BR112018077159B1 - TEST SYSTEM, AND, METHOD FOR CONDUCTING A TEST - Google Patents

Abstract

The present invention relates to a microfluidic test system and associated reading device, as well as individual components thereof. The present invention also relates to methods for conducting the assays, using a disposable system and associated reading device, as well as kits for conducting the assays.

Description

FIELD OF INVENTION

[001] The present invention relates to a microfluidic test system and associated reading device, as well as the individual components themselves. The present invention also relates to methods for conducting assays, for using a disposable system and associated reading device, as well as kits for conducting assays.

BASICS OF THE INVENTION

[002] Microfluidic cartridges are routinely used to perform a variety of assays, both biological and chemical and/or physicochemical, and assay results are often determined using an associated reader device into which the cartridge has been inserted.

[003] Movement of liquid within the cartridge is often required in order to ensure that a sample is able to make contact with reagents that are deposited within the cartridge and that are capable of reacting with one or more target analytes that may be present in a sample. Following reaction with one or more reagents, it is often desirable that the sample be removed from the area where the reaction occurred so that an additional reaction can occur, or simply to allow detection of any reaction products, which may be difficult. can be achieved when the sample remains in place, due to optical interference, for example.

[004] Movement of fluid in the cartridge may occur by capillary action alone, or control of fluid in the cartridge may be effected by an active force provided, for example, by the use of micropumps and valves that may be present in the cartridge, or by means of mechanisms present in the reader device that are designed to interact with the cartridge and pump a fluid into and/or out of the cartridge in order to control the movement of liquid within the cartridge itself - see, for example, EP2613881. Alternatively, a combination of capillary action and an active force can be used.

[005] US7238324, for example, describes a microfluidic device that employs both capillary action and the application of an external pump. A sample can naturally enter the microfluidic cartridge through a first orifice by capillary action and flow to a sensing chamber where the assay takes place. Following the test reaction, liquid is introduced into the chip through a second hole for use by an external pump. The purpose of this liquid is to wash the original sample, leaving only the reaction products behind that can be detected. However, this means that a separate liquid must be introduced into the chip from the outside and through an additional hole, which may be obstructed and/or subject to contamination. Additionally, the liquid can become contaminated or degrade over time. Thus, there is a need to provide microfluidic cartridges in which it is not necessary to introduce fluids, other than the sample, from the outside of the cartridge and/or which have holes, other than the sample hole through which the sample must be introduced. Some designs are known to include liquid-filled bags within the cartridge itself in order to provide adequate reagents/wash buffers, etc., but this significantly increases the complexity and cost of the cartridges and the liquid reagents may also be susceptible to degradation.

[006] US5821399 describes a system and cartridge that employs electrical conductivity to measure samples and fluid between samples. Flushing fluid is provided in a fluid-filled bag inside the cartridge, which can be transported inside the cartridge and detected for its electrical conductivity by the reader. The difference in electrical conductivity between a sample and the rinse fluid or air segment can easily be determined.

[007] WO2013154946 describes a microfluidic system that uses a combination of capillary action and gas pressure to control the movement of liquid samples within a microfluidic device. Initially a liquid sample that is introduced into the device is transported by capillary action partially along a capillary channel. As the liquid advances, the gas pressure at the distal gas-liquid interface increases by an amount sufficient to stop the movement of the liquid. To initiate further movement of the liquid sample, a pump connected to a distal portion of the capillary channel decreases the gas pressure acting at the distal gas-liquid interface of the liquid sample by an amount sufficient to allow the liquid sample to move by capillary action further. along the capillary channel of the microfluidic device.

[008] US5096669 describes a disposable device for use together with a reader device. A sample may initially be drawn into the disposable device by means of capillary action and further movement of the sample within the device may be effected by the reader automatically pressing a gas balloon comprising a flexible membrane, within the device, so as to cause the fluid sample flows over the sensors and the concentration of a chemical species is determined.

[009] WO2013/096801 describes a lateral flow detection system, which may include fluidic resources. In one embodiment, a cartridge is described that includes the lateral flow device that includes chromatographic media and immobilized antibodies, but additionally includes a gas balloon that is actuated by a pump in the reader. Depression of the gas balloon serves to move the fluid sample within the fluidic channels of the cartridge and to the capture zone. Following appropriate capture, the fluid sample is further pushed by the action in the gas balloon into a washing chamber. The use of a flushing fluid is described to wash components of the fluid sample, such as red blood cells, that may interfere with detection. However, combining lateral flow capabilities with microfluidic capabilities provides a degree of complexity and lateral flow testing is generally by qualitative or semi-quantitative measurement rather than being quantitative.

[0010] EP2613881, a previous application from the current applicant, describes a microfluidic cartridge and associated reader. A fluid port in the cartridge is designed to form a fluid-tight seal with the reader so that gas can be transported into and through the cartridge by the reader.

[0011] WO03/049860 describes a complex device for chemical or biochemical analysis, comprising first and second layers separated by a fragile third layer. Upon breaking of the fragile third layer, fluid present in the second layer is able to enter the fluidic network of the first layer. Many chambers are provided which must be sequentially compressed in order to provide various reagents and conduct any particular assay.

[0012] US2015/0004680 describes biosensor cartridges for detecting one or more components in a sample. Each cartridge comprises, in addition to microfluidic channels, an air pump chamber and a reagent pump chamber that comprise pores on its upper surface. When an associated sensor employs optical detection, wash buffers are also provided in the cartridge. The chambers are described as separate pockets that are inserted into the cartridge recesses. The reagent and buffer bags are filled with suitable liquid.

[0013] There is also a need to be able to test fluid, such as blood samples, from subjects more quickly and with less complexity. There is also a desire for subjects to test themselves at home.

[0014] Typically, when a subject presents to a local medical clinic, or even a hospital, relatively large fluid or blood samples are taken to be analyzed and, depending on the tests performed, numerous separate vials or samples may be required. Also, where testing is not performed at the time of sample collection, it is often necessary to store the sample in a manner that minimizes degradation or loss of a particular analyte to be detected. Some tests are time sensitive, and the time it takes to conduct a test may result in the disease progressing undesirably when the subject could have been treated.

[0015] There are also diseases and conditions where it would be desirable for the subject to be able to test for themselves on a regular basis and to be able to self-medicate based on the test results. In this way, the subject can be directed based on a test result, possibly with input from a healthcare professional, as to which medication should be taken.

[0016] Furthermore, clinical analysis of samples has traditionally been conducted using clinical analytical techniques that require the use of specialized laboratories with large-scale machines to perform the analyses. In recent years, there has been a push to develop bench-sized or even portable devices that are capable of conducting such tests. However, the ability of such devices to handle only small sample volumes and/or perform a variety of different types of analyzes is limited. Furthermore, it would be desirable if a single reader could perform a variety of different panel tests, so that the user would not have to have numerous different readers in order to be able to conduct different types of tests.

[0017] US8435738, for example, describes a modular system that is capable of conducting numerous assays from a single blood sample. However, it is clear that individual and separate modules are provided to perform different functions and the sample is transported to each module by means of a sample handling system. It is also apparent that the system comprises a housing that is designed to handle sample preparation as well as performing the various assays, but it is uncertain what happens to each sample once it has been analyzed.

SUMMARY OF THE INVENTION

[0018] The present invention is based in part on studies by the present inventors of controlling the movement of a liquid sample within a microfluidic cartridge and also how to effectively remove the sample and/or wash bound components from an analyte detection zone in the cartridge. , without having to introduce a fluid from the outside of the cartridge, or use a wash or other liquid(s) that would be present in the cartridge or associated reader. The inventors have developed a “dry” microfluidic cartridge that includes a gas-filled chamber that can be used to facilitate very precise controlled movement of the liquid sample in the cartridge and to remove the sample and unbound material from an analyte detection zone in the cartridge. such that any detectable elements which may include complexes comprising the analyte or analyte reaction product can be detected in a gaseous environment. Importantly, the cartridges of the present invention do not require additional liquids, other than the sample itself, to be present in the cartridge and/or introduced into the cartridge. An associated reader device is additionally provided for use with the cartridge.

[0019] In one area, the present invention prescribes methods and cartridges that use only a gas, such as air within a chamber that is present in a microfluidic cartridge, to control the movement of the liquid sample in the cartridge and also optionally remove the sample liquid and unbound material from a detection area before detection is performed.

[0020] In another alternative and/or complementary area, the present invention provides cartridges, readers and methods for performing a multiplicity of different assays using a single cartridge and associated reader.

[0021] In a first aspect, an independent microfluidic system is provided for use in conducting an assay on a liquid sample, the microfluidic system comprising: a sample inlet port for receiving the liquid sample, the sample inlet port connected to at least one microfluidic channel, wherein each microfluidic channel(s) comprises one or more reagents deposited therein for use in conducting the assay and a zone of detection for use in detecting any analyte that may be present in a sample or analyte reaction product thereof; and each/said microfluidic channel(s) is/are additionally fluidically connected to a gas-filled compressible chamber downstream of each/said detection zone, and wherein the system is formed of three layers, which are sandwiched together to define each/said microfluidic channel(s) and said gas-filled chamber, and wherein compression or decompression of said chamber causes gas to be expelled or drawn into the chamber, which, in turn, causes movement of the liquid sample within said/each microfluidic channel.

[0022] Typically, although not exclusively, the system may be in the form of a cartridge, which is designed to be inserted into an associated reader device. For the sake of brevity, reference will be made below to the system being in the form of a cartridge, but this should not be considered limiting.

[0023] For the avoidance of doubt, the present invention does not require the use of liquid-filled bags that must be present in or provided with the cartridge and/or the ability to transfer fluid (liquid or gas) from an associated reader to the cartridge . In this regard, the cartridge of the present invention should be considered independent. The cartridges of the present invention prior to application to a sample are substantially free of liquid and can be considered dry. The only fluid prior to application of the liquid sample, which may be, or is, present in the cartridge, will be a gas, typically air. Advantageously, the only liquid required in the tests of the present invention is the liquid sample itself.

[0024] In certain embodiments, said one or more reagents may be deposited at a first location in each/said microfluidic channel(s). In other embodiments, said one or more reagents may be deposited in more than one location in each/said microfluidic channel(s). At least one of said one or more reagents may be deposited in the detection zone or, alternatively, no reagent is deposited in the detection zone. The reagents may initially be provided in a liquid form which is allowed to dry by evaporation or other means. In terms of the present invention, when said reagents are initially presented in a liquid form, which is subsequently dried, it is to be understood that the term “dry” means that less than 10%, 5%, or 1% of the liquid initial remains after drying.

[0025] In certain embodiments, the detection zone may be downstream of where said one or more reagents were deposited.

[0026] The expression “downstream” in the context of the present invention is in reference to where the sample is applied in the system and the direction of flow of the sample.

[0027] Optionally, following reaction of the liquid sample with said one or more reagents deposited within said/each microfluidic channel(s) and transferring the sample and other reagents to the detection zone, gas is expelled from the chamber optionally serves to remove liquid from the detection zone within the microfluidic channel so that any analyte or analyte reaction products captured in the detection zone can be detected in a substantially liquid-free environment. Thus, in one embodiment, the present invention provides a cartridge and method in which detection occurs in a substantially liquid-free environment. Furthermore, the inventors observed that it is only necessary to displace the liquid from the detection zone, using a corresponding volume of gas from the chamber. Thus, it is not necessary to use a conventional washing step, which may employ significant volumes of a fluid or fluids, which is/are designed to prevent and/or minimize signal interference. Thus, the present invention advantageously uses a gas, in a much smaller volume, to remove liquid and material in the liquid from the analyte detection zone. This is very different from what would be understood in terms of carrying out a conventional washing step.

[0028] The microfluidic cartridge may comprise a plurality of microfluidic channels, wherein all of the plurality of microfluidic channels are in fluidic communication with a single sample inlet port. The sample orifice may be in communication with a microfluidic channel that is divided into said plurality of microfluidic channels in accordance with the present invention. Each of said plurality of microfluidic channels may be in fluidic communication with a respective gas-filled chamber, or two or more microfluidic channels may be in fluidic communication with a single gas-filled chamber. In accordance with the present invention, each chamber may be separately or independently controlled, thereby allowing independent control of sample movement within an individual microfluidic channel, or may control sample movement in a plurality of said microfluidic channels at the same time. In addition to being in fluidic communication with one or more microfluidic channels in the cartridge, in some embodiments, said gas-filled chamber(s) do not connect with any other feature that may be present in the cartridge. microfluidic or associated reader. For example, the only opening/exit of the gas-filled chamber may be the opening with said microfluidic channel(s). Thus, said gas-filled chamber(s) may be sealed in terms of not having a valve, orifice or otherwise being in communication with the outside. of the cartridge. In one embodiment, the sample inlet port is connected to a first end of each/said microfluidic channel(s) and the second end of each/said microfluidic channel(s) is/are connected to said opening(s) of one or more of said gas-filled chambers. In such an embodiment the gas-filled chamber(s) may be considered downstream of the sample inlet port and at an opposite end of each/said channel(s). is) microfluidic(s) in the sample inlet port.

[0029] Unless context dictates otherwise, the expression “fluidic communication” is understood to mean that a fluid, including a gas or a liquid, is capable of being transported between the relevant parts.

[0030] Optionally, the microfluidic cartridge may additionally comprise one or more collection features designed to receive fluid waste and/or excess liquid sample. For the avoidance of doubt, some embodiments of the present invention specifically exclude one or more collection features, which may be advantageous.

[0031] The cartridge design of the present invention can easily be adapted to perform numerous different assays and, consequently, can be considered a testing platform for a variety of similar and/or different assays. The cartridge and channel(s) disposed therein may be formed in any of the ways known to those skilled in the art, which may include techniques of photolithography, wet etching, laser ablation, injection molding , die punching, embossing and printing. In accordance with the first aspect of the invention, the cartridge and the channels and other features arranged therein are formed by a sandwich of three separate substrates - a first, second and third substrate, such as a top and bottom substrate with a base substrate. medium disposed between the top and base substrates. The three layers can be sealed together by applying heat or using an adhesive. Furthermore, the middle layer may itself be in the form of an adhesive layer that is capable of adhering the top and bottom substrates.

[0032] In one embodiment, the three substrates are flat. Typically, the first and third substrates (e.g., top and bottom and optionally the second, e.g., middle) are substantially uniform in nature. That is, the thickness of said substrate is uniform and does not vary across the surface of the substrate.

[0033] In a specific embodiment, the base substrate is adhered to the medium substrate that has channels already arranged therein. The reagents required to perform the assay are deposited in their specific deposition zones on the base substrate, and are held in place, once reconstituted by the sample, by the channel walls (formed by the middle adhesive layer) and prevented from spreading throughout the channel formed by features printed on the base substrate, e.g. hydrophobic ink. In this way, reactants are prevented from spreading out of their deposition zone by the features on all four sides. The reagents are then dried and the final top layer of substrate is adhered to the middle layer to produce fully formed cartridges. Many other methods of depositing assay reagents into the cartridge can be considered by those skilled in the art.

[0034] The cartridges of the present invention may be formed by a continuous band or roll-to-roll process known in the art from a roll of flexible polymer film, plastic sheet or metal.

[0035] Advantageously, the inventors have observed that when the cartridge is formed from a sandwich of three separate flat substrates, it is not necessary for the top and bottom substrates of the cartridge to be of different thicknesses and/or have portions of different thickness or formed of other materials. Thus, the top and bottom layers can have a uniform thickness and be formed from a single material. This simplifies cartridge manufacturing and associated costs. The material used to form the top and optionally base layers may be flexible, but in the gas channel and chamber it is reasonably rigid but somewhat resilient. Surprisingly, the substrate forming the top and base surfaces, especially the top outer surface of each gas-filled chamber can be resilient, even though the thickness of the top surface is uniform across the surface of the substrate.

[0036] The adhesive that can be used to seal the layers together can also combine to facilitate the compressibility of each/said chamber. Thus, the compressible nature of the chamber may in part be due to the adhesive being resilient, as well as the top and optionally base substrate being resilient. Contrary to the prior art, the gas-filled chambers of the present invention need not comprise a flexible membrane or sheet forming an outer surface of the chamber, which is produced from a different material and/or is of a different thickness or flexibility compared to the substrate employed. to form the rest of the top and optionally base layer. Thus, the top layer and optionally the base layer is/are produced from a single material of uniform thickness across the layer. Ideally, the top and bottom layers are produced from the same material and are of uniform thickness. This simplifies cartridge manufacturing, which is an important cost consideration.

[0037] The cartridge can be formed from any suitable material, such as polycarbonate, polyolefins, such as cyclic olefin copolymers (COCs), polyester, polystyrene, PMMA, etc. and the/each substrate may be formed from a single or plurality of material(s). In embodiments comprising three substrates, the middle substrate comprises a pattern cut into the substrate corresponding to certain features of the cartridge, such as said channel(s), gas-filled chamber(s), residue collector and similar. By applying and sandwiching (such as by heat sealing, glue, clamping and the like) appropriately cut top and base substrates, to sandwich the middle substrate between the top and base substrates, a cartridge can be provided in which channels and other features are willing. Each layer can be provided separately and sandwiched together, or the three layers can be connected to each other and the sandwich formed by folding the layers over each other to form the cartridge. The top and/or base substrates may be formed from or coated with a material that is different from the middle substrate and/or adhesive material applied to any of the substrates to facilitate adhesion of the three substrates together. Features on the top and/or base substrate can be designed for co-location with features on a reader device (as will be discussed below), which can facilitate correct location of the cartridge in the reader.

[0038] In a convenient embodiment, the assay readings of the present invention are designed to be detected optically. In this regard, an associated reader will include optical detection means, such as a spectrometer or fluorimeter, which is designed to detect electromagnetic radiation emitted by the detection area of the cartridge. For fluorescent detection, the spectrometer or fluorimeter in the reader will detect fluorescence emitted by the material in the detection zone. Thus, at least a portion of the first or third layer (e.g., top or bottom layers) of the cartridge that is designed to face toward the spectrometer or fluorimeter in the reader must be optically transmissible in an appropriate range of the electromagnetic radiation spectrum. . In the case of fluorescence detection, at least a portion of the first or third layer (e.g., top or bottom) of the cartridge must be optically transmissible in the range encompassing an excitation wavelength and a detection wavelength. For example, at least the first and third layer portion (e.g., top or bottom) of the cartridge must be optically transmissible in the range 200 - 1,200nm

[0039] When the first and third layers (e.g., top and/or base) are produced from a single material, it will be appreciated that the entire first and third layer (e.g., top and/or base) will be optically transmissible and not just a portion of it. However, paint and/or masks may be employed to prevent or minimize electromagnetic radiation of appropriate wavelengths from arriving or scattering outside the detection zone. In one embodiment a portion of the third layer (e.g., base) that encompasses the detection zone or portion may therefore be coated with a material that is designed to maximize the emission of any fluorescent signal into the optical detection means of the reader. .

[0040] The first and third substrates (e.g., top and bottom) can be connected to a hinge that allows the two substrates to fold adjacent to each other with the middle substrate being disposed between them. Alternatively, hinges may be provided between the first and second substrates (e.g., top and middle) and the second and third substrates (e.g., middle and bottom), so that the first, second and third substrates (e.g., e.g. top, middle and bottom) fold adjacent to each other and can be formed from a single sheet of substrate.

[0041] Importantly, said gas-filled chamber(s) is/are designed to co-locate with a feature or features on the reader that are designed to make contact with an external surface. of said gas-filled chamber(s) (i.e., the top and/or base substrates when in the form of a three-substrate sandwich, which shall be provided to the reader in a substantially horizontal manner ) and be able to control force, e.g. compression, to be applied to/removed from an external surface of the chamber. Applying a force to the outer surface of each chamber causes the chamber to be deformed and gas within the chamber to be expelled from the chamber into a microfluidic channel. Conversely, a subsequent reduction in force, e.g. decompression, applied to/each chamber causes the chamber to deform less and optionally return to an undeformed state, such that the gas is drawn back into the chamber from a channel. microfluidic.

[0042] It will be appreciated that without application of a force, the gas-filled chamber will typically comprise a maximum volume of gas. Upon application of force, gas will be expelled from the gas-filled chamber, thereby reducing the volume of gas within the chamber. A subsequent reduction in the force applied to the chamber will allow gas to be drawn back into the chamber, with a consequent increase in the volume of gas within the gas-filled chamber.

[0043] The gas within each chamber is typically air, although other gases or gas mixtures may be introduced. For example, if any of the reagents that are deposited within each microfluidic channel(s) are subject to oxidation or otherwise have a shorter shelf life when present in air, the cartridge and channels and chambers associates may be filled with an inert gas such as nitrogen, or the like. Generally reference will be made to the gas being air, but this should not be considered limiting.

[0044] Typically in use, the cartridge may be provided or inserted into the reader prior to sample application and a force applied to said/each chamber in order to expel gas from said/each chamber and into said/each microfluidic channel. The cartridge can be considered “primed” for sample application.

[0045] A sample, such as a blood sample, or any other liquid sample, can be introduced into the cartridge through the sample inlet port. The sample can be introduced directly by placing the sample in contact with the inlet hole. Alternatively, the sample may first be collected using a sample collection device and sample collection device such as a probe, micropipette, capillary tube or the like, placed in contact or inserted with/in the sample inlet port to so that the sample can be introduced into the microfluidic cartridge and channel. In some embodiments, such as when performing nucleic acid analysis, it may be desirable to perform any analysis in a closed system. Thus, the sample collection device designed to introduce the sample into the microfluidic cartridge can serve the dual purpose of introducing the sample and sealing the sample inlet hole once the sample collection device has been inserted into/brought into contact with /with the sample inlet hole.

[0046] After the sample is contacted with/introduced into the sample inlet port of the cartridge, the sample may initially be drawn into the microfluidic channel via capillary action. Alternatively, the sample can be actively pulled into the microfluidic channel by reducing the force applied to said/each chamber so that gas is drawn into the chamber which in turn pulls the liquid sample into and along said/each channel. microfluidic(s).

[0047] In one embodiment the liquid sample is initially drawn along a single microfluidic channel, which divides into a plurality of microfluidic channels, each of said plurality of channels being capable of performing one or more assays. In this way, a single sample can be provided which is in turn divided into numerous portions or aliquots.

[0048] Once a sample has been introduced into the cartridge and said/each microfluidic channel(s), such as through capillary action and/or actively pulling the sample through the cartridge, further transport of fluid into and through of the cartridge and associated channel(s) is carefully controlled/facilitated by means of controlled pressure as applied to said/each gas chamber which causes gas to be introduced into and/or expelled from said/each chamber(s) filled with gas. Gas that is drawn back into said/each chamber will typically serve to pull a liquid sample along said/each microfluidic channel(s) toward said/each chamber because of a vacuum effect and expelled gas of said/each chamber will push liquid within said/each microfluidic channel out of said/each chamber, towards the inlet orifice and optionally towards the fluid waste collector, when present.

[0049] As previously mentioned, a collection feature is completely optional. According to the present invention, it is possible through appropriate fluid and gas chamber control management to ensure that once a sample is introduced into the cartridge, the sample or other liquid cannot be expelled through the sample orifice. Before sample application, each chamber can be compressed to a maximum extent so that it is not possible to expel the sample through the sample inlet hole. Advantageously, compression of each gas-filled chamber prior to sample application means that after conducting any test and releasing any compression pressure in each gas-filled chamber, the liquid sample is further drawn into the cartridge and possibly to the gas-filled chamber, outside the sample inlet port. This can be seen as a useful safety feature in terms of isolating any sample following assay by the user.

[0050] Fluidic movement can be controlled very precisely by force control means in the reader. Additionally, the position of fluid within each channel can optionally be detected by the reader by means, such as electrodes, positioned along the microfluidic channels that are in contact with the reader and can feedback the position of any liquid and/or fluid in each/said microfluidic channel, thereby allowing the reader to very carefully determine the position and/or rate of liquid movement by applying force/pressure to the gas/air filled chamber.

[0051] As identified, in use, the sample is applied to the cartridge through a sample inlet port such as through direct contact by the subject/patient or other means, for example, a pipette, capillary tube or the like. In a preferred embodiment, the sample inlet port is an opening in a side or face (e.g., top face) of the cartridge. Desirably, the cartridge may be in the form of a generally thin flat device comprising top and base faces and four edges. In this arrangement, the sample inlet hole may be formed on one of the edges of the cartridge or on the top face, so that a user need only bring the sample into contact with the opening formed on the edge or top face in order to to allow sample capture in the cartridge. In use, the user places the fluid sample in contact with the orifice/opening and, in certain embodiments, by virtue of the dimensions of said channel(s) in the cartridge, fluid is drawn into the cartridge by capillary action. The dimensions of the sample orifice/aperture may be smaller than the dimensions of the channel(s). Thus, when expelling fluid from said/each microfluidic channel an optional fluid waste collector provides a large area of voids in which waste fluid sample and any unreacted reagents/label can be directed to and into the waste collector, in instead of out through the sample inlet port.

[0052] In certain embodiments, a waste collector is not provided. Since it is not necessary to remove the sample, it is only necessary to remove the sample and unreacted reagents/label from the detection zone within said/each microfluidic channel. Careful control of liquid movement can be effected so that the gas expelled from said/each gas-filled chamber is sufficient to simply remove the sample and any unreacted/undesirable reagents/label out of the detection zone. Initial maximum compression of said gas-filled chamber, prior to application of liquid sample, ensures that it is not possible to push the sample beyond the sample inlet port. Such careful control of liquid movement within the microfluidic channel means that a waste collector and/or large volumes of a flushing fluid may not be required, resulting in a simplification in manufacturing/use and a reduction in cost.

[0053] Said microfluidic channel(s) in the cartridge may also comprise one or more fluid stop features, which are designed to prevent the sample from passing through the stop feature, due to capillary action alone. That is, the sample must be actively forced beyond said stop feature(s) and/or further along said microfluidic channel(s) by the action of a force, such as the compression and/or decompression force acting on the gas-filled chamber, which serves to pull or push the liquid sample in the cartridge. The stopping feature may be a hydrophobic material (e.g., printable conductive or non-conductive ink) or a process or material that changes the surface properties of a channel surface thereby creating a hydrophilic/hydrophobic differential (e.g., by means of laser ablation, surface classification, removal of surface material, evaporated metallic materials, etc.), which is designed to support/be a wall feature or is clad on a wall (e.g. top, side(s) and /or base) of a channel. In an embodiment where the channels are formed by virtue of three substrates being sandwiched together thereby forming the channels, the hydrophobic material can be applied to the top and/or base substrates such that when the three substrates are sandwiched together, the hydrophobic stop material forms a feature on the top and/or base surface of said channel (the walls of which are formed by the middle layer). Alternatively, or additionally, a small one-way vent may be provided adjacent to or within the channel, which vent is capable of allowing air to be vented out of the cartridge or into a void in the cartridge, but not allowing air or liquids to enter the cartridge. said microfluidic channel(s). Liquid entering the cartridge by capillary action will fill to but not beyond the vent without additional force being applied.

[0054] In an embodiment where multiple channels are provided in order to perform separate and/or repetitive tests, a fluid stop feature may be provided in each channel downstream of the sample inlet port. In this way, a sample would initially enter the cartridge through the sample inlet port, but would be prevented from filling the length of each microfluidic channel by a fluid stop feature. In order to initiate each assay, the sample must be actively pulled past the fluid stop and along each microfluidic channel in order to bring said one or more reagents into contact, drawing gas back into said/each gas chamber. . Advantageously, this ensures that each assay can be started at the same time or at different times as required, and also serves to minimize problems that may arise due to sample differences, such as blood hematocrit values and consequently viscosity differences, for example.

[0055] It is also preferred that a stop feature be located upstream of the fluid waste collector, when present, so that the sample, upon initial application, does not flow into the waste chamber. When sufficient force is applied to said/each gas-filled chamber in order to actively push the liquid sample within said/each channel(s), liquid can pass the stop feature upstream of the fluid waste collector and enter the waste collector. This stopping feature may also be designed in such a way that, although it prevents fluid from entering the collector upon initial contact, the sample may eventually wet over this stopping feature and flow into the collector, but only once the sample has filled the sample channels. Once these channels are full, the capillary force on the stop feature increases and excess sample can flow over the stop feature and into the collector. In this way, the collector can act as an overflow for applying excess sample and the fluid stop feature can act as a timing gate, controlling this liquid movement. In other embodiments this stopping feature is not required. The collector can be filled with sample and act as a reservoir from which the sample can be drawn by reducing the force on the gas-filled chamber(s) in order to transfer that sample from the collector to the sample channel(s).

[0056] In one embodiment, the waste collector is designed to be an area of cartridge voids into which used fluid/sample, or fluid that is not required or considered undesirable, can be evacuated. For example, whole blood contains many proteins and other agents that can interfere with assay reactions and/or detection of captured analyte through fluorescence detection, for example. The present invention allows the initial binding and/or reaction of any analyte to be carried out in the sample (e.g., whole blood), but all or substantially all of the unbound material present in the liquid sample and the liquid remaining following the reaction may subsequently be be evacuated from the detection zone, optionally to the waste chamber, allowing further reactions and/or detection to be carried out in an environment substantially free of liquid or gas.

[0057] However, as mentioned previously, it may not be necessary to include a waste collector. Advantageously, the present inventors have observed that gas which is expelled from said/each gas chamber upon application of a force to said/each gas chamber is sufficient to push/transport the liquid sample and unbound/unreacted material out of the zone. detection. Thus, only captured, bound or immobilized material is retained in the detection zone in a substantially liquid-free environment, and advantageously detection of any such material is easily conducted.

[0058] As well as each/said microfluidic channel(s), the cartridge of the present invention may comprise one or more electrode features that contact said channel(s) and , consequently, the sample once introduced into the cartridge. The electrodes are designed to make contact with electrical contacts on the reader, allowing a variety of readings to be taken where appropriate. For example, one or more electrodes on the cartridge may be designed to detect correct loading of the cartridge and the reader may signal to the user whether or not the cartridge was a) correctly inserted into the reader and/or b) the sample loaded into the cartridge correctly, e.g. example, for a fluid stop feature. The electrode(s) may also perform one or more electrical measurements on the sample itself. For example, when the sample is a whole blood sample, the electrode(s) may conduct a hematocrit measurement of the sample, which may be important in determining an accurate concentration of the analyte to be detected. Conductivity and/or impedance measurements can be determined depending on the sample being studied. Thus, the cartridges of the present invention can not only detect whether or not an analyte is present in a sample by binding/reacting with any analyte, but electrical measurements on the sample can also be conducted. The electrode(s) can also be used to confirm that removal of the sample from the detection area by gas expelled from the gas-filled chamber has occurred correctly, as there will be a significant change in conductivity which is being detected when a liquid is present or absent. Electrodes can also be provided to the gas-filled chamber in order to signal the degree of compression of each/said chamber.

[0059] The sample to be applied to the cartridge can be any suitable liquid sample. It may, for example, be a fluid sample obtained from a subject, such as whole blood, plasma, saliva, semen, sweat, serum, menstrual flow, amniotic fluid, tears, a tissue swab, urine, cerebrospinal fluid, mucosal sample and the like. It should be realized that the assay systems of the present invention can be applied in the area of human healthcare, including large and growing IVD markets (e.g., cancer, cardiology, drugs of abuse detection, and infectious disease, including bacterial, fungal, and viral). Assays can also be used to test drugs and drug action. However, the system can also be applied in environmental scenarios where it is desirable to detect, for example, toxic agents or infectious agents such as bacteria, fungi or viruses. Thus, samples from rivers or lakes or swabs from solid surfaces can be taken in order to obtain a liquid sample to feed into the cartridge. The test systems can also be used for veterinary applications. Essentially any assay in which a sample can be provided in a liquid form or rendered into a liquid form can be used in the present invention, for example, a breath sample can be obtained in a liquid and the liquid used in accordance with the invention. Swabs can also be taken from surfaces and placed in a liquid to provide a liquid sample.

[0060] The sample may, for example, include materials obtained directly from a source, such as a whole blood sample, as well as materials pretreated using techniques such as filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of agents interference, etc. These steps can be performed before the sample is introduced into the cartridge or can be performed by the cartridge itself.

[0061] The sample can be introduced before the cartridge is inserted into the reader device or after the cartridge has been inserted into the reader. In some embodiments, the cartridge will be inserted into the reader device before the sample is applied and a force applied to the gas-filled chamber in order to expel gas from said/each chamber. This can effectively make the cartridge ready for sample application. Reducing the force applied to said/each chamber will draw gas back into the chamber and in turn will pull the sample into and along said/each microfluidic channel(s). The cartridge may also be designed so that the sample can initially be introduced via capillary action. In this way, a stop feature, as previously described, can be provided to limit sample ingress into said microfluidic channel(s). Further transport of the sample as a result of the expulsion or introduction of gas from/into said/each chamber(s) filled with gas. In order for the sample to initially be introduced by means of capillary action, it is necessary that gas, which is present in said microfluidic channel(s), be displaced by the sample. This can be achieved through a valve or similar that exits the microfluidic channel to the outside of the cartridge. In one embodiment, the valve is a one-way valve that is designed only to allow gas to exit the cartridge and not to allow a gas or liquid to be introduced into the cartridge.

[0062] The valve may be a small hole or slit, for example, positioned adjacent to or in close proximity to a hydrophobic stop feature designed to prevent further transport of the sample within said/each microfluidic channel(s) by capillary action alone. Each valve may be in fluidic communication with said/each microfluidic channel through a channel of smaller size than said/each microfluidic channel itself (such as less than 50%, 25% or 20% of the width of said/each microfluidic channel itself). microfluidic channel). In use, when the sample is removed from the detection zone, along said/each microfluidic channel, following the reaction process taking place, the sample is favorably directed to the sample inlet port and/or waste collector. fluid when present, rather than toward the valve, by virtue of the dimensions of said/each microfluidic channel being larger than the channel connecting the microfluidic channel to the valve. Additionally, upon initial sample application, a small amount of sample can fill the smaller channel and act as a barrier for fluid to flow further between the valve and the microfluidic channel after sample application. Without being bound by any theory, it is expected that the barrier is caused by the relatively greater capillarity of the smaller channel compared to the/so-called larger main microfluidic channel. The small amount of sample may remain in the smaller channel and in effect seal the valve following capillary filling. In this way, the valve has an effect only during initial capillary filling, and then liquid transport in the cartridge is effected or controlled by gas being drawn into or expelled from said/each chamber.

[0063] In a further aspect there is provided a valve system for use in accordance with the present invention, the valve system comprising: a vent or slit opening in a top or base surface of a test system in accordance with present invention; and a microfluidic channel of smaller size than the/said microfluidic channel of the test system, which smaller microfluidic channel is in fluidic communication with the vent or slide opening and the/said microfluidic channel of the test system.

[0064] Conveniently, the valve system is positioned so as to be adjacent to a capillary stop of/said microfluidic channel, so that, upon the sample being introduced into the assay system, the sample fills by capillary action up to the capillary stop. and a portion of the sample also at least partially fills the smaller microchannel. The portion of the sample that at least partially fills the smaller microchannel acts as a barrier to further transport of fluid along the smaller microchannel and export of fluid through the vent or slit.

[0065] Desirably, a cartridge of the present invention can be designed to conduct a plurality of assays (repetitions of the same assay and/or different assays) on a single liquid sample. The dimensions of the cartridge and associated channels are such that all such assays are ideally performed on a liquid sample, such as a blood sample obtainable by a finger prick, that is less than 100μL, 50μL, such as less than 40μL, 30μL or even 20μL or less. In this way it is possible to conduct an assay or assay within a single channel of a cartridge that employs less than 10μL, such as less than 7μL, 5μL, or even 2μL or less, of liquid sample, such as blood. This is considerably less than required for analysis performed in hospitals using larger benchtop analyzers or other known point of care platforms.

[0066] The analyte to be detected can be any desired analyte and can include proteins, peptides, antibodies, nucleic acid, microorganisms (such as bacteria, fungi and viruses), chemical agents, biochemicals, toxins, pharmaceuticals, enzymes, metabolites , cellular fractions, antigens and the like. For example, the present system can be adapted to detect any type of analyte that can bind to a suitable binding agent, or react with a suitable reagent(s), the product of which is capable of being detected and optionally bonded to a suitable binding agent. The binding agent can be any suitable agent that is capable of specifically binding to the analyte or reaction product to be detected. For example, if the analyte is a protein or peptide, the binding agent may be a receptor or antibody that is capable of specifically binding to the protein/peptide. Instead, an antibody can be bound by a protein/peptide that the antibody is specifically designed to bind to. Nucleic acids can be linked by other nucleic acids that are capable of specifically hybridizing to the nucleic acid of the analyte. Microorganisms can be bound by antibodies that specifically bind to proteins on the surface of the microorganism. Chemical agents, toxins, pharmaceuticals, metabolites can be linked by chemical moieties that are capable of reacting or binding to the above-mentioned chemical analytes through appropriate binding reactions, or affinities. Many types of bonding techniques are well known to those skilled in the art.

[0067] Furthermore, said reagent can be an enzyme or an enzyme substrate. For example, analytes such as glucose through well-described enzymatic methodologies can be detected, as a reaction product formed following the enzyme reaction with glucose, which can be detected using electrochemical, or optical detection techniques known to those skilled in the art. . Such measurements can be made as stand-alone measurements or in combination with other analytes to be detected in the sample.

[0068] It should be noted that reference here to analyte/analyte-binding agent complexes includes complexes in which the analyte is unmodified from its form observed in the liquid sample, or where the analyte has been modified through reaction with an additional reagent and , consequently, can be considered a reaction product of the analyte.

[0069] The binding agent may itself be affixed directly or indirectly to a wall or surface of said microfluidic channel(s) in the cartridge, by suitable attachment to the wall or surface, e.g. through physical adsorption, covalent chemical coupling, non-covalent chemical bonding (e.g., biotin-avidin), or a combination of any of the foregoing. In a preferred embodiment, the binding agent is in the form of a magnetic or paramagnetic particle, comprising a binding moiety, and the binding moiety may be linked directly or indirectly, for example, by non-covalent chemical bonding (e.g., biotin-avidin ) to the particle surface. Additional modalities may also include physical adsorption, covalent chemical coupling, non-covalent chemical bonding (e.g., biotin-avidin) or any combination thereof to the surface of a magnetic agent, such as a magnetic particle. The magnetic agents/particles which are functionalized to comprise the binding agent attached thereto may simply be deposited within said microfluidic channel(s) of the cartridge, so that upon the sample being applied to the cartridge and being drawn into and along said channel(s), the functionalized magnetic agents/particles are resuspended by the liquid sample and consequently come into contact with any analyte in the sample. The deposition area or areas for binding and/or other reagents may be specifically defined using hydrophobic stop or other features at either or both ends of the deposition area, by the previously described techniques in order to optionally separate such area or areas. of the detection area/zone. Where appropriate, this can ensure that high background readings are not obtained due to reagent components (e.g. fluorescent latex particles) being dried in the detection/measurement zone area.

[0070] As also mentioned previously as the binding agents, the cartridge may and/or alternatively comprise one or more reagents deposited within said microfluidic channels(s), which reagents may facilitate detection of the analyte or captured analyte. For example, said one or more reagents may include a label that has been adapted to specifically bind to the analyte, thereby facilitating its detection, or an enzyme that reacts with an analyte in order to generate an analyte reaction product. Thus, in accordance with the present invention, the assays described herein can be used to detect an analyte or an analyte reaction product thereof.

[0071] Bound analyte can be detected directly by providing the bound analyte that is capable of generating a detectable signal, or upon binding of the analyte a reaction can occur, in order to generate a reaction product and the reaction product can be detected . However, in a preferred embodiment, bound analyte is contacted with a label that is capable of binding the bound analyte and a label/binding agent/analyte complex is subsequently detected. The label can itself be linked to an additional binding moiety that is also capable of specifically binding to the binding agent/analyte complex. Typically, the label is capable of binding to a different portion of the analyte to which the first binding agent binds, or is capable of binding to a region of the binding agent/analyte complex that is formed only upon generation of a complex such as that.

[0072] Bound analyte can be transported to the label within a different region of the microfluidic channel by means of gas being drawn back into the fluid-filled chambers, which as a result pulls the fluid sample further along the channel. microfluidic in the direction of said/each gas-filled chamber.

[0073] Desirably, the binding agent and any detection/labeling agent are in a dry state when deposited in the microfluidic channel(s) of the cartridge, so that they are capable of long-term storage and are reconstituted by the liquid sample by means of the liquid sample flowing into and along the microfluidic channel(s).

[0074] In one embodiment, the binding and/or detection/label agent that is designed to facilitate detection of the analyte, is initially located downstream (in terms of the direction, the sample flows into the cartridge following introduction) of a first stop feature. In this way, said binding agent and/or detection agent does not initially come into contact with the sample upon application of the initial sample and capillary filling into the cartridge. Only when a force applied to said/each gas-filled chamber is reduced and gas is drawn back into the gas-filled chamber, the sample is further pulled along said/each microfluidic channel and brought into contact with the binding agent and /or detection agent.

[0075] In one embodiment, transport of the sample along the microfluidic channel may occur in a plurality of stages. For example, following initial sample application and capillary filling, the sample may be drawn along a first portion of said/each microfluidic channel, by a controlled reduction in the force being applied to said/each gas chamber and gas being drawn back into said/each gas-filled chamber in a controlled and precise manner. The first portion of said/each microfluidic channel may comprise said binding agent, for example. Thus, introducing the fluid sample into the first portion allows the binding agent to react with any analytes that may be present in the liquid sample. Then the sample and binding agent may be drawn into a second portion of said/each microfluidic channel, by a further controlled reduction in force being applied to said/each gas chamber, so that more gas is drawn into said/each. gas-filled chamber which, in turn, draws the sample and binding agent into the second portion of said/each channel. An additional reagent or label, for example, may be present in the second portion and the sample and binding agent come into contact with it. In this way, numerous separate steps or stages in relation to a particular assay can be easily carried out and each step/stage may require a period of time that is different from the other. It should be realized that more than two stages, such as three, four or more stages can easily be considered, with each stage being effected by a further controlled reduction in force as applied to/each gas-filled chamber. Advantageously, each gas chamber can be independently controlled. In this way, it is also possible for a plurality of different types of assay to be conducted using a single cartridge of the invention. In this way, each separate channel is provided with the reagent(s) necessary to conduct a particular assay or assays and the reader is programmed to perform the required number of gas chamber compression/decompression steps for each private rehearsal. In this way, the cartridges and associated reader of the present invention are capable of conducting, substantially simultaneously, numerous different discrete assays that may require different reagents, reaction time periods, numbers of steps, etc.

[0076] Although the foregoing description discusses the pulling or pushing of the liquid sample in a stepwise manner, it will be appreciated that due to the controllable nature of the force control means, it is possible to reversibly compress and decompress a gas-filled chamber to time or degrees variables, such as to allow for the pulling and pushing of the liquid sample at any point in time and to allow for a mixing effect. Thus, for example, when the liquid sample is transported to a region of the microfluidic channel that includes one or more reagents that is/are to be reconstituted by the liquid sample, upon reaching the area in which said one or more reagents are deposited, the liquid sample may be pushed and pulled back and forth using small compressions/decompressions of each/said gas-filled chamber(s), for a period of time, to facilitate reconstitution and/or mixing of said one or more reagents in the sample liquid.

[0077] Necessary control and implementation of the methods and assays of the present invention can be facilitated by the use of a suitable microprocessing device and associated software in the reader.

[0078] In another embodiment, after the initial binding phase between the sample and the binding agent, such as a magnetic particle, the binding agent-analyte complexes formed in the liquid sample can be transported to a region downstream of the channel, where the label is located in dry form within the microfluidic channel. The liquid sample resuspends/rehydrates the label and allows binding of the label to the binding agent-analyte complexes. This transport of the liquid sample and any reconstituted material is by virtue of decompression in each/said gas-filled chamber which draws gas back into each/said gas-filled chamber. Drawing gas/air back into each gas-filled chamber causes a vacuum effect that serves to pull the liquid sample along said microfluidic channel. This method can allow greater control of rehydration of deposited reagents and homogeneity of reagent dispersion.

[0079] In another embodiment, the binding agent and the label are deposited in the same region of said/each microfluidic channel. The sample rehydrates these reagents substantially at the same time allowing the binding and labeling reactions to occur at the same time. In this embodiment all reagents can make contact with the sample, the reader then accumulates the analyte-magnetic particle label complexes within the detection area through the application of a magnet/electromagnet. Unlike other prior art devices, the magnet/magnetic force can be designed in such a way as to simply accumulate or concentrate the magnetic particles in the detection zone. Thus, the magnet/magnetic force may not serve to pull or move the magnetic particles longitudinally along said/each microfluidic channel, but instead, concentrate and maintain any complexes in one area of the detection zone. In one embodiment, the magnetic particles may be initially deposited within the microfluidic channel at a location that is opposite to where the magnetic force is to be applied. For example, magnetic particles may be deposited in or along the base of a channel and the magnet or magnetic force must be brought into contact/applied to the top surface of the cartridge. In this way, the magnetic particles will be pulled laterally (or perpendicular to the liquid sample flow within the channel) through the channel on application of the magnetic force. The process of actively pulling magnetic particles through the liquid sample is expected to increase the number of possible capture events that can occur between the functionalized magnetic particles and analyte that may be present in the liquid sample.

[0080] In one embodiment an electromagnet is provided which is positioned to be in line with the detection zone of the cartridge once correctly inserted into the reader. Particle-analyte-label complexes can be pulled into the detection zone by controlled fluid movement and only once in the detection zone is electromagnetic force applied. Additionally, the electromagnet can be adapted to provide a magnetic field focused on the detection zone. This may serve to concentrate the particle-analyte-label complexes in a defined portion of the detection zone, rather than throughout the detection zone. Alternatively to the magnet being present in the reader, it is possible to provide a magnet or appropriate electromagnetic field generating circuit system within the cartridge itself. For example, the circuit system that generates the electromagnetic field may be positioned adjacent to the detection zone and include electrical connectors or the like that are/are intended to make contact with corresponding connectors on the reader. Once connected together, the reader is capable of providing the electrical signals necessary to generate the electromagnetic force.

[0081] In order to facilitate detection of bound analyte, it may be desirable to remove spent liquid sample from said/each detection area in which bound analyte is to be detected. When required, the present invention achieves this by using the gas that is present in said/each gas-filled chamber by removing/pushing the spent or reacted liquid sample from said/each detection zone of the microfluidic channel, and, when present, toward to and from the waste chamber. The bound analyte, such as a magnetic particle-analyte-label complex can then be detected and/or quantified in a substantially liquid-free or substantially gaseous environment. It should be understood that the bound analyte may still be “wet,” that is, there may be some residual liquid coating surrounding or otherwise associated with the bound analyte, but the bound analyte, as understood by those skilled in the art, reader is not present in a bulky liquid. For example, the bound analyte may remain hydrated (e.g., is not considered to be in a “dry” state) during detection even though it is not present in a bulk liquid.

[0082] Advantageously, the present invention, through careful control of the movement of gas in and out of said/each gas chamber is capable of precisely controlling the rate of movement of liquid along each channel, in any direction. For example, it may be desirable for the reconstitution of dry reagents that are deposited within said/each channel to occur quickly, but the removal of liquid sample and unbound material after any necessary reaction(s) occurs, occurs relatively slowly. Thus, the reader and associated force control means are capable of varying or altering the speed of gas expulsion/intake into said/each gas chamber, which has a corresponding effect on the speed/rate of liquid movement in said/each gas chamber. each channel. Different assays may require different speeds of reconstitution and/or liquid removal and this may also be independently controlled by force control means in combination with programming or associated software.

[0083] Furthermore, through fine control of the force control means, it is possible to carefully control very small volumes of gas expulsion/intake into said/each gas chamber, with a corresponding small movement of the liquid sample. For example, it is possible that the volume of gas that is expelled or introduced into said/each gas-filled chamber is in increments of less than or equal to 500nL, such as less than or equal to 200nL, 100nL, or even 50nL, 25nL, or 15nL, 10nL or even less. Such small volumes of gas movement result in correspondingly very small linear movements of the liquid in said/each channel. In embodiments of the invention where detection is carried out in a substantially liquid-free environment, the inventors have found that it is possible to use such very small volumes of gas to remove liquid sample and/or uncaptured material just from the detection zone, or even portion of the same and, consequently, provide the captured analyte or analyte reaction product in a substantially liquid-free environment in which bulk liquid and uncaptured material have been removed by the gas. This is very different from what would be considered a conventional washing step in the art, which would use large volumes of a fluid, typically a liquid, to wash the detection zone of bound sample/analyte, etc., before performing the detection step. . In fact, the use of air in the present invention may not be considered a wash, but instead simply removal of the liquid sample and material not captured therein. Thus, where liquid sample and/or uncaptured material needs to be removed from the detection zone, the present invention is capable of using a volume of gas that is substantially equivalent (or just slightly greater, e.g., 15nL, 25nL, 50nL, 100nL or 200nL) in volume to the volume of the detection zone or portion thereof where detection occurs, as this is sufficient to remove the liquid sample from the detection zone or portion, leaving the analyte or analyte reaction product in a substantially free of liquid. In a conventional washing step, many wash volumes compared to the sample volume would generally be required.

[0084] Furthermore, in general, only a small proportion, such as less than 50%, 40% or 25% of the volume of said/each gas chamber(s) may be required for control of liquid sample transport in said/each channel and/or removing liquid sample from each/said detection zone, when required.

[0085] Each cartridge can be designed to perform single analyte detection or multiple analyte detections. Furthermore, each cartridge can comprise more than one microfluidic channel system, so that more than one assay can be performed using a single cartridge. It is also possible to perform more than one test per microfluidic channel. In this way, each cartridge may be capable of performing many repetitions and/or distinctly different assays of a single liquid sample, as said/each gas chamber is independently controllable.

[0086] Desirably, the cartridges can be easily mass produced. The cartridge may be provided in a strip, wherein a number of cartridges are initially connected together, for example, such as by means of a perforated seal. This way, the user can easily remove a cartridge from the strip before use.

[0087] Once the cartridge has been loaded with a sample, any captured analyte can be detected using a suitable optical device or other means present in the reader device. The present invention provides such a reader and an important aspect of the present invention is the provision of at least one force control means which is/are present in the reader and which is/are designed to control the force applied to an external surface of said one or more gas-filled chambers, so as to expel/introduce gas from/to said/each gas-filled chamber. A reduction in the force applied by the force control means will result in gas being drawn back into said/each gas-filled chamber. An advantage of the present invention is that the cartridges can themselves be initially “dry”, that is, contain little or no bulk liquid in the cartridge prior to sample application. This not only simplifies the manufacture of the cartridges themselves, but also improves shelf life and allows many of the cartridges of the present invention to be stored at room temperature, with little degradation of the chemical or biological components in the cartridge, prior to use.

[0088] In a further aspect, there is provided a reader device for use with a microfluidic system of the present invention, the reader device comprising: force control means for controlling compression or decompression of a gas-filled chamber of the microfluidic system; and detection means for enabling detection of a desired analyte in a liquid sample introduced into the microfluidic cartridge, or analyte reaction product thereof; wherein the force control means comprises a piezoelectric bending actuator that is designed to directly or indirectly effect compression or decompression of the gas-filled chamber through displacement of the actuator.

[0089] Pierre Curie discovered the piezoelectric effect in 1883. He noticed that certain materials, such as quartz crystals, produce a voltage when they are mechanically stressed.

[0090] On the contrary, these material shapes are deformed when a voltage is applied to them. As a result, they can be used as transducers, which convert an electrical signal into mechanical vibration.

[0091] Several materials have piezoelectric properties; the most commonly used being Lead Zirconate Titanate (PZT). Modification of the manufacturing process and chemical composition of the ceramic can alter the performance of the piezoflexor. When the PZT layer is joined to a specific substrate sheet (e.g. a thin metal sheet), any electrical activation of the PZT sheet leads to a plane movement of the sheet relative to the substrate and thereby induces an internal mechanical tension resulting in in a bending movement of the composite structure similar to a thermobimetal

[0092] Piezoflexors are well known in the art. Typically, a piezo ceramic crystal can be coated with silver on both sides and bonded to a brass, nickel alloy, or stainless steel strip.

[0093] The ceramic can be configured with or without feedback. Feedback can be used in conjunction with an external circuit to monitor the operation of the piezoflexor and adjust the input signal to maintain a consistent output frequency.

[0094] Flexors can be produced in a wide variety of geometries, cut from a PZT bilayer or multilayer structure. The piezoflexors of the present invention may take the form of strip-shaped flexors. For strip-shaped flexors, one end of the strip is mounted fixed, with the other end moving freely: For this assembly, the maximum displacement of a strip-shaped flexor is obtained and the specified data for displacement, stiffness and resonance refer to this situation. The displacement depends on the length of free movement of the strip. Typically approximately 5-10% of the total flexor length is provided for assembly purposes. Assembly can be done by stapling or using adhesives such as epoxies, cyan acrylates, etc.

[0095] The piezoelectric flexor may be initially polarized, or cause an associated foot or finger to come into contact with an external surface of a gas-filled chamber. In this way a maximum force as applied to the gas chamber can be provided initially, which causes gas to be expelled from the gas chamber. Upon suitable electrical signaling, the piezoelectric flexor can be induced to flex away from the outer surface of the gas chamber, resulting in a decrease in the force applied to the chamber and a resultant drag of gas into the chamber. Since each gas chamber is in fluidic communication with said microfluidic channel(s), those skilled in the reader technique easily understand how the gas that is expelled or drawn into a respective gas chamber causes a movement corresponding direction on the liquid sample in the respective microfluidic channel.

[0096] The reader may include a receiving hole into which the cartridge is to be inserted. The reader can be adapted to ensure correct insertion of the cartridge and this can take a variety of forms. For example, the cartridge may initially be located in a support mechanism that enters the reader, such as may be found in computers for loading CDs and the like. Alternatively, the receiving hole may be sized to allow the cartridge to be received and an internal stopping member may be found in the reader which the cartridge supports once correctly inserted. Additionally, or alternatively, features found in or cut into the surface of the cartridge can be designed for co-location with features found in the reader, and only once the cartridge is correctly located in the reader can the cartridge be controlled by the reader. Different sized receiving holes may be provided, or a single receiving hole suitably shaped to accept different sized cartridges that are designed to perform a specific number of assays, for example.

[0097] The reader can be configured, possibly through appropriate software, to perform a variety of different types of assay. The user may be provided with a kit comprising assay cartridges and optionally sample collection devices. The cartridges may comprise a bar code or other surface features that the reader device is capable of determining, which may serve to inform the reader of the type of cartridge that has been inserted into the reader and, accordingly, which assay or assays are to be conducted. and, consequently, how the reader supposedly works and/or to provide details to the patient, for example. In this way, a single type of reader can be provided that is capable of receiving a variety of different cartridges that can conduct different assays and/or assay panels.

[0098] In the embodiment where the binding agent is attached to the surface of magnetic agents, such as magnetic microspheres, it is understood that the reader will comprise a permanent magnet or electromagnet. The magnet will be designed to be in close proximity to the magnetic agents, or the electromagnet induced to apply a magnetic field in order to concentrate and maintain the magnetic particles in a particular area of said microfluidic channel of the cartridge. This area can be the detection area. In one embodiment, an electromagnet is employed, which is turned on only once the magnetic particles have been transported to the detection zone. Through appropriate design it is also possible to control or focus the magnetic field of the electromagnet to ensure that the magnetic particles are focused and maintained in a small region of the detection zone. This can serve to accumulate the magnetic particles in a small area and increase the signal that can be detected.

[0099] Concentration of magnetic particles in a particular area can serve to facilitate detection of any captured analyte and/or increase detection sensitivity. Furthermore, trapping the particles by means of the magnetic field also allows the undesirable/used fluid sample surrounding the bound analyte to be removed by gas expelled from said/each gas-filled chamber(s), thereby leaving the captured analyte free of potentially interfering agents/contaminants that may be present in the initial sample. The permanent magnet or electromagnetic field can be reduced or increased, such as by moving a permanent magnet closer to, or further away from, the cartridge, or increasing or decreasing the intensity of the applied field. This may serve to allow the magnetic particles to “relax” or become less concentrated in a particular location, whilst still being held at a certain point by the magnetic field or otherwise. This can further facilitate reactions to be carried out on the particles, which can be conducted more efficiently compared to if the magnetic particles were densely concentrated. It may also be preferred in certain applications that detection is performed when the particles are less “concentrated” or relaxed.

[00100] In use, the magnet can be used to retain any bound agent once the magnetic field has been applied to the sample. Gas may be expelled from said/each gas-filled chamber in order to transport the liquid sample and any unbound components present in the sample out of said/each detection area and/or to allow other reagents such as a detection agent to be placed in contact with the captured analyte. Careful control of the speed of movement of gas and a corresponding liquid sample and removal of any unbound components is necessary in order to ensure that the force of the gas is not sufficient to pull out the magnetically bound material. Thus, the speed of gas being expelled from said/each gas-filled chamber can be carefully controlled. In certain embodiments it may be desirable to pull the liquid sample and reagents, etc., beyond the detection zone before applying a magnetic field/force. Thus, any capture of the magnetic particles occurs only once, the liquid sample is pushed back through the detection zone by the gas being expelled from the gas chamber.

[00101] In another embodiment, magnetic particles can be coated in a binding reagent designed to remove interferences from the sample. The magnetic particles would bind this interferent present in the sample and the magnetic particles can then be kept in a specific location separate from specific capture/detection reagents and/or detection zone to allow the reaction to take place and be measured in the absence of the specific interferer(s).

[00102] The reader of the present invention further comprises detection means for detecting any analyte captured in the sample cartridge. The detection means may be any suitable means depending on the particular assay. For example, the detection means may be a fluorimeter or spectrophotometer, which may be used to detect a fluorescent signal, once appropriately excited, from the labeled or unlabeled bound analyte or reaction product. The bound analyte/reaction product may fluoresce naturally once light of an appropriate wavelength has been used to excite the analyte/product, or an additional label may be used to separately bind the bound analyte and the label detected by means fluorescent. Other labels that may be employed, and consequently detection means adapted in this manner, include radiolabels, phosphorescent labels, colloidal metal particles, bioluminescent labels, colorimetric labels, electrochemical labels and the like. Furthermore, as mentioned previously, the analyte or reaction product thereof, or bound analyte or reaction product itself can be directly detected using techniques such as Raman spectroscopy and the like. In some embodiments, the detection means are designed to optically detect the analyte or analyte reaction product, or a captured analyte/analyte reaction product and/or label affixed to any of the aforementioned fractions.

[00103] Detectable labels can be used alone, or together with a microparticle or microsphere, such as a metal oxide, polysaccharide or latex particle. Many types of latex and other particles are known in the art.

[00104] The reader comprises force control means comprising one or more previously discussed piezoflexors for contacting said/each gas-filled chamber of the cartridge and reducing or increasing a force applied to said/each filled chamber(s). s) of gas, increasing or decreasing the flexure formed by the flexor. Where more than one gas-filled chamber is provided, a separate independently controlled piezoflexor may be provided for each gas-filled chamber. The force control means may include a finger or foot that is designed to contact and apply force to a surface on the outside of said/each chamber. In this way, the piezoflexor acts on the finger or foot, so that the finger/foot acts on the gas-filled chamber. In use, before the force control means makes contact with a surface on the outside of said/each chamber, the chamber will be in a maximum volume, gas-filled state. Upon contact with the surface of said/each chamber filled with gas and a force being applied, gas within said/each chamber will be expelled. Increasing the applied force will result in additional gas being expelled from said/each chamber. Conversely, a reduction of the force applied to said/each gas-filled chamber by the force control means will result in gas being drawn back into said/each chamber.

[00105] The finger/foot may be designed to make contact with a central portion of a surface on the outside of said/each chamber. Typically, the finger/foot may be in contact with only a portion of the total outer surface of said/each gas-filled chamber. For example, in use, the finger/foot may contact the top surface of a cartridge and be sized to contact between 10 and 50% of the top surface area overlapping the gas chamber. The finger/foot that is in contact with the surface of the cartridge is raised and lowered, or forced into contact and relaxed from the surface of the gas chamber using the piezoflexor as described. Speed and degree of bending and, consequently, action of the force control means can be carefully controlled in order to be able to control the speed and quantity of gas that is expelled from or drawn into said/each gas-filled chamber.

[00106] The reader may include other features, such as a heating device to allow assays to be conducted at a particular temperature, as well as appropriate electrical circuitry and software to allow the reader to be programmed to perform one or more different assays. .

[00107] In a further aspect, a testing system is provided comprising an independent microfluidic system and an associated reader device, wherein: the independent microfluidic system comprises: a sample inlet port for receiving a liquid sample to be tested, the sample inlet port connected to at least one microfluidic channel, wherein each microfluidic channel(s) comprises one or more reagents deposited therein for use in conducting of an assay and a detection zone for use in detecting any analyte that may be present in a sample or analyte reaction product; and each/said microfluidic channel(s) is/are in fluidic communication with a gas-filled compressible chamber downstream of each/said detection zone, wherein the microfluidic system is formed of three layers, which are sandwiched together in order to define each/said microfluidic channel(s) and said gas-filled chamber, and wherein compression or decompression of said chamber causes gas to be expelled or drawn into the chamber, which, in turn, causes movement of the liquid sample within said/each microfluidic channel; and a reader device for use with the microfluidic system, the reader device comprising: force control means for controlling compression or decompression of the gas-filled chamber of the microfluidic system; and detection means for enabling detection of a desired analyte in a liquid sample introduced into the microfluidic cartridge, or analyte reaction product thereof; wherein the force control means comprises a piezoelectric bending actuator that is designed to directly or indirectly compress or decompress the gas-filled chamber through displacement of the actuator.

[00108] In a further aspect, a method is provided for conducting an assay on a liquid sample, the method comprising: a) providing a microfluidic system as described here to a reader device as described here; b) compressing one(s)/said gas-filled chamber(s) of the microfluidic system, in order to expel gas from said/each gas-filled chamber(s); c) introducing a liquid sample to the microfluidic system and allowing the sample to be drawn into said/each microfluidic channel(s) by capillary action, and/or partially decompressing said/each filled chamber(s) of gas such that the gas is drawn into said/each chamber(s) thereby causing the liquid sample to be drawn into said/each microfluidic channel(s); d) allow one or more reagent(s) to react with any analyte present in the liquid sample; e) optionally partially further decompressing said/each gas-filled chamber(s) of the microfluidic system, such that the liquid sample is further drawn along said/each microfluidic channel(s) toward to said/each gas-filled chamber(s) and optionally contacting the liquid sample with an analyte binding agent and/or one or more additional reagent(s); f) optionally capturing any analyte or analyte reaction product and compressing said/each gas-filled chamber(s) such that the gas expelled from said/each chamber(s) causes the sample to be liquid and uncaptured material is pushed away from any captured analyte or analyte reaction product; and g) detect any analyte or analyte reaction product, or captured analyte or analyte reaction product.

[00109] It should be noted that step e) of the previous method can be performed as a single or multiple steps. Thus, depending on the tests to be performed, step e) may be a single step so that the decrease in force applied to said/each gas-filled chamber is a single decrease in force and the sample is pulled to a single location in said/each microfluidic channel. Alternatively, there may be multiple steps, such as 2, 3, or 4 steps, where successive decreases in force are applied to said/each chamber, resulting in the sample being pulled to any number of successive locations within said/each microfluidic channel depending on the number of times a decrease in strength is carried out. Thus, the present invention allows tests to be conducted where a single step or multiple steps are required.

[00110] Force as applied to the gas-filled chamber may be provided by force control means as discussed previously comprising a piezoflexor and optionally a finger or foot in association therewith.

[00111] The rate at which an increase or decrease in the application of force in said/each gas-filled chambers can be varied in order to increase or decrease the speed of liquid movement in said/each channel. For example, the decrease in force applied in step e) may be faster than the rate of increase in force applied in step f) when required.

[00112] The capture of analyte/analyte-binding agent complexes can be attributed, for example, to the analyte-binding agent being bound to a surface of the microfluidic channel, or captured by virtue of being magnetic and applying a magnetic force to the complexes formed. . The magnetic particles that are employed to form the complexes may initially be deposited on a surface of said microfluidic channel(s) that is opposite to the surface of the cartridge with which the magnet is placed in close contact. , or the applied magnetic force. The effect of this is that the magnetic particles are pulled laterally through said microfluidic channel(s) perpendicular to the flow of liquid through said channel(s), which increases and /or intensifies contact of the magnetic particles with the analyte or analyte reaction product, thereby increasing the sensitivity of the assay.

[00113] It is possible for more than one embodiment of the previous methods to be performed using a single cartridge. Thus, for example, a method that includes the previous step f) can be carried out in a channel within a cartridge of the present invention and a method that does not include step f) can be carried out in a separate channel. Additionally, or alternatively, step e) can be performed alone or multiple times in the aforementioned channels and/or additional channels. In this way, multiple different types of assays can be conducted using a single cartridge comprising a plurality of assay channels.

[00114] The present invention is further based on the development of an assay system comprising a disposable microfluidic cartridge that is capable of conducting numerous different assays on a single sample and an associated reader that is capable of detecting and/or determining levels of a plurality of analytes from the single sample and provide an output to a user. The present invention also allows a variety of disposable cartridges to be received by the reader, each of said variety of disposable cartridges being capable of performing a distinct panel of different assays. In this way, a single reader can be provided that is capable of being used to provide results from a variety of distinct panels from different assays. In this regard, each cartridge can be specifically adapted to the number and types of assays that can be performed. For example, different sample volumes may be required for particular assays and this can be addressed independently through appropriate sizing of each channel and/or chamber. Thus, by increasing or decreasing the size of any particular channel, it is possible to increase or decrease the volume of sample that is introduced into each particular channel. Furthermore, the size of any chamber that is connected to one or more channels can be increased or decreased as necessary depending on the type of assay, number of steps and/or volume of sample being introduced into said channels. This is easily determined by those skilled in the art.

[00115] Thus, in a further aspect, an independent disposable microfluidic system is provided for use in conducting a plurality of distinct assays, the microfluidic cartridge comprising: a sample inlet port for introducing a liquid sample into the microfluidic cartridge; multiple microfluidic channels; each of said microfluidic channels being adapted to receive a portion of the liquid sample and being capable of conducting one or more assays on said sample portion using one or more reagents that are present within each of said microfluidic channels prior to introduction of the sample. liquid; and wherein liquid movement within each microfluidic channel is independently controllable by compression and/or decompression of two or more gas-filled chambers of the microfluidic system, which chambers are each in fluidic communication with one or more of said microfluidic channels.

[00116] It should be understood that the previous additional aspect of the invention may be in addition to or as an alternative to the aspects and modalities previously described above. Thus, all features described in relation to the previous aspects of the invention may apply equally to the immediately preceding aspect and, consequently, may be included as limiting or optional features.

[00117] It is also possible to carry out tests using the cartridges of the present invention which, in addition to having channels that are in fluidic communication with a chamber or chambers, additionally comprise one or more channels that are not in fluidic communication with any gas chamber or chambers. . A non-limiting example of such an assay is described in the examples section below.

[00118] The microfluidic cartridge of the present invention is independent in the sense that in addition to the sample itself, all other physical reagents necessary to conduct each assay are present in the microfluidic cartridge before each assay process is performed. Therefore, other reagents, such as reactive species, buffers, washing liquids, etc., are not introduced into the cartridge during the assay process. Typically, the only liquid that enters the cartridge is the liquid sample itself. Any reagent that may have been deposited in said/each channel may have initially been applied by means of a liquid, but this will have been dried and the cartridge, prior to conducting any particular assay, may be considered dry with no or substantially no liquid present. .

[00119] Application of heating/cooling and/or magnetic force may be provided to the cartridge of an associated reader, as discussed below, but this should not be considered a chemical reagent.

[00120] It should be understood that multiplex assay in the context of the present invention means that a microfluidic cartridge is capable of not only performing a plurality of assays on a single sample introduced into the cartridge, but that the cartridge is capable of performing a plurality of types of distinctly different trials. For example, each microfluidic cartridge of the present invention is capable of performing at least two, three, four, five or more of the following types of assays: immunoassay, nucleic acid assay, receptor-based assay, competition assay, cytometric assay, colorimetric test, enzymatic test, electrophoretic test, electrochemical test, spectroscopic test, chromatographic test, microscopic test, topographic test, calorimetric test, turbimetric test, agglutination test, viscometric test, coagulation test, clotting time test, synthesis test protein, histological assay, culture assay, osmolarity, chemical, biochemical, ion, gas, or absorption assay. In certain embodiments a particular type of assay may be performed in order to detect different analytes. For example, more than one immunoassay can be performed in order to detect different analytes. Said more than one immunoassay can be performed in a single and/or multiple microfluidic channels.

[00121] In one embodiment, a microfluidic cartridge of the present invention is designed to conduct a panel of assays related to a particular disease or condition. Exemplary test panels may include panels testing cardiac conditions, adrenal gland condition, renal function, liver function, neurological function, diabetes, pregnancy and pregnancy conditions, a metabolic condition, and illicit drugs.

[00122] For example, a microfluidic cartridge that is designed to assay for markers associated with cardiac conditions may comprise an assay or assays designed to detect and/or determine a level of one or more of the following: lipid profile - which can detect low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides and/or total cholesterol, for example; Apolipoproteins – the protein component of lipoproteins – are not included in a standard lipid profile but can be tested separately. Abnormal levels can promote atherosclerosis, and may increase the risk of coronary artery disease (CAD) and stroke; homocysteine - is an amino acid (protein building block). High blood levels can promote atherosclerosis and DKA, as well as blood clots that can lead to a heart attack or stroke; troponin; BNP; C-reactive protein (CRP) is a substance that reflects low levels of systemic inflammation and is increased in people at risk for DKA; and cardiac markers such as cardiac enzyme studies measure certain enzymes, such as CK-MB, or troponins, or cardiac hormones such as brain natriuretic peptide, which are released when the heart is stressed or diseased or damaged, such as from a stroke cardiac.

[00123] Subjects who experience stress or other conditions may undergo an adrenal function panel, which may include one or more of the following: Aldosterone controls salt, potassium, and water balance in the body and helps regulate blood pressure. Overproduction (hyperaldosteronism) or underproduction (hypoaldosteronism) of this hormone may be caused by tumors or other abnormalities in the adrenal glands (primary; e.g., adrenal cancer) or may result from problems on the outside of the adrenal glands (secondary); Cortisol is a glucocorticoid hormone that helps control the metabolism of carbohydrates, proteins, and fats; mediates the body's response to stress; and regulates the immune system. Cortisol oversecretion, most often caused by a benign adrenal tumor, results in Cushing's syndrome. Undersecretion may indicate a form of adrenal insufficiency known as Addison's disease. Both blood levels and urine levels (known as Cortisol-free) are normally measured; 18-Hydroxycortisol, a product of Cortisol metabolism, is an unusual steroid produced in excessive amounts in patients with primary hyperaldosteronism. Measuring blood levels of this hormone can help determine whether hyperaldosteronism is caused by a tumor called adrenal adenoma, or by overgrowth (hyperplasia) of adrenal tissue; levels are significantly higher in people with an adenoma; and DHEA-S, or dehydroepiandrosterone-sulfate - a sex hormone (androgen) synthesized by the adrenal gland - is a precursor to testosterone. In women, the adrenal glands are the main, and sometimes only, source of androgens. Elevated DHEA-S levels are associated with virilism (male body characteristic), hirsutism (excessive hair growth), amenorrhea (absence of menstruation), and infertility. Adrenal abnormalities such as tumors can lead to abnormally high levels of DHEA-S.

[00124] Kidney function tests are used to help determine the cause of symptoms such as jaundice that may be due to liver disease. They are also used to screen for potential liver damage, for example in alcoholics or people exposed to the hepatitis virus, and also to monitor changes in abnormal kidney function. Thus, a kidney function microfluidic cartridge of the present invention may include one or more of the following: Enzyme testing: The liver is the site of many biochemical reactions that are controlled by numerous enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase ( AST), alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT). Elevated levels of liver enzymes in the bloodstream may indicate liver damage; however, they do not necessarily point to a specific liver disease. Although enzyme tests can be ordered individually, they provide more information when performed in combination, since levels of many enzymes in the liver can be elevated in diseases that affect other organs; Bilirubin, the main pigment in bile, is a breakdown product of hemoglobin, an iron-containing substance in red blood cells. Normally, only a small amount of bilirubin circulates in the blood. Elevated blood levels can result from many forms of liver and biliary tract disease, including hepatitis and bile duct obstruction. The presence of excess bilirubin in the blood produces a yellowish discoloration of the skin and eyes called jaundice; Albumin is a major protein that, like most proteins in the bloodstream, is synthesized by the liver. A decreased serum albumin level (the liquid portion of blood that remains after whole blood clots) is an indication of chronic liver disease; Prothrombin time (PT) is a blood clotting study that can be performed to evaluate liver function. Because prothrombin is one of the clotting proteins that is synthesized by the liver, an abnormal PT may reflect liver dysfunction; Viral hepatitis tests may be done in people with abnormal liver enzymes whose medical history and/or symptoms raise suspicion of the disease. (Symptoms include low-grade fever, malaise, loss of appetite, and fatigue, but they are not always present.) The three most common types of this virus found in the U.S. are hepatitis A, B, and C (known as HAV, HBV, and HCV); they are all detected by testing for the presence of specific antigens or antibodies found only in the blood of infected individuals. Different antibody/antigen tests may be performed depending on which type of hepatitis is suspected. Furthermore, the presence of particular antibodies can signal whether the infection is in an acute or chronic stage.

[00125] Test panels are often employed to consider a subject at risk of developing diabetes or confirm that a subject has type I or II diabetes. Much like a lipid panel described previously, a diabetes panel microfluidic cartridge can be designed to conduct one or more of the following assays: Complete Blood Count (CBC) tests for blood disorders such as infection or anemia; Fasting glucose is used to detect both hyperglycemia and hypoglycemia, to help diagnose diabetes, and to monitor glucose levels in people with diabetes; Hemoglobin A 1 c can detect prediabetes, diagnose it, or see if diabetes is under control; and Diabetic Urinalysis will determine whether albumin (protein) is found in your urine (if so, it is possible that one of the subject's kidneys is not functioning properly).

[00126] It is also possible to test for illicit drugs or pharmaceuticals that are considered prohibited for use by sports men and women. A microfluidic cartridge of the present invention designed to detect and/or determine a level of an illicit drug in a subject, may be designed to assay one or more of the following: amphetamines; Barbiturates; Buprenorphines; Benzodiazepines; Cocaine; Ecstasy; Methamphetamines; Heroin (Opiates/Morphine); Methadone; Tricyclic Antidepressants; Cannabis and/or other psychoactive agents.

[00127] It will be appreciated that the test panels described above are merely exemplary and should not be considered limiting. In accordance with the present invention, particular test panels may be considered and a cartridge in accordance with the present invention provided in order to conduct the particular test panel.

[00128] Although each panel of assays is conducted within a microfluidic cartridge of the present invention, results of each assay need to be detected and/or determined. This is performed by a reader as described here

[00129] The reader may include a cartridge determination means, which may be a barcode/QR code or similar reader present in the reader that is designed to read a barcode/QR code or other type of code on the cartridge. microfluidic. The code transfers to the reader information regarding the type of microfluidic cartridge and assays to be conducted, so that the reader is prepared to perform and detect/determine the results of the particular microfluidic cartridge. In a simpler embodiment, said reader receiving ports may be designed to accept only a particular type of microfluidic cartridge, much like a lock and key. Thus, each receiving hole can accept only one particular cartridge type whereby introduction of a cartridge into a specific receiving hole instructs the reader which type of cartridge was inserted and the tests to be conducted. A user could also enter details into the reader so that the reader is instructed what test to perform, but this may be less desirable as it may lead to user error.

[00130] The reader of the present invention is constructed so that it is capable of receiving a plurality of different microfluidic cartridges. "Different" is understood to mean that the cartridges of the present invention can be adapted to conduct a particular panel of tests, rather than the cartridges clearly appearing visually different. That is, two cartridges when placed side by side may appear quite similar, but one cartridge may be adapted to perform a panel assay suitable for detecting heart disease and another cartridge may be adapted to perform a panel assay suitable for detecting diabetes. , for example.

[00131] Thus, in a further aspect, a multiplex assay platform is provided for use in conducting multiple panels of assays, the multiplex assay platform comprising a plurality of microfluidic cartridges, each cartridge being capable of conducting a defined panel of assays. in a sample and a reader constructed to be capable of receiving and verifying each of said plurality of microfluidic cartridges, whereby the reader is configurable to detect and/or determine levels of a panel of analytes that may be present in the sample.

[00132] In use, a subject will be predetermined by testing with a particular testing panel, or a patient will visit a healthcare professional, such as a doctor, nurse, or other medical professional, and the healthcare professional will identify the subject as needing a appropriate panel testing is conducted. The patient or healthcare professional will select a cartridge that is configured to perform the desired panel of assays and insert that chosen cartridge into the reader. The reader will determine from the features present on the cartridge which panel of assays the cartridge is designed to conduct and the reader will configure itself appropriately in order to be able to run the assays and detect and/or determine panel analyte levels. particular present in the subject's sample. A sample will be provided or obtained from the subject and the sample introduced into the cartridge inlet port. The test panel will be conducted on the sample by the reader and cartridge working together and upon completion of the tests, the reader will detect and/or determine the levels of analyte that are present in the sample. The reader will then provide the panel test results to the subject and/or healthcare professional.

[00133] As well as healthcare professionals, the user may be a law enforcement official, or sports doping testing official, for example, where the subject is an individual being tested for inappropriate drug use, for example.

[00134] The present invention will now be further defined by reference to the following enumerated clauses: 1. A microfluidic cartridge for use in conducting an assay on a liquid sample, the microfluidic cartridge comprising a sample inlet port connected to at least one channel microfluidic, wherein each/said microfluidic channel(s) comprises one or more reagents deposited therein for use in conducting the assay and a detection zone, each/said Microfluidic channel(s) additionally fluidically connected to a gas-filled compressible chamber, wherein compression or decompression of an external surface of the chamber causes gas to be expelled or drawn into the chamber respectively , which, in turn, causes a reciprocal movement of the liquid sample within said/each microfluidic channel.

[00135] 2. The microfluidic cartridge according to clause 1, wherein, following reaction of the liquid sample with said one or more reagents deposited within said/each microfluidic channel, gas expelled from the chamber serves to remove liquid from the detection zone within said/each microfluidic channel, so that any analyte or analyte reaction product within said/each detection zone can be detected in a substantially liquid-free environment.

[00136] 3. The microfluidic cartridge according to clauses 1 or 2, comprising a plurality of microfluidic channels, wherein each of said plurality of microfluidic channels is in fluidic communication with the sample inlet orifice.

[00137] 4. The microfluidic cartridge according to clause 3, wherein each of said plurality of microfluidic channels is connected to a respective gas-filled chamber, and/or two or more microfluidic channels are connected to a gas-filled chamber. gas.

[00138] 5. The microfluidic cartridge according to any preceding clause wherein the sample orifice is connected to a first end of said microfluidic channel(s) and a second end of the (s)and said/each microfluidic channel(s) is connected to one or more of said gas-filled chambers.

[00139] 6. The microfluidic cartridge according to any preceding clause additionally comprising one or more collection features designed to receive fluid waste and/or excess liquid sample.

[00140] 7. The microfluidic cartridge according to any preceding clause wherein the cartridge and the channels and other features arranged therein, are formed by a sandwich of three separate flat substrates comprising a top substrate, a base substrate and substrate of the medium arranged between the top and base substrates.

[00141] 8. The microfluidic cartridge according to clause 7 wherein each layer has a uniform thickness and is formed from the same material, optionally each layer having the same uniform thickness.

[00142] 9. The microfluidic cartridge according to any one of clauses 7 or 8 wherein the cartridge is formed from a continuous strip or roll-to-roll process.

[00143] 10. The microfluidic cartridge according to any one of clauses 7 - 9 wherein the flat substrates are sealed together by application of heat and/or the use of adhesive.

[00144] 11. The microfluidic cartridge according to clause 10 wherein the flat substrates are sealed together using an adhesive that is resilient and facilitates the compressibility of each/said chamber.

[00145] 12. The microfluidic cartridge according to any preceding clause wherein said microfluidic channel(s)/each microfluidic channel(s) in the cartridge comprises one or more fluid stopping features, which are designed to prevent sample and/or other fluids from passing through said stop feature(s) due to capillary action alone.

[00146] 13. The microfluidic cartridge according to any preceding clause comprising a one-way valve that is designed only to allow gas to exit the cartridge upon a liquid sample being introduced into the cartridge by capillary action, yet not allowing fluid to be introduced into the cartridge through the valve.

[00147] 14. The microfluidic cartridge according to clause 13, wherein the valve is positioned adjacent to a stop feature that is designed to prevent further transport of the sample within the microfluidic channel by capillary action alone.

[00148] 15. The microfluidic cartridge according to clause 14 wherein the valve is located within a microfluidic channel of smaller size than said/each microfluidic channel and which is in fluidic communication with one of said microfluidic channels.

[00149] 16. The microfluidic cartridge according to any preceding clause comprising one or more electrode features in contact with said channel(s) for use in measuring or detecting liquid present in the said(s)/each channel(s).

[00150] 17. The microfluidic cartridge according to any preceding clause further comprising an analyte binding agent deposited within said channel(s), wherein optionally the analyte binding agent is bound to a surface of the said channel(s).

[00151] 18. The microfluidic cartridge according to clause 17 in which the binding agent is affixed to a magnetic or paramagnetic particle.

[00152] 19. The microfluidic cartridge according to clauses 17 or 18 wherein the binding agent or magnetic/paramagnetic particle is deposited within said/each microfluidic channel(s) of the cartridge, such that Upon the sample being applied to the cartridge and being drawn into said channel(s), the binding agents or magnetic/paramagnetic particles are resuspended by the liquid sample.

[00153] 20. The microfluidic cartridge according to any one of clauses 17 - 19 wherein the binding agent or magnetic/paramagnetic particles are deposited in an area of said microfluidic channel(s)/each microfluidic channel(s). s) defined by features at either end of the deposition area designed to limit the movement of magnetic/paramagnetic particles when initially deposited in said/each channel.

[00154] 21. The microfluidic cartridge according to any one of clauses 19 or 20 in which the magnetic/paramagnetic particles are deposited on a surface of said/each channel that is opposite the surface of the cartridge with which a magnet or magnetic force is placed in close proximity.

[00155] 22. The microfluidic cartridge according to any preceding clause wherein the cartridge further comprises one or more additional reagents deposited within said microfluidic channel(s), which additional reagents facilitate detection of analyte present in the sample.

[00156] 23. The microfluidic cartridge according to clause 22 wherein said one or more additional reagents includes a label that has been adapted to specifically bind to an analyte to be detected to facilitate analyte detection.

[00157] 24. The microfluidic cartridge according to clauses 22 or 23 wherein analyte is bound to the analyte binding agent in a first area of said microfluidic channel(s), before being transported to an additional area or areas of said microfluidic channel(s), in which said one or more additional reagents and/or label are deposited, by means of gas being pulled back into said/each gas-filled chamber.

[00158] 25. The microfluidic cartridge according to any preceding clause wherein said cartridge is capable of holding a plurality (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thereof and /or different assays on a single sample.

[00159] 26. The microfluidic cartridge according to any preceding clause in which the sample volume applied to the cartridge is less than 50μL, such as less than 40μL, 30μL, or 20μL.

[00160] 27. A kit comprising a microfluidic cartridge in accordance with any preceding clause, together with a sample collection device.

[00161] 28. The kit according to clause 26 wherein the sample collection device is adapted to be inserted into the sample inlet hole of the cartridge and then provide a seal to the inlet hole.

[00162] 29. The kit according to clause 27 for use in conducting a nucleic acid detection assay.

[00163] 30. A reader device for use with a microfluidic cartridge of any one of clauses 1 - 26, or kit according to clauses 26 - 29, the reader device comprising: a receiving hole for introducing the microfluidic cartridge into the device reader; force application means for contacting an external surface of said/each gas-filled chamber of the cartridge and being capable of exerting a variable force on said/each gas-filled chamber, whereby an application of initial force to the surface of said/each gas-filled chamber will result in gas being expelled from said/each gas-filled chamber and along said/each microfluidic channel out of said/each chamber; and a reduction in force applied to said/each gas-filled chamber will result in gas within said/each microfluidic channel being drawn back towards and against the gas-filled chamber; and detection means for enabling detection of an analyte or reaction product of the desired analyte present in a liquid sample introduced into the microfluidic cartridge.

[00164] 31. The reader device according to clause 30 comprising a receiving hole adapted to receive cartridges of different dimensions, each differently sized cartridge designed to perform a defined number of tests.

[00165] 32. The reader device according to clause 31 wherein the receiving hole is so adapted to ensure correct insertion and identification of each differently sized cartridge.

[00166] 33. The reader device according to clauses 30 - 32 further comprising a permanent magnet to be placed in close proximity to (or electromagnet which is designed to apply a magnetic field to), a cartridge according to clauses 18 - 26 which was introduced into the reader in order to concentrate and maintain the magnetic/paramagnetic particles in the detection zone of said/each microfluidic channel of the cartridge.

[00167] 34. The reader device according to clauses 30 - 33 wherein the means of applying force is in the form of a finger or a foot that is designed to contact and apply force to an external surface of a chamber of the cartridge.

[00168] 35. The reader device according to clause 34 wherein said finger/foot is designed to bring into contact only a portion of the total outer surface of the gas-filled chamber.

[00169] 36. The reader device according to clause 35 wherein each finger/foot is sized to make contact between 10 and 50% of the outer surface of each chamber.

[00170] 37. The reader device according to any one of clauses 30 - 36 wherein the means of applying force is designed to be raised and lowered into contact with the surface of the cartridge using a motor present in the reader.

[00171] 38. The reader device according to clause 37 wherein the motor is capable of raising and lowering the force applying means at a variable rate so that the gas in the cartridge can be drawn into and/or expelled from the dictates/each chamber filled with gas at different rates.

[00172] 39. The reader device according to clauses 30 - 38 wherein the detection means is an optical detection device, such as a fluorimeter or spectrophotometer.

[00173] 40. The reader device according to any one of clauses 30 - 39 further comprising heating and/or cooling means to allow tests to be conducted at a particular temperature, or plurality of temperatures.

[00174] 41. A method for conducting an assay on a liquid sample, the method comprising: a) introducing a microfluidic cartridge in accordance with any one of clauses 1 - 26 into a reader device in accordance with any one of clauses 30 - 40 ; b) applying a force to a/said gas-filled chamber of the microfluidic cartridge, in order to expel a portion of gas from/said chamber; c) introducing a liquid sample into the microfluidic cartridge and allowing the sample to be drawn into the microfluidic channel(s) by capillary action, or decreasing the force applied to said chamber(s) filled with gas, so that air is drawn into said chamber(s) causing a liquid sample to be drawn into the microfluidic channel(s); d) decreasing the force applied to said chamber(s) of the microfluidic cartridge, so that air is drawn into said chamber(s) and causes the liquid sample to be further pulled into said microfluidic channel(s) to allow contact with an analyte binding agent and optionally one or more additional reagent(s); e) allowing any analyte/analyte-binding agent complexes or reaction product of analyte/analyte-binding agent complexes to be formed and captured in a detection zone of said microfluidic channel(s) (s); f) optionally increasing the force applied to said gas-filled chamber(s) of the microfluidic cartridge, such that gas is expelled from said chamber(s) causing that liquid is expelled from at least a portion of said microfluidic channel(s) where the analyte/analyte-binding agent complexes are captured, such that the analyte/binding agent complexes of captured analyte are present in a substantially liquid-free environment; and g) detecting any analyte or analyte reaction product optionally captured in said substantially liquid-free environment.

[00175] 42. The method according to clause 41 in which step d) is carried out as a single or multiple steps, whereby the sample is drawn to another or a number of successive locations respectively within said/each microfluidic channel corresponding to the number of times a decrease in force is performed.

[00176] 43. A method for conducting an assay on a liquid sample, the method comprising: a) introducing a microfluidic cartridge comprising a gas-filled compressible chamber or chambers into a reader device comprising means for compressing/decompressing the ) said chamber(s); b) applying a force to said gas-filled chamber(s) of the microfluidic cartridge, in order to expel a portion of gas from said chamber(s) camera(s); c) introducing a liquid sample into the microfluidic cartridge and allowing the sample to be drawn into a microfluidic channel or channels of the microfluidic cartridge by capillary action, or decreasing the force applied to said filled chamber(s) of gas, so that air is drawn into said chamber(s) causing a liquid sample to be drawn into the microfluidic channel(s); d) decreasing the force applied to said chamber(s) of the microfluidic cartridge, so that air is drawn into said chamber(s) and causes the liquid sample to be drawn additionally to said microfluidic channel(s) in order to allow contact with an analyte binding agent and optionally one or more additional reagent(s) that is present(s). s) on the said channel(s); e) allow any analyte/analyte-binding agent complexes or reaction product of analyte/analyte-binding agent complexes to be formed and captured in a detection zone of said microfluidic channel(s). s); optionally increase the force applied to said gas-filled chamber(s) of the microfluidic cartridge, such that gas is expelled from said chamber(s) causing the liquid is expelled from at least a portion of said microfluidic channel(s) where the analyte/analyte-binding agent complexes are captured, such that the analyte/analyte-binding agent complexes captured are present in an environment substantially free of liquid; and g) detect any analyte or analyte reaction product captured in the substantially liquid-free environment.

[00177] 44. The method according to clauses 41 or 43 wherein the analyte/analyte-binding agent complexes or reaction product of the analyte/analyte-binding agent complexes to be formed comprise magnetic or paramagnetic particles

[00178] 45. The method according to clause 44 wherein the magnetic particles which are employed to form the complexes are initially deposited on a surface of said microfluidic channel(s) which is opposite to the surface of the cartridge in which a magnet is placed in close contact, or a magnetic force applied, so that the magnetic particles are drawn laterally through said microfluidic channel(s).

[00179] 46. The method according to clauses 43 -45 wherein step d) is carried out as a single or multiple steps, whereby the sample is drawn to another or a number of successive locations respectively within said/each microfluidic channel corresponding to the number of times a decrease in force is performed.

[00180] 47. The method according to any one of clauses 41-46 wherein the volume of gas that is expelled from said chamber(s) causing liquid to be expelled from at least a portion of the (s)/said microfluidic channel(s) where the analyte/analyte-binding agent complexes are captured, is sufficient to cause the liquid to be removed from the detection zone, but not further along the microfluidic channel(s).

[00181] 48. A self-contained disposable microfluidic cartridge for use in conducting multiplex assays, which is a plurality of distinct assays, the microfluidic cartridge comprising: a sample inlet port for introducing a sample into the microfluidic cartridge and multiple microfluidic channels, each which of said microfluidic channels being adapted to receive a portion of the sample and being capable of conducting one or more assays on said portion of the sample, such that the microfluidic cartridge is capable of detecting and/or determining multiple different levels of analyte in the sample and Conduct multiple different types of assay on the sample using reagents that are present in the cartridge prior to introducing the sample.

[00182] 49. The self-contained disposable microfluidic cartridge according to clause 44 for use in a method according to any of clauses 41 - 46.

[00183] 50. The self-contained disposable microfluidic cartridge according to clause 48 further comprising the features as defined in clauses 1 - 26.

[00184] 51. The self-contained disposable microfluidic cartridge according to any one of clauses 48 - 50 that is capable of performing at least two, three, four, five or more of the following types of assays: immunoassay, nucleic acid assay, assay receptor-based, cytometric assay, colorimetric assay, enzymatic assay, electrophoretic assay, electrochemical assay, spectroscopic assay, chromatographic assay, microscopic assay, topographic assay, calorimetric assay, turbimetric assay, agglutination assay, viscometric assay, coagulation assay, clotting time, protein synthesis assay, histological assay, culture assay, or osmolarity assay.

[00185] 52. The self-contained disposable microfluidic cartridge according to any one of clauses 48 - 51 that is capable of conducting a panel of separate assays that are designed to test for a cardiac condition, an adrenal gland condition, a liver, diabetes or illicit drugs.

[00186] 53. The self-contained disposable microfluidic cartridge according to clause 52 for use in detecting a cardiac condition and wherein the separate assay panel is for detecting levels of lipid, apolipoprotein; homocysteine; C-reactive protein (CRP); and/or cardiac enzymes.

[00187] 54. The independent disposable microfluidic cartridge in accordance with clause 52 for use in detecting an adrenal condition, and wherein the separate assay panel is for detecting aldosterone, cortisol, 18-hydroxycortisol, and/or DHEA-S .

[00188] 55. The self-contained disposable microfluidic cartridge according to clause 52 for use in detecting a liver condition and wherein the separate assay panel is for detecting a level of one or more liver enzymes, bilirubin, albumin, prothrombin and/or the presence of a virus or viruses.

[00189] 56. The self-contained disposable microfluidic cartridge in accordance with clause 52 for use in detecting subjects at risk of developing diabetes or confirming subjects with diabetes and wherein the separate assay panel is for detecting lipid levels, complete counts of blood, fasting glucose levels, hemoglobin A1 c and/or albumin.

[00190] 57. The self-contained disposable microfluidic cartridge in accordance with clause 52 for use in detecting illicit drugs, wherein the assay panel is for detecting Amphetamines; Babituria; Buprenorphine; Benzodiazepines; Cocaine; Ecstasy; Methamphetamines; Heroin (Opiates/Morphine); Methadone; Tricyclic Antidepressants; and/or Cannabis.

[00191] 58. A multiplex assay platform for use in conducting multiple panels of assays, the multiplex assay platform comprising a plurality of microfluidic cartridges in accordance with any one of clauses 48 - 57, each cartridge being capable of conducting one panel of assays defined on a sample and a reader constructed to be capable of receiving and verifying each of said plurality of microfluidic cartridges, whereby the reader is configurable to detect and/or determine levels of a panel of analytes that may be present in the sample.

[00192] 59. A multiplex assay platform for use in conducting multiple panel assays in accordance with clause 58 for use with a reader device in accordance with any one of clauses 30 - 40.

[00193] The present invention will now be further described by way of example and with reference to the following figures showing: Figure 1: shows a microfluidic cartridge according to the present invention; Figure 2: shows in detail portion A as identified in Figure 1; Figure 3: shows a reader according to the present invention; Figure 4: shows the internal mechanisms of the reader shown in Figure 3; Figure 5: shows a plan view of an internal portion of a reader comprising a force control means of the invention; Figure 6: shows a sectional view along line A - A of figure 5; Figure 7: shows schematics of exemplary cartridge formats that are capable of running different numbers of trials per cartridge; Figure 8: Shows a comparison chart that detects C-peptide according to the present invention and Siemens Centaur C-peptide assay. N=350; Figure 9: Shows a trend graph comparison of detecting C-peptide according to the present invention vs. Siemens Centaur C-peptide assay. N=294; Figure 10: shows a comparison graph that detects D-Dimer according to the present invention and the HemolL D-Dimer HS 500 clinical analyzer test; Figure 11: shows a comparison chart that detects CRP according to the present invention and the Siemens Dimension CRP clinical analyzer test; Figure 12: shows a comparison chart that detects hsCRP according to the present invention and the Siemens Dimension hsCRP clinical analyzer test; Figure 13: shows a dose response curve of Plasmodium falciparum (P.f) HRP2 analyte injected into the blood and run according to the present invention; Figure 14: shows a schematic of reagents used in a multistep troponin I assay; Figure 15: shows a schematic representation of the steps involved in a multistep troponin I assay; Figures 16(a) and (b): show graphs of Troponin I measured in healthy subjects using a multistep assay according to the present invention compared to the Siemens Centaur troponin Ultra test; Figure 17 shows a comparison of a response to the C-peptide assay conducted in accordance with the present invention before and after air removal of buffer; Figure 18 shows a comparison of a response to the C-peptide assay conducted in accordance with the present invention on a blood sample, before and after removal of the liquid sample by air; and Figure 19 shows a comparison chart of the method using a cartridge of the present invention comprising a channel without a gas chamber to control liquid filling and/or removal in order to perform an INR test and the Roche CoaguCheck INR test.

[00194] Figure 1 shows a microfluidic cartridge (1) according to the present invention, to perform 4 separate tests on a single sample. The cartridge (1) comprises a liquid sample inlet port (3) connected to a microfluidic channel (4) which divides into a plurality of separate channels (5 and 7). Each channel (5) extends within the cartridge (1) and is fluidically connected to the gas-filled chambers (10). The additional channel (7) that is not connected to a gas-filled chamber is a control channel for use in multiple control measurements. In use, a sample of fluid fills the channels (5 and 7) and this can be detected by electrodes (not shown) that are in electrical contact with corresponding electrical contacts on the reader. When the reader detects an appropriate signal that a sample has been loaded into the cartridge (1), the reader can begin testing. A collector (13) for receiving liquid is also provided. Immediately upstream of the collector there is a liquid stop (15) that prevents liquid from entering the collector immediately (13) by capillary action alone. Thus, in initial sample application using a capillary application, the liquid sample does not pass the liquid stop (15)

[00195] Describing each channel (5) in more detail, there are printed features (20, 22, 24, 26) that are designed to limit the movement of any reagent that is deposited within each channel (5) during the manufacturing process . Adjacent to the printed feature (20) and restated by section A, as shown in more detail in Figure 2, is a smaller (e.g., 0.1 - 0.2mm) channel (50) that extends perpendicularly outward from the test channel (e.g. 0.75-1 mm) (5). Inside each channel (50) is a unidirectional valve (0.1 mm by 0.9 mm) (52) which is designed to allow gas or air present in each channel (5) to exit the cartridge (1) by applying a liquid sample. Thus, upon application of a sample to the cartridge by capillary application, the sample fills the channel (4) displacing the air present in the channels (5) which leaves the cartridge through the one-way valves (52). The sample fills by capillary action until the sample is approximately adjacent to each side channel (50). Located above the printed feature (20) is a first reaction zone (28) of each assay channel (5) into which one or more binding and/or reacting agents designed to react with and bind a particular analyte has been deposited. or reaction product thereof that may be present in a liquid sample to be tested. For example, deposited in the first zones (28) of said channels (5) may be magnetic particles functionalized with an antibody designed to specifically bind to a first epitope of an analyte to be detected. Deposited with a second zone (30) of each channel may be fluorescently labeled latex particles functionalized with an additional antibody designed to specifically bind a different analyte epitope to be detected. Located distal/proximal to zones (28, 30) is a detection zone (32) where label/analyte/magnetic particle complexes can be detected.

[00196] Located distal/proximal to the detection zones (32) are gas-filled chambers (10), which are designed to co-locate with a force application feature present within a reader device (as will be described later) of the present invention, so that the force application feature is capable of applying a force to the gas-filled chambers (10) so as to cause gas within the chambers (10) to be expelled from the chambers (10) and into the gas channels. test (5). A decrease in the force applied to the chambers (10) causes air to be drawn back into the chambers (10) of the test channels (5).

[00197] In use, the cartridge (1) is inserted into a reader (100) as shown in Figure 3. The reader has a lockable door (102), which can be opened in order to access a cartridge reception hole ( 103) from the reader. Once a cartridge has been inserted into the reader (100) and a sample applied to the cartridge (1), the door (102) can be closed. The reader houses numerous features that are designed to make contact with the cartridge (1) and/or facilitate the performance of the assay of the present invention as will be described in more detail. The top surface of the reader (100) comprises a touch screen monitor (104) that allows a user to interact with the reader (100) as well as receive information regarding the performance of any assay.

[00198] Figure 4 shows the internal resources of the reader (100). The reader includes a rechargeable battery (110) to power the reader and its various functions as will be described. Power to charge the battery (110) is provided via a DC socket (106). The reader (100) additionally includes a heater (111) for heating the cartridge (1) when required; optical blocks (112) containing the optics necessary to detect a fluorescent signal from the cartridge (1); a movable magnet (113) which is designed to immobilize magnetic particles in the detection zone (32) of the cartridge; and a lever mechanism (114) that is designed to contact the chambers (10) of the cartridge (1) and apply a force in order to cause air to be expelled from the chambers (10).

[00199] In use, a cartridge (1) is inserted into the reader (100) until the cartridge comes into contact with an alignment feature (122) on the reader (100). Correct insertion of the cartridge (1) is detected by electrodes that are present on the cartridge with corresponding contacts that are present on the reader. This signals to the reader that a cartridge (1) has been correctly inserted and the start of a test process can begin. A motor (120) is signaled to activate a rack and pinion mechanism. The gear (124) is rotated clockwise so as to cause a rack mechanism (126) of a lever (128) to move vertically upward. This movement causes the other end (132) of the lever (128), in the shape of a finger, to move downwards and into contact with chambers (10) of the cartridge (1). Continuous operation of the motor causes the rack mechanism (126) to move upward, with a corresponding downward movement of the other end (132) of the lever (128), so that an increasingly greater force is applied to the chambers (10 ) of the cartridge (1), expelling gas from the chambers (10). Once the desired amount of gas has been expelled from the chambers (10), the end (132) of the lever (128) remains in contact with the gas-filled chambers (10) in order to prevent gas from being drawn back into the chambers (10). the cameras (10). At this point, the user will be advised by a message on the monitor (104) that a sample can now be applied to the cartridge (1).

[00200] A sample comes into contact and is introduced into the cartridge (1) through the inlet hole (3). The sample fills the channels (4, 5 and 7) by capillary action, as previously described, with air being released through valves (52). Following capillary filling, a portion of the liquid sample is electrically detected in channels (5 and 7), signaling the reader to continue. The motor is then induced to rotate the gear mechanism (124) in a counterclockwise direction which in turn causes the rack mechanism (126) at one end to move in the downward direction and the other (132) of the lever (128) upwards, so that the force applied to the chambers (10) of the cartridge (1) is reduced. This reduction in the force applied to the chambers (10) causes air to be drawn back into the chambers (10), which, in turn, pulls the sample into the first zones (28) of the channels (5). The motor (120) and associated lever movement are capable of carefully controlling the reduction in force applied to the chambers (10) that control how far the liquid sample is drawn into the first zones (28). This can also be controlled via electrode-sensed feedback. The liquid sample entering the first zones (28) of the channels (5) causes functionally derived magnetic particles present in the first zone (28) to be resuspended by the sample. The motor (120) is stopped for a period of time in order to allow any desired analyte that may be present in the liquid sample to bind to the functional analyte binding moieties on the surface of the magnetic particles to form analyte/particle complexes. magnetic. After a set period of time, the motor is activated again and a further reduction in force is applied to the chambers (10) causing more air to be drawn back into the chambers (10), which in turn pull the sample and analyte/magnetic particle complexes for the second zone (30) of the channels (5). The second zone (30) of each channel (5) contains functionally derivatized fluorescently labeled latex particles that are capable of binding to analyte/magnetic particle complexes in order to form a latex particle/analyte/magnetic particle complex sandwich. After an additional period of time, the force applied to the chambers (10) is further reduced and the liquid and associated complexes present therein are drawn into a detection zone (32).

[00201] Once the liquid sample and associated complexes have been drawn into the detection zone (32), the magnet (113) is driven by a motor (150) and associated gear (152) and rack (154) so that the magnet is placed in close proximity to the detection zones (32) of the cartridge, so that the magnetic complexes are attracted to the magnet and held in place in the detection zone (32) by the magnetic force of the magnet (113). Next, the motor (120) is reapplied in order to cause the lever mechanism (114) to increase the force applied to the gas-filled chambers (10) causing air to be expelled once more from the chambers (10) that results in liquid sample and magnetically unbound material that is present in the detection zone (32) being pushed out of the detection zone (32) and along the channel (5) with a portion of the liquid exiting the collector (13). . It may not be necessary to expel all of the liquid through the collector (13) and in fact it may only be necessary to remove the liquid from the detection zone (32), so that the resulting magnetically bound complexes are present in an essentially airy environment. This can be particularly advantageous in terms of not using extra sample volume to perform a wash as occurs with lateral flow products and no requirement for a strip buffer bag or inlet buffer dispensing system.

[00202] The motor (120) is capable of operating at a variable speed and so it is easily possible for the extraction of air into the chambers (10) and the expulsion of air from the chambers (10) to occur at different rates, with a flow rate corresponding variable of the liquid present in the channel (5) and associated zones (28, 30 and 32).

[00203] Following removal of the liquid from the detection zones (32), the captured complexes are present in an essentially liquid-free environment and can be detected using a detector that is present in the optical block (112). The detector may be in the form of a spectrophotometer, for example, which is capable of detecting the fluorescent label present in the captured latex particle/analyte/magnetic particle complexes.

[00204] In an alternative embodiment to that shown and described in relation to Figure 4, piezoelectric flexors can be employed to control force applied to the gas-filled chambers of the cartridge. Figure 5 shows a force control means (200). The force control means (200) comprises a series of piezoelectric flexors (202) which are fixed to a first end (201) by a clamping block (204). Each piezoelectric flexor is also electrically coupled to the first end for electrical connections (206) that control the electrical signal provided to each flexor (202). As can be seen, each flexor (202) is connected to its own set of electrical connections (206), so that each flexor is independently controllable. As shown in Figure 6, the other end (208) of each flexor (202) rests on the top surface (209) of a foot (210) which in use is designed to make contact with the outer surface of a pressure chamber. gas from a microfluidic cartridge (220) of the present invention.

[00205] Figure 6 shows a sectional view along line A-A of figure 5 so that various parts of the force control means (200) and how they function can be better understood. In Figure 6 the force control means (200) is shown in conjunction with a microfluidic cartridge (220) when correctly inserted into the reader such that the gas-filled chamber of the microfluidic cartridge is directly positioned below the foot (210) of the force control means (200). force control (200). The base surface (212) of the foot (210) is shaped to contact a portion of the gas chamber of the microfluidic cartridge (220) and through appropriate control is applied to the foot (210) by the piezoflexor (202), the foot (210) is capable of applying a variable force to the gas chamber of the microfluidic cartridge (220).

[00206] As shown in Figure 6, the piezoflexor (202) is in its unformed rigid state. In this embodiment, the force control means (200) is constructed so that the piezoflexor (202) is capable of exerting maximum force on the foot (210), so that the base surface (212) of the foot (210) pushes downward and compress the gas-filled chamber, causing the gas within the chamber to be expelled from the chamber.

[00207] Although not shown, applying an electrical charge to the piezoflexor (202) will cause the piezoflexor (202) to flex and the end (208) of the piezoflexor (202) to flex upward. This upward flexion of the piezoflexor (202) reduces the force applied to the foot (210), which in turn causes the foot (210) to reduce the force applied to the gas-filled chamber of the cartridge (220). Reducing the force applied to the gas-filled chamber provides a decompression in the gas-filled chamber and a corresponding entry of gas back into the chamber. By appropriate electrical signaling, it is possible to flex and relax the piezoflexor (202) resulting in the gas-filled chamber being decompressed or compressed in this manner, and gas being expelled or drawn into the chamber.

[00208] Many piezoflexors are known in the art and may be suitable for use in the present invention. Those versed in the art will choose a flexor that is suitable for a particular purpose. The present inventors have employed a variety of such piezoflexors with displacements of up to several millimeters and response times in the millisecond range. A programmable voltage amplifier can be used to control each piezoflexor. Suitable amplifiers include a 32-channel, 14-Bit DAC with Full Scale Output programmable voltage from 50V to 200V (AD5535) or Quad-Channel 12-Bit High Voltage voltage output DAC (AD5504) available from Analog Devices ( Norwood, MA 02062, USA). Forces of 1N - 2N may be achievable.

[00209] The foregoing provides a description of specific embodiments of the present invention, but the present invention is designed to be in the form of a platform that can easily be adapted. For example, the position of the vent may be changed to allow capillary filling at different positions within the channel (5), or a vent omitted altogether and sample filling occurring by active filling after the gas is expelled from the chamber (10) and the sample by extraction into the cartridge (1) and channels (5,7) by air returning to the gas chambers after a release in the pressure being applied to the chambers (10)

[00210] Additionally, the reader can be designed to utilize multiple test formats with a family of strip sizes defined by product requirements. The strip can be designed to be manufactured in 2, 4, and 10 channel shapes, for example, for specific product configurations and panel testing (see Figure 5 showing different strip sizes). The availability of different strip sizes allows the present system to dispense multiplexed tests through mixed technologies to meet the specific user requirements of the Point of Care market at a framework of increased performance and reduced cost compared to established products in targeted markets.

[00211] Referring to figure 7 showing strips of different sizes, the channel 2 cartridge is designed for a single assay with controls, the channel 4 cartridge for panels of 2-3 analytes with controls, and the channel 10 cartridge allows complex trials of mixed technologies and products that require high multiplexing capacity (e.g. illicit drugs) are carried out. The described platform has a highly flexible sample and assay architecture and reader control and measurement capabilities allowing forward compatibility for new opportunities to be explored as new test panels or test types are identified or move to the Point of Care.

[00212] Although the primary measuring technology is fluorescence, the platform also incorporates electrochemical measurement and other methodologies can easily be incorporated. This is discussed in further detail below.

[00213] To dispense multiple test types and formats on a single platform, a set of flexible core technology capabilities and controls have been developed that can be used as required and in sequences that do not require different assay format steps. The system architecture design principles are: Magnetic Particle Capture Phase Control of liquid movement Removal of liquid from the detection zone Label detection in air Multichannel multiplexing Intrachannel multiplexing Dynamic range Internal controls Electrochemical measurements Heating and temperature control Pretreatment of Sample

[00214] This platform architecture allows many different test types and technologies to be formatted into the system. Each core technology principle is discussed below.

Magnetic Particle Capture and Liquid Control

[00215] The use of particle capture is known to improve capture kinetics. For immunoassays, the platform of the present invention uses paramagnetic particles as the capture surface. Different paramagnetic particle sizes can be used to optimize performance for each type of test. Paramagnetic particles ranging from 100 to 1,000nm were used during assay development. The particle capture phase is combined with a fluorescent particle label phase. Similarly, the fluorescent particle phase can be varied in size depending on assay sensitivity and range requirements. Typical sizes of fluorescent particles can be in the range 40nm-4,000nm

[00216] Some assays, such as C-Reactive Protein (CRP), require relatively high concentrations of analyte to be measured and utilize conjugated direct fluorophore-labeled antibody in combination with magnetic particles, although high-sensitivity assays generally utilize fluorescent particle labels in combination with magnetic particles. Importantly, both the capture and label phases are mobile in the sample to trigger capture events. This is further helped by the fact that undesirable flow within the strip is minimized. During channel filling, the sample flows over the dry test reagents. Reagent dissolution and therefore flow front effects are minimized by using formulations that allow good channel filling but result in slower controlled dissolution. After the initial sample filling event, the flow is stopped so that the sample is prevented from flowing further for a period of time. This allows very consistent dissolution and subsequent binding efficiency to occur since there are no matrix-dependent flow errors affecting the sample volume or binding kinetics interrogated.

[00217] Performing reagent dissolution and analyte capture in a fixed, static, optionally mixed volume, opposed to a variable flow system (e.g., Side Flow, Screening) significantly improves assay precision and accuracy.

[00218] For more complex assays, such as Troponin (as described elsewhere), the assay is most efficiently performed as a multi-step procedure using multiple reagent zones. In this case, the meter's functionality of being able to compress the gas chambers (10) to expel gas and remove liquid from the detection zone is also used to control the movement of fine liquid in the cartridge (1) and channels. associates. Before a sample is applied to the cartridge (1), the gas-filled chambers (10) are compressed by the meter expelling gas from the chambers (10) and the test channels. The chambers (10) remain compressed by the meter during sample application and sample filling is by capillary action or completely under gas-driven fluidic control. The high-resolution motor or piezoflexor in the meter allows very controlled incremental release or increase of pressure in the gas chambers (10) with the rate and amount of pressure changing specific to a particular test. This feature provides numerous important advantages including the ability to mix using fine positive and negative bending of any piezoflexors.

[00219] Sample filling time can have a significant effect on the performance of a product to introduce reagent dissolution variability, fluid front effects and the sample volume interrogated. Fluidic control reduces variation in filling time by directly controlling the sample filling rate. Fluidic control allows the sample to be moved at a controlled time into different zones within each channel, allowing sample pretreatments and multi-step assays to be performed (described here). Fluidic control and isolation is also a necessity for closed systems as required for NAT tests (see below).

Liquid Removal from the Detection Zone

[00220] Liquid movement and control are achieved by compressing or releasing the gas chambers (10) in the test cartridge using a motor and force applicator, or piezoelectric flexing mechanism that makes contact with the fluid chambers (10). The resulting gas movement from each chamber (10) allows fine control of sample and reagent movement including removal of unbound label from the detection zone (32) of the test channel and optionally into the collector area (13).

[00221] The fluid control function built into each cartridge leads to numerous important differentiating advantages.

[00222] Firstly, the described system provides a very effective separation of bound and unbound test components using gas control of liquid movement. This is important because it completely avoids the complexity and cost of a strip liquid reagent pouch or meter replaceable liquid wash reagent packets.

[00223] Secondly, the present invention additionally allows the use of laminate manufacturing technology with very low cost and cartridge manufacturability using high production, high control continuous strip production systems.

[00224] Thirdly, the removal of the sample and the unbound label from the detection zone (32) by the use of gas means that the measurement of fluorescent labels can be made in a gaseous environment, essentially free of liquid.

In-air Label Detection

[00225] Gas label measurement results in several significant technical advantages for making fluorescent measurements compared to standard testing protocols of prior art products.

[00226] Use of an essentially gaseous environment significantly decreases the finishing effect of a liquid sample thereby removing a primary source of variation effect and test matrix. For example, the presence of blood cells and plasma proteins terminates the fluorescence signal by reducing the sensitivity and increasing the variability of the fluorescence measurement. Measuring fluorophores in a gaseous or air environment allows the use of fluorophores that would not necessarily have been chosen due to sample completion. This allows for simpler optical designs, optimization of fluorophores for each assay, and multiplexing within a single channel. As described, for example, below and with reference to Figures 15 and 16, detection in air provides a significant improvement in sensitivity compared to detection in buffer or whole blood.

[00227] In summary, the use of gas to remove the sample and unbound label approach reduces assay variation by decreasing the sample matrix effect of terminations and gives access to a greater range of fluorophores for assay optimization. This translates to assay design flexibility, assay speed and unparalleled performance.

Multichannel Multiplexing

[00228] The platform of the present invention has multichannel and intrachannel multiplexing capabilities. Panel tests can be dispensed using multiple channels on a single strip combined with an optical scanning head to measure the label, e.g., fluorescence intensity in each channel. The number of channels can be varied depending on the product requirements.

[00229] This allows the development of panel tests with each channel containing a different assay, e.g., cardiac panel, metabolic panel, etc. Since individual assays are spatially distinct on separate channels, each assay can be configured with unique reagents on the multichannel strips. This leads to numerous key advantages:

[00230] Firstly, each assay can use an ideal formulation including reagents, buffers, pH, etc. for: reagent dissolution, anticoagulation, neutralization of matrix effects (HAMA etc.), optimal sensitivity, linearity, range and assay stability. It is not necessary to find compatible optimization for multiple assay reagent sets or compromise assay performance in order to develop panel products. Each assay can exist in its own optimal formulation in an individual channel and maintain its respective high assay performance.

[00231] On the contrary, multiplexing tests within a single channel inherently compromises the performance of individual tests as the reagent formulation must be compatible with all assays. Individual assay requirements often conflict, for example something as fundamental as pH will significantly affect assay performance.

[00232] Multichannel multiplexing translates into flexibility, simplicity and speed of developing the panel test design of the panel test and maintaining unique test performance across panels.

[00233] Second, the multichannel approach allows the present platform to realize new panel products that combine different assay technologies and different transduction methods in a single strip.

[00234] There is increasing evidence that measuring molecule families can be advantageous over measuring a single molecule from this family. For example, natriuretic peptides used in congestive heart failure stratification are generally separated for BNP and NT-proBNP testing. Multichannel multiplexing allows measurement of proBNP, BNP, NT-proBNP and other forms of natriuretic peptide on one strip and avoiding antibody epitope passing within the peptide family. In contrast, intrachannel multiplexing leads to greater nonspecificity of molecule family measurements. The currently described multichannel approach is applicable to the troponin testing market whereby different troponin isoforms can be measured in separate channels to improve diagnosis of myocardial infarctions.

Intrachannel multiplexing

[00235] Where ratio-metric measurements are required, for example, HbA1 c and ion measurements in blood, intrachannel multiplexing is necessary in order to obtain the most accurate assay performance. The present platform achieves this by measuring more than one fluorophore in a single channel.

[00236] The combination of multi- and intra-channel multiplexing allows for flexible and powerful product combinations with internal controls that will improve accuracy and confidentiality.

Dynamic Range

[00237] The large dynamic range of an analyte to be measured can often be a limitation of assay performance. For example, a troponin test needs to be very sensitive, but at the same time it needs to be able to measure high concentrations in order to monitor changes observed in patients with myocardial infarction. Dynamic range often leads to nonlinearity across the required measurable range, which impacts precision and accuracy.

[00238] The multichannel design allows challenging tests with large dynamic ranges to be divided into multiple channels on the strip covering high sensitivity and high concentrations of the required measurable range in a linear manner.

[00239] For troponin (forms I and/or T), one channel may contain reagents optimized for measuring 0 - 100pg/mL although another channel may contain reagents optimized for measuring 50-1,000pg/mL and an additional channel optimized for 500 - 50,000pg/mL. The sensitivity and range each have their own calibration parameters with the sample concentration assigned by the confidence interval of the two results.

Internal controls

[00240] The present platform incorporates internal control features to verify the validity of the test results obtained. Each test type requires unique internal assay controls as well as several generic features. All tests can have fill detection to ensure proper sample application and used cartridges cannot be retested. Where required, the cartridge incorporates a hematocrit measurement to adjust for those tests affected by hematocrit variation. Specific channel controls can be implemented to incorporate low and high controls that are used to calibrate remaining blood matrix variables and/or independently verify the test result. Depletion controls can be used to check for human anti-mouse antibodies (HAMA) or other sample-dependent variables.

[00241] A microprocessor and associated software can control time, temperature, fluid control, etc. for each particular assay, as these may have different requirements on a single cartridge.

Electrochemical Measurements

[00242] Although the primary detection methodology described is fluorescence, other optical measurements can be made and/or electrochemical measurements can also be made on the present platform to incorporate traditional electrochemical test formats (e.g., glucose testing). Furthermore, both electrochemical and fluorescent measurements can be made on the same strip, for example, a diabetes panel of a fluorescent C-peptide immunoassay coupled with electrochemical glucose measurement. Conventional ion selective electrode (ISE) measurement approaches for ion gases and blood can also be ported on the present platform. The combination of optical, such as fluorescence and electrochemical transduction technologies allows provision of a wide variety of different panel tests.

Heating and Temperature Control

[00243] Temperature is a significant variable in most tests. For some assays, temperature effects can be compensated for using a temperature correction algorithm. However, this is often problematic in determining how much individual cartridge batches and fixed compensation can become a source of error in itself. Characterizing temperature profiles across all process and matrix variables can significantly impact the product development cycle. In some products such as PT/INR and molecular testing, adequate temperature control is critical to functionality and test performance. This platform allows the incorporation of an integrated heating capacity that provides the ideal temperature requirements for each type of test. Typical operating temperatures are used for immunoassays (34°C), PT/INR (37°C) and nucleic acid detection (>37°C) etc. Heating capacity can be optimized to dispense a strip range and controlled pretreatment temperatures for maximum flexibility in testing protocol.

Sample Pretreatment

[00244] Strip sample movement control allows sample pretreatment before the sample is presented to specific assay reagents. This approach can be applied to immunoassays, for example, to remove interferents such as HAMA species or lipid panels to remove undesirable fractions for specific lipid measurements (e.g., HDL). Strip fluidic steps mimic the capabilities used by clinical analyzers to optimize product performance by allowing sample matrix and interferences to be quickly resolved during product development.

Exemplar Test Descriptions and Test Data

[00245] One-step immunoassay Summary Test Sequence: 1. Insertion of the cartridge into the reader 2. Compression of the cartridge's gas chamber by the reader 3. Application of sample to the cartridge, filling by capillary action or by filling controlled by the reader. 4. Wetness of cartridge fill detection electrodes determines test start timing. 5. The sample rehydrates the dried reagents that contain: a. anti-analyte antibody (epitope 1) functionalized paramagnetic particle phase b. fluorescent label/anti-analyte antibody functionalized particle phase (epitope 2) 6. The reagents mix and bind the analyte contained in the sample forming the immunoassay sandwich complex (fluorescent label/particle-analyte-paramagnetic particle). 7. The binding reaction occurs for a defined amount of times (typically 2 minutes). 8. A magnetic field is applied to the strip located in the optical detection zone that accumulates the paramagnetic particles there, forming a band of particle-analyte-label complex in each channel. 9. A liquid sample and unbound label removal step is then performed by the reader initiating a force being applied to the gas chambers of the cartridge. This compressive force expels the gas from the gas chambers through the test channel resulting in the liquid sample and unbound fluorescent label/particles being expelled from the detection zone and optionally from the channel and into the sample residue collector. The magnetic field is applied throughout this step keeping the particle-analyte-label paramagnetic complexes in the local detection zone by the magnetic field although the sample is expelled from this area. 10. The optical meter head sweeps across the strip and the fluorescence intensity for each channel is measured. The fluorescence intensity is proportional to the analyte concentration. Each strip batch and analyte channel is calibrated separately so that the fluorescence intensity is transformed into analyte concentration.

[00246] Example performance data sets for step one immunoassays are shown in Figures 8 -13:

C Peptide

[00247] C-peptide is a short 31-amino acid polypeptide that connects insulin's A chain to its B chain to the proinsulin molecule. Proinsulin is cleaved into insulin and C-peptide in equimolar concentrations. In the diagnostic context, C-peptide is used as a surrogate biomarker for insulin and is used to monitor β-cell function (insulin production) in diabetic patients. The present inventors ran a comparison of the present test against the commercially available ADVIA Siemens Centaur benchtop system (see Figure 8).

[00248] Table 1 below shows the percentage of results that are in a given polarization of the reference system for the C Peptide range as indicated. This shows that the present assay typically obtains around 95% results in 20% of the reference system. Table 1. C-Peptide Assay Accuracy

[00249] A polarization analysis of the present system vs. The Siemens Centaur reference system was performed for samples above 0.5 ng/mL (294 points), this is plotted in (Figure 9) compared to an established commercially available clinical analyzer. The percentage bias of each point to the reference system value is plotted vs. the reference value. The graph shows the present assay system has clinical accuracy comparable to an established laboratory system.

D-dimer

[00250] D-dimer is a fibrin degradation product (FDP), a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. The D-dimer molecule contains two cross-linked D fragments of fibrin protein.

[00251] D-dimer concentration is used to help diagnose thrombosis. It is an important test performed on patients suspected of having thrombotic disorders. Although a negative result virtually excludes thrombosis, a positive result may indicate thrombosis but does not exclude other potential causes. Its main use, therefore, is to exclude thromboembolic disease where the probability is low.

[00252] The inventors performed a dose response analysis using the currently described methodology and compared results with those of a HemolL D-Dimer HS 500 (a commercially available clinical analyzer) (see Figure 10)

C-reactive protein (CRP)

[00253] C-reactive protein (CRP) is an annular (ring-shaped) pentameric protein, found in blood plasma, whose levels rise in response to inflammation. It is an acute phase protein of hepatic origin that increases following the secretion of interleukin-6 by macrophages and T cells.

[00254] CRP has diagnostic utility for numerous types of disease that can be summarized as follows: 1. State of inflammation in type 1 diabetic patients 2. Administration of antibiotics to control infection and general status of infection 3. Cardiovascular disease 4 .Certain cancers

[00255] A graphical method of comparison is shown in Figure 11. The required reportable range is 5-20μg/mL.

High Sensitivity CRP (hs-CRP)

[00256] High sensitivity CRP (hs-CRP) is used in assessing the risk of developing cardiovascular disease. General guidelines are as follows: 1. Low: hs-CRP level below 1.0 mg/L 2. Medium: between 1.0 and 3.0 mg/L 3. High: above 3.0 mg/L L

[00257] A method comparison chart is shown in Figure 12. The data demonstrates that the present platform is well capable of measuring the required hs-CRP concentrations.

HRP2 from Plasmodium falciparum malaria

[00258] The malaria parasite Plasmodium falciparum secretes the histidine-rich protein II (HRP2) used as a biomarker to detect the presence of the malaria parasite Plasmodium falciparum (Pf). The present platform was used to demonstrate the measurement of HRP2 in blood samples. HRP2 protein was injected into bloods and measured on the present platform and the Standard Diagnostic (SD) malaria Pf rapid test.

[00259] The lowest HRP2 concentration measured on the present platform was 0.25ng/mL. In comparison, using the SD test a very weak band was observed at 5ng/mL. Lower concentrations may not be measured. The present platform's test result of 0.25 ng/mL took 7 minutes versus the recommended test time of 30 minutes required for SD testing to measure the concentration of 5ng/mL. The 30-minute assay time is required for concurrent testing to wash the label of unbound gold solution and any lysed blood to resolve very low concentrations. There are also additional user actions to apply a buffer to the strip to perform this washing step.

[00260] The data was analyzed and results are summarized in Figure 13. The present assay was able to measure significantly lower HRP2 concentrations than the SD test with much faster test times. This assay has the sensitivity to meet the requirements for a rapid test to monitor residual infection in a population Malaria eradication program.

Multistep Immunoassay – e.g. Troponin

[00261] The present platform is configurable to perform multistep assays allowing stepwise binding reactions to occur to optimize binding kinetics, test time and sensitivity.

[00262] In the high-sensitivity Troponin assay, the antibody particle binding steps and label/particle binding steps are decoupled to significantly improve the binding rate and capture efficiency of the paramagnetic antibody particle binding step. analyte for very low Troponin concentrations. Subsequent stepwise binding of the label particle and the paramagnetic particle using high-affinity anti-fluorescein isothiocyanate and Biotin-Streptavidin functionalized particles, respectively, allows for greater capture and transduction of the bound Troponin complex.

Summary Test Sequence:

[00263] 1. Insertion of cartridge into reader 2. Compression of gas chamber by reader 3. Application of sample to cartridge, filling by capillary action at first vent stop feature where first reagents are located (labeled antibodies) 4. Resolubilization of reagent and incubation and analyte-antibody binding time. 5. A small decompression of the chamber results in the liquid sample being drawn further along the channel locating the sample reagent mixture over a secondary reagent. 6. Reagent resolubilization and incubation and analyte-antibody-particle label binding time. 7. A second small decompression of the chamber results in the sample being moved further along the channel localizing the sample reagent mixture over a third reagent 8. Reagent resolubilization and incubation and analyte-antibody-particle-particle label binding time paramagnetic. 9. A magnetic field is applied to the cartridge located in the optical detection zone which accumulates the paramagnetic particles at this location forming a band of the antibody-analyte-particle-paramagnetic particle label complex in each channel. 10. Sample liquid and unbound label is removed from the detection zone by recompressing the chambers expelling the sample and unbound label from the optical detection zone 11. The optical head of the reader scans through the strip and the fluorescence intensity for each channel is measured. The fluorescence intensity is proportional to the Troponin analyte concentration.

Troponin I (TnL) assay - reagents are identified in Figure 14

[00264] Step 1: This is passive capillary filling. The TnL assay uses two capture antibodies each of which is labeled with a biotin group. The label antibody is labeled with a Fluorescein Isothiocyanate (FITC) group. The biotin and FITC molecule groups serve as immunogenic tags for the second and third step.

[00265] Step 2: The sample from step one is moved to a secondary reagent deposition area by fluidic reader control (chamber decompression). This deposition contains latex particles coated with anti-FITC antibody. The anti-FITC latex particles will bind the FTIC-labeled antibody (which is bound to the complex). This reaction is fast.

[00266] Step 3: The sample is moved to the third deposition zone by fluidic reader control. The third deposition area contains streptavidin-coated magnetic particles. Streptavidin paramagnetic particles will rapidly bind biotin-labeled antibodies that are bound to the Tnl complex. Paramagnetic particle accumulation is followed by removal of unbound sample/label. Fluorescent optical scanning is then performed. The fluorescence intensity is proportional to the Tnl concentration.

[00267] A schematic of the previous method is shown in Figure 15

[00268] Step 1 is capillary filling, step 2 & 3 are under fluid reader control.

[00269] This approach is very attractive since it has generic application and greatly simplifies the assay reagents and, more importantly, leads to excellent assay performance (see exemplary results shown in Figures 16(a) and (b)), in sensitivity of this method over a wide concentration range. For example, anti-FITC latex is a generic label for other assays (e.g. BNP), similarly streptavidin paramagnetic particles are also generic across assays. Batch-by-batch production of reagents will become much easier for challenge assays such as Tnl.

[00270] To show the significance of performing optical detection, such as fluorescent detection, in air, the inventors additionally performed C-peptide assays in order to show the response when conducted in buffer or whole blood, compared to air. Figure 17 shows a response to the C-peptide assay in buffer using the present system before (white circles) and after (black triangles) air stripping. It can be seen that without liquid removal by air there is a high background due to the unbound label still being present in the detection area. This leads to poor accuracy and sensitivity at low analyte concentrations. Following liquid removal by air, this unbound label is efficiently removed, leaving a very low background allowing a highly sensitive measurement to be made. Figure 18 shows a response to the C-peptide assay in whole blood using the present system before (black circles) and after (white triangles) removal of liquid by air. It can be seen that without liquid removal by air there is a high background due to the unbound label still being present in the detection area and no visible slope due to blood sample interference, terminating the fluorescent measurement. Following liquid removal by air, this unbound label and whole blood sample are efficiently removed, leaving the binding reagents in an essentially liquid-free environment without interfering with the blood cells or unbound label. This produces a very low background and allows a highly sensitive measurement to be made.

[00271] It is possible that the present cartridges also run assays that do not require a bladder to run the assay, for example, in determining the prothrombin time (PT) and international normalized ratio (INR) of a blood sample. PT and INR are tests that evaluate the extrinsic coagulation pathway (PT/INR). They are used to determine blood clotting tendency, measure warfarin dosage, liver damage, and vitamin K status.

[00272] A PT/INR measurement method comparison chart is shown in Figure 19 which was generated using a channel that does not have any fluid control provided for a gas chamber. In this regard, the sample would fill by capillary action alone. For the avoidance of doubt, PT/INR measurements can also be made using a channel with an associated gas chamber, which allows fluidic control of the sample to normalize filling rates. Compared to previously described immunoassay examples, the channel is enlarged in the detection area of the strip to allow a greater volume of sample to be assayed. Additionally, INR/PT specific reagents are deposited in this area. The reagents contain all components required to initiate the extrinsic coagulation cascade and a specific thrombin fluorophore substrate that is converted from a non-fluorescent form to a fluorescent form by thrombin. Capillary refill resuspends reagents and allows detection of thrombin activity. The measured thrombin activity (fluorescent intensity) is used to determine the PT/INR result.

Claims (16)

1. Test system comprising an independent microfluidic system and an associated reader device, characterized by the fact that: the independent microfluidic system comprises: a sample inlet port (1) for receiving a liquid sample to be examined, the inlet port of sample (1) connected to at least one microfluidic channel (5), wherein each/said microfluidic channel(s) (5) comprises one or more reagents deposited therein(s). s) for use in conducting an assay and a detection zone (32) for use in detecting any analyte that may be present in a sample or analyte reaction product; and each/said microfluidic channel(s) (5) is in fluidic communication with a gas-filled compressible chamber (10) downstream of each/said detection zone (32), wherein The microfluidic system is formed by three layers, a top layer, a middle layer and a base layer, which are sandwiched together in order to define each microfluidic channel(s) (5) and the gas-filled chamber (10), and wherein compression or decompression of the chamber (10) causes gas to be expelled or drawn into the chamber (10), which in turn causes movement of the liquid sample within of said/each microfluidic channel (5); and a reader device (100) for use with the microfluidic system, the reader device (100) comprising: force control means (200) for controlling compression or decompression of the gas-filled chamber of the microfluidic system; and detection means for enabling detection of a desired analyte within a liquid sample introduced into the microfluidic cartridge, or analyte reaction product thereof; wherein the force control means (200) comprises a piezoelectric bending actuator (202) which is designed to directly or indirectly compress or decompress the gas-filled chamber (10) through displacement of the actuator (202). 2. Test system according to claim 1, characterized by the fact that the independent microfluidic system is in the form of a cartridge (1) that is inserted into the reader device (100). 3. Test system according to any one of claims 1 or 2, characterized by the fact that the independent microfluidic system is free of liquid before applying the sample. 4. Test system according to any one of claims 1 to 3, characterized in that the top and bottom layers and optionally the middle layer are flat and optionally of uniform thickness. 5. Test system according to any one of claims 1 to 4, characterized in that the top layer and optionally the bottom layer are resilient. 6. Test system according to claim 4, characterized by the fact that the flat substrates are sealed together by application of heat and/or by the use of adhesive, optionally the adhesive is resilient and facilitates the compressibility of each/said chamber . 7. Test system according to any one of claims 1 to 6, characterized in that the microfluidic system comprises a one-way valve (52) that is designed only to allow gas to exit the system when a liquid sample is introduced into the system by capillary action, while not allowing fluid to be introduced into the system through the valve. 8. Test system according to claim 7, characterized by the fact that the valve (52) is positioned adjacent to a stop feature (20) designed to prevent further transport of the sample within the microfluidic channel (5) by action of capillarity alone, and optionally the valve (52) is located within a microfluidic channel (50) of smaller size than said/each microfluidic channel (5) and which is in fluidic communication with one of the microfluidic channels (5). 9. Assay system according to any one of claims 1 to 8, characterized by the fact that it further comprises an analyte binding agent deposited within the channel(s), wherein the analyte binding agent is optionally bound to a surface of the channel(s) and more optionally the binding agent is affixed to a magnetic or paramagnetic particle. 10. Assay system according to any one of claims 1 to 9, characterized in that the system further comprises one or more additional reagents deposited within said microfluidic channel(s), additional reagents these that facilitate the detection of analyte present in the sample, optionally said one or more additional reagents including a label that has been adapted to specifically bind to an analyte to be detected to facilitate analyte detection. 11. Test system according to any one of claims 1 to 10, characterized by the fact that the piezoelectric flexor (202) is in the form of a strip, bar, rod or similar comprising a first immobilized end (201) and a second non-immobilized end (208), wherein the second non-immobilized end (208) is free to flex away from the gas-filled chamber in appropriate electrical signaling, optionally further comprising a foot (210) that is capable of engaging with an outer surface of the gas-filled chamber (10), wherein an upper surface of the foot is in contact with the piezoelectric flexor (202) and wherein the foot (10) is capable, through the action of the piezoelectric flexor (202) , to compress or decompress the gas-filled chamber (10). 12. Assay system according to any one of claims 1 to 11, characterized by the fact that it further comprises detection means to allow detection of a desired analyte or analyte reaction product present within a liquid sample introduced into the microfluidic system , optionally the detection means being an optical detection device, such as a fluorimeter or spectrophotometer. 13. Test system according to any one of claims 1 to 12, characterized in that the reading device (100) additionally comprises a receiving orifice (103), optionally adapted to receive differently sized systems, each system differently sized designed to perform a defined number of tests. 14. Test system according to any one of claims 8 to 12, characterized in that it additionally comprises a permanent magnet (113) to be placed in close proximity to the microfluidic system that has been introduced into the reader device (100), in order of concentrating and maintaining the magnetic/paramagnetic particles in the detection zone (32) of said/each microfluidic channel (5) of the system. 15. Method for conducting a test on a liquid sample, characterized by the fact that it comprises: a) providing a test system as defined in any one of claims 1 to 14; b) compressing an external surface of one/said gas-filled chamber(s) (10) of the microfluidic system, so as to expel gas from said/each gas-filled chamber(s) gas (10); c) introducing a liquid sample to the microfluidic system and allowing the sample to be drawn into said microfluidic channel(s) (5) by capillary action, and/or partially decompressing said(s) /each chamber(s) filled with gas (10) so that gas is drawn into said/each chamber(s) (10), thereby causing a liquid sample to be drawn into said(s)/each microfluidic channel(s) (5); d) allowing one or more reagents to react with any analyte present in a liquid sample; e) optionally partially decompress said/each gas-filled chamber(s) (10) of the microfluidic system, so that the liquid sample is further drawn along said/each channel(s) microfluidic device(s) (5) in the direction of said/each gas-filled chamber(s) (10) and optionally being in contact with a liquid sample with an analyte binding agent and/or one or more other reagent(s), optionally this step being performed as a single step or multiple steps, whereby the sample is pulled to another or a number of successive locations respectively within said/each corresponding microfluidic channel (5). to the number of times a decrease in strength is carried out; f) optionally capturing any analyte or analyte reaction product and compressing said/each gas-filled chamber(s) (10), such that gas expelled from said/each chamber(s) (10) cause a liquid sample and uncaptured material to be pushed away from any captured analyte or analyte reaction product; and, g) detect any analyte or analyte reaction product, or captured analyte or analyte reaction product. 16. Method according to claim 15, characterized by the fact that the volume of gas that is expelled from the chamber(s) (10) causing liquid to be expelled from at least a portion of the (s)/said microfluidic channel(s) (5) where the analyte/analyte binding agent complexes are captured, is sufficient to cause the liquid to be removed from the detection zone (32) or portion thereof, but not yet along the microfluidic channel(s) (5).