCR reaction chamber for a microfluidics PCR device, in which the heating elements are configured to generate a convection current in which the liquid cycle cycles between about 55 °C, 72 °C and 95 °C.

According to a first aspect of the present invention, there is provided a microfluidic device having a microfluidic heating chamber and optionally a controllable microfluidic channel configured to deliver a liquid to the microfluidic heating chamber. The microfluidic heating chamber typically comprises a first region having a first heating element configured to heat to a first temperature in the presence of an RF magnetic field. The microfluidic chamber typically comprises a second region having a second heating element disposed in a spaced-apart disposition to the first heating element and configured to heat to a second temperature in the presence of an RF magnetic field. Exposure of the microfluidic heating chamber to the or each RF magnetic field creates a temperature gradient within the chamber which, during use, causes liquid within the chamber to circulate by convection.

The speed of fluid circulation in each camber, where conventionally controlled by gravity, can now optionally be controlled by the frequency of rotation of the disc. The angular acceleration of the chamber dictates the centrifugal force and therefor essentially the force of gravity. This creates a far higher degree of control over the fluids residence time at a given temperature, further to this at high spin rates the small differences in fluid density (Temperature) will be amplified to create very defined temperature boundaries. These combined effects can dramatically increase the efficiency of temperature cycling.

In one embodiment, the heating chamber comprises a third region disposed between the first and second regions. This allows a gradual change in temperature of the fluid as it moves between the heating elements. In one embodiment, the first and second heating elements are configured to heat to the first and second temperature, respectively, in the presence of the same RF magnetic field. For example, the heating elements can comprise different materials, for example different metals or alloys, that allow the same field frequency achieve different temperatures in the heating elements.

In another embodiment, the first and second heating elements are configured to heat to the first and second temperature, respectively, in the presence of different RF magnetic fields. In one embodiment, the device is a circular planar disc configured for mounting on a support. Microfluidic devices of this type are well known in the art and are described in, for example, (Ducree et al., 2007; Tang et al., 2016b) In one embodiment, the microfluidic device is configured for performing a polymerase chain reaction (PCR), wherein the microfluidic heating chamber is the PCR reaction chamber. Generally, in this embodiment, the first temperature is about 55 °C and the second temperature is about 95 °C.

In one embodiment, the microfluidic device of the invention additionally comprises one or more of: a microfluidic cell separation chamber; a microfluidic cell lysis chamber; and a nucleic acid capture support.

In one embodiment, the DNA capture support comprises surface activated nanoparticles., for example surface activated polystyrene nanoparticles.

In one embodiment, the microfluidic cell-lysis chamber comprises a heating element configured to heat to a cell-lysing temperature in the presence of an RF magnetic field that is different to the or each RF magnetic field that actuates the heating chamber.

In one embodiment, the microfluidic device of the invention additionally comprises a controllable valve.

In one embodiment, the controllable valve comprises a heating element configured to heat in the presence of an RF magnetic field, and a barrier configured to rupture is response to heating of the heating element.

In one embodiment, the barrier is a pressure-responsive barrier and the heating element is disposed within a fluid, wherein the heating element is configured to heat the fluid in the presence of an RF magnetic field to cause an increase in pressure in the fluid sufficient to rupture the pressure-responsive barrier.

In one embodiment, the barrier is a heat-sensitive barrier, and the heating element is disposed in contact with the barrier, wherein heating the element causes the heat-sensitive barrier to rupture.

The device of the invention, or the layers of the device, may be formed from a suitable polymeric material, for example a thermoplastic resin. Examples of thermoplastic resins include cyclic olefin copolymers, polymethylmethacrylate, polycarbonate, polystyrene, polyoxymethylene, perfluoralkoxy, polyvinylchloride, polypropylene, polyethylene terephthalate, polyetheretherketone, polyamide, polysulphone, and polyvinylidine chloride. The invention also provides an analytical system comprising a microfluidic device according to the invention.

In one embodiment, the analytical system additionally comprises an induction coil configured to generate an RF magnetic field.

In one embodiment, the analytical system comprises a sensor configured to detect a parameter of a liquid within the heating chamber of the microfluidic device.

In one embodiment, the sensor is an optical sensor configured to detect an optical parameter of the liquid within the heating chamber.

In one embodiment, the analytical system comprises a microfluidic device in the form of a circular planar disc; and a rotatable platform configured to support the microfluidic device, wherein the rotatable platform comprises the induction coil.

In one embodiment, the analytical system comprises an electrical current supply module operably connected to the induction coil, in which the electrical current supply module is typically adjustable to vary the electrical current supplied to the induction coil.

In one embodiment, the analytical system comprises a motor operably connected to the rotatable platform for rotation of the platform, in which the motor is typically adjustable to vary the speed of rotation of the platform.

The invention also provides a method of amplifying a nucleic acid that employs a microfluidic polymerase chain reaction (PCR) device according to the invention, the method comprising the steps of: providing a sample of nucleic acid to the PCR reaction chamber of the microfluidic PCR device; and generating one or more RF magnetic fields to heat the first element to first temperature(generally about 55 °C) and the second element to a second temperature (generally about 95 °C), whereby a convection current is generated in the PCR reaction chamber causing the liquid to cycle between first and second temperatures (generally about 55 °C and about 95 °C).

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below. Brief Description of the Figures

Figure 1 A is a top plan view of a microfluidic device according to the invention shown sitting on an induction coil.

Figure 1 B is an exploded view of part of the microfluidic device of Figure 1 showing detail of the microfluidic heating chamber.

Figure 2 is a side elevational view of a section of the microfluidic device and induction coil of Figure 1 .

Figure 3 is a side view of an analytical system according to the invention prior to assembly of the microfluidic device and induction coil. Figure 4 is a side view of the analytical system of Figure 3 showing the microfluidic device and induction coil assembled.

Detailed Description of the Invention All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full. Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties,

method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method/process steps.

Definitions:

"Microfluidic device" means a device comprising at least one microfluidic channel typically having a diameter of less than 1000 microns. The term includes devices configured to perform continuous flow microfluidics, droplet based microfluidics, digital microfluidics, and for application in nucleic acid arrays and amplification, and immuno-assays for clinical and research applications. Typically, the device comprises a plurality of microfluidic channels, one or more reservoirs for liquids, and one or more reaction chambers. In one embodiment, the microfluidic device is a passive device, in which fluid transport on the chip is effected by means of an external force (for example rotary drives applying centrifugal forces). In one embodiment, the device may comprise active micro-components such as micropumps. In one embodiment, the microfluidics device may be configured to provide for both active and passive transport of fluids within the device. Microfluidics devices are well known in the literature, and are described in WO2012/164086, US6719682, US2009/166562, WO2006/044841 , and US2006/078462. In one embodiment, the microfluidic device is configured to perform an enzyme linked immunosorbent assay (ELISA). In one embodiment, the microfluidic device is configured to perform nucleic acid amplification typically be means of polymerase chain reaction (PCR). As used herein, the term "microfluidic heating chamber" refers to a reaction chamber forming part of a microfluidics device and configured to contain and heat a volume of fluid. Typically, the microfluidic chamber has a volume of less than 10 mis, 5 mis, 2 mis, 1 mis, 0.5 mis, 0.2 mis, or 0.12 mis. In one embodiment, the microfluidic heating chamber comprises a heating element disposed on a base of the chamber. In one embodiment, the microfluidic heating chamber comprises a heating element disposed on a side of the chamber. In one embodiment, the microfluidic heating chamber comprises a heating element disposed on a top of the chamber.

As used herein, the term "heating element" refers to an element that heats in the presence of a RF magnetic field. Such elements are well known and are employed, for example, in Chen et al in the context of sterilisation of bacteria in a chamber of a microfluidic chip. The heating element may be made of a metal or a metal alloy. Examples of metals include copper, aluminium, nickel, iron, cobalt, tungsten. In one embodiment, the heating element is a micropatterned heating element. In one embodiment, the heating element is printed on to a substrate. In one embodiment, the heating element comprises an array of heating elements. In one embodiment, the heating element is an inductor-capacitor circuit optionally comprising a spiral coil as an inductor.

As used herein, the terms "first" and "second" as applied to heating temperatures typically refer to different temperatures that are capable of creating a temperature gradient in the heating chamber which is capable of generating a fluid (i.e. a liquid) convention current within the heating chamber. For example, the first temperature could be 55 °C and the second temperature could be 95 °C. In one embodiment, the first and second heating elements are disposed on opposite ends of the heating chamber, and a third region is disposed between the first and second heating elements. In one embodiment, the third region does not comprise a heating element. In another embodiment, the third region comprises a heating element. As used herein, the term "RF magnetic field" refers to a rapidly alternating magnetic field capable of creating eddy currents in the heating element resulting in heating of the material of the heating element by Joule heating or, in the case of

ferromagnetic or ferromagnetic heating elements, by magnetic hysteresis losses. The rapidly alternating magnetic field is typically generated by an electromagnet and an electronic oscillator that passes a high frequency alternating current through the electromagnetic. The electronic oscillator is typically adjustable to vary the frequency of the alternating current, and thereby vary the frequency of the RF magnetic field (field frequency). Joule heating in the electric element is maximised when the field frequency matches the resonant frequency of the heating element.

As used herein, the term "polymerase chain reaction" or "PCR" refers to a process of nucleic acid amplification that involves use of nucleic acid primers, nucleic acid polymerase, and cycles of heating and cooling the nucleic acid for DNA melting and enzymatic replication, resulting in exponential amplification of the target nucleic acid. It is described more fully in Bartlett et al (Bartlett, J. M. S. ; Stirling, D. (2003). "A Short History of the Polymerase Chain Reaction". PCR Protocols. Methods in Molecular Biology. 226 (2nd ed.). pp. 3-6. doi:10.1 385/1 -59259-384-4:3. ISBN 1 - 59259-384-4). Microfluidic PCR devices are also known in the literature, for example, (Czilwik et al., 2015b; Jung et al., 2012b; Miao et al., 2015a).

As used herein, the term "microfluidic cell separation chamber" refers to a chamber of a microfluidic device configured to receive a cell containing fluid and at least partially separate cells from the fluid to provide a cell-enriched fraction and a cell- depleted fraction. In one embodiment, a force is applied to the fluid in the cell to effect cell separation, for example a centrifugal force. This may be achieved to providing the separation chamber in a disk-type microfluidic device, and then rotating the device on a turntable to generate sufficient centrifugal force to separate cells from the fluid. Examples of microfluidic cell separation chambers are described in the literature, for example in (Burger & Ducree, 2012; Ducree et al., 2007) As used herein, the term "microfluidic cell lysis chamber" refers to a chamber of a microfluidic device configured to receive a cell containing fluid and treat the cells to cause lysis of the cells. In one embodiment, the cell-containing fluid is heated. In one embodiment, a cell lysis reagent is added to the chamber to cause lysis of the cells. Examples of suitable reagents include detergents (i.e. non-ionic and/or ionic detergents) Examples of microfluidic cell separation chambers are described in the literature, for example in (Baek et al., 2010; Burger & Ducree, 2012; Kido et al., 2007; Tang et al., 2016b) As used herein, the term "nucleic acid capture support" refers to a support that is configured to bind nucleic acid and release the nucleic acid for example by elution. Examples of nucleic acid capture supports include functionalised filters,

functionalised (activated) nanoparticles, silica based separation supports, which are described in the following literature references: (Miao et al., 2015b; Tang et al., 2016b)

As used herein, the term "controllable valve" refers to a valve forming part of the microfluidic device which is remotely actuable to open a fluid channel within the device. For example, the device may comprise two reaction chambers for performing separate reactions (i.e. cell lysis and DNA capture), a microfluidic channel providing fluid communication between the chambers, and a controllable valve disposed within the microfluidic channel. The valve generally comprises a barrier, for example a sacrificial membrane, and means for sacrificing the

membrane. Examples of controllable valves, that are remotely actuable is response to various stimuli (including pressure, force, light, etc) are described in the literature, for example (Gorkin III et al., 2012; Nwankire et al., 2014; Siegrist et al., 2010; Tang et al., 2016b)

As used herein, the term "induction coil" refers to a coil of copper wire with Direct or alternating current passing through it, coil dimensions range from flat circular coils, helical coils or U-shaped coils. The coil generates a magnetic field which is directly related to the strength and direction of current passing through the wire. Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Referring to the drawings, and initially to Figures 1 and 2, there is illustrated a microfluidic device according to the invention indicated generally by the reference numeral 1 shown sitting on an induction coil 2. The device 1 comprises a planar circular disk having four heating chambers 3 disposed within the disk. Although not illustrated, the device will additionally include an architecture of connecting microfluidic channels for the purpose of controllably transporting liquids around the disc.

In more detail, and referring to Figure 1 B and Figure 2, the heating chamber 3 is a rectangular chamber having a longitudinal aspect generally parallel to a radius of the disc, and a first heating element 5 disposed at one of the chamber (towards the edge of the disc) and a second heating element 6 disposed at an opposite end of the chamber (towards the centre of the disc). The first and second heating elements are spaced apart by an intermediate region 7. The first heating element 5 is composed of a copper foil which is disposed positioned on the base of the chamber, and the second heating element 6 is composed of an aluminium foil disposed on the base of the chamber. In the embodiment shown, the heating elements are contained within chambers disposed beneath the heating chamber and in fluid communication with the heating chamber. In this embodiment:

• Metal pieces are generally 0.5mm thick, 1 5mm in length and 1 0mm in width · Each chamber is about 1 mm in height, 30 mm in length and 1 5mm in width.

• Heating element 1 (55 °C) is radially closer to the centre of rotation then

heating element 2 (95 °C). Heating element 1 is copper, heating element 2 is aluminium.

Distance from heating element to induction coil is 10mm during rotation. Frequency of alternating current is 20kHz

A thermal camera is typically used to measure the temperature gradient across the chamber, this is done by sinking the frame rate of a short expositor thermal camera to the spin rate of the disk to obtain a static image of the chamber.

Speed of circulation can generally be measured using a short exposure camera and coloured dye's which are carried by the convection current. A strobe light is slinked to the spin rate of the disk to provide a static image of the chamber.

These are general parameters based on the current prototype, with a more powerful device the size of the heating elements can be reduced.

Referring to Figures 3 and 4, an analytical system according to the invention is described and comprises a microfluidic device 1 , induction coil 2 mounted on a rotatable spindle 8, motor 9 operably connected to the rotatable spindle 8 for rotation thereof, and induction control module 1 1 for controlling the AC supplied to the induction coil 2. The disc is configured for mounting to the induction coil and rotation therewith and is secured to the spindle using appropriate securement device such as a screw 10. The speed of the motor can be varied to vary the frequency of rotation of the device 1 , typically between 0 Hz and 80 Hz. The induction control module is adjustable to vary the frequency of the alternating current supplied to the induction coil, and thereby vary the field frequency that the disc is exposed to.

In use, the microfluidic device (disc) 1 is placed on the induction coil 2 and secured in position using the screw 10. The microfluidic device is then operated to perform microfluidic operations within the disc; for example, in the case of a PCR microfluidic device, the microfluidic operations may be chosen to perform separation of cells from the cell containing fluid, lysis of the cells, extraction of DNA from the cell lysate and subsequent release of partially purified DNA, and then transport of a DNA-containing liquid and PCR reagents to one of the heating chambers 3 (which in such an embodiment would PCR reaction chambers). One, some, or all, of the microfluidic operations may involve application of centrifugal forces to move liquids or reagents between chambers and/or actuate valves within the device, which centrifugal forces are generated and controlled by spinning the disc using the motor. Once the DNA and PCR reagents are in the PCR reaction chamber, the induction control module is actuated to cause the induction coil generate an RF magnetic field of defined field frequency of 20 kHz, which causes the heating elements 5 and 6 to heat to 55 °C and 95 °C, respectively. This generates a temperature gradient within the chamber, which results in a convection current being generated which circulates the DNA and the PCR reagents from the first region (55 °C), through the intermediate region, to a second region (95 °C), back to the intermediate region, and then back to the first region. This results in the thermal cycling of the liquid in the chamber between 55 °C and 95 °C while the first and second heating elements are actuated, allowing cyclical rounds of DNA melting and strand elongation, resulting in exponential amplification of the target DNA. Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

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