Fluid control

Abstract

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

Claims

1. An assay system comprising a self-contained microfluidic system and an associated reader device, wherein (a) the self-contained microfluidic system comprises: a sample input port for receiving a liquid sample to be assayed, the sample input port connected to at least one microfluidic channel, wherein each/said microfluidic channel(s) comprises one or more reagents deposited therein for use in conducting an assay and a detection zone for use in detecting any analyte which may be present in a sample or analyte reaction product; and each/said microfluidic channel(s) is in fluid communication with a compressible, gas-filled chamber downstream from each/said detection zone, wherein the microfluidic system is formed from three layers, which are sandwiched together in order to define each/said microfluidic channel(s) and said gas filled chamber, and wherein said chamber is configured to be compressed or decompressed to expel gas from or draw gas into the chamber, which in turn causes movement of the liquid sample within said/each microfluidic channel; and (b) the reader device for use with the microfluidic system comprises: a 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 within a liquid sample introduced into the microfluidic cartridge, or analyte reaction product thereof, wherein the force control means comprises a piezoelectric bending actuator configured to directly or indirectly compress or decompress the gas filled chamber through displacement of the actuator.

2. The assay system according to claim 1 wherein the assay system is for use in conducting a plurality of distinct assays, wherein the microfluidic cartridge comprises 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 portion of sample using one or more reagents which are present within each of said microfluidic channels prior to liquid sample introduction; and wherein fluid 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 fluid communication with one or more of said microfluidic channels.

3. The assay system according to claim 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, in order that any analyte or analyte reaction product within said/each detection zone can be detected in a substantially liquid free environment.

4. The assay system according to claim 1, wherein the top and bottom layers are planar and of uniform thickness.

5. The assay system according to claim 4 wherein the planar substrates are sealed together by application of heat and/or the use of adhesive.

6. The assay system according to claim 5 wherein the planar substrates are sealed together using an adhesive which is resilient and facilitates with the compressibility of each/said chamber.

7. The assay system according to claim 1 wherein said/each microfluidic channel(s) in the system comprises one or more fluid stop features, which are designed to prevent the sample and/or other fluids from passing through said stop feature(s) by virtue of capillary action alone.

8. The assay system according to claim 1 comprising a one-way valve which is designed only to allow gas to exit the system upon a liquid sample being introduced into the system by capillary action, whilst not permitting fluid from being introduced into the system via the valve.

9. The assay system according to claim 8 wherein the valve is located within a microfluidic channel of smaller dimension than said/each microfluidic channel and which is in fluid communication with one of said microfluidic channels.

10. The assay system according to claim 1 further comprising an analyte binding agent deposited within said channel(s).

11. The assay system according to claim 10 wherein the binding agent is deposited within said/each microfluidic channel(s) of the system, such that upon the sample being applied to the system and being drawn into said/each channel(s), the binding agent is suspended by the liquid sample.

12. The assay system according to claim 11 wherein the binding agent is attached to a magnetic or paramagnetic particle.

13. The assay system according to claim 12 wherein the binding agent or magnetic/paramagnetic particles are deposited within an area of said/each microfluidic channel(s) defined by features at either end of the area of deposition designed to limit movement of the magnetic/paramagnetic particles when initially deposited in said/each channel.

14. The assay system according to claim 1 wherein the system further comprises one or more additional reagents deposited within said/each microfluidic channels(s), which additional reagents facilitate detection of analyte present in the sample.

15. The assay system according to claim 14 wherein said one or more additional reagents includes a label which has been adapted to specifically bind to an analyte to be detected for facilitating analyte detection.

16. The assay system according to claim 1, wherein the piezoelectric bender is in the form of a strip, bar, rod or the like comprising a first immobilised end and a second non-immobilised end, wherein the second non-immobilised end is free to bend away from the gas filled chamber, upon suitable electrical signaling.

17. The assay system according to claim 16 wherein the piezoelectric bender is in the form of a strip.

18. The assay system according to claim 17 further comprising a foot which is capable of engaging with an external surface of the gas filed chamber, wherein a top surface of the foot is in contact with the piezobender and wherein the foot is capable, through action of the bender, of effecting the compression or decompression of the gas filled chamber.

19. The assay system according to claim 16 further comprising optical detection means for enabling detection of a desired analyte or analyte reaction product present within a liquid sample introduced into the microfluidic system.

20. The assay system according to claim 19 further comprising a receiving port adapted for receiving different sized assay cartridges, each differently sized assay cartridge designed to carry out a defined number of assays.

21. The assay system according to claim 16 further comprising a permanent magnet to be brought into close proximity to an assay cartridge which has been introduced into the reader, in order to concentrate and hold the magnetic/paramagnetic particles in the detection zone of said/each microfluidic channel of the cartridge.

22. The assay system according to claim 16 wherein the piezobender is designed to be bent and relaxed using electrical circuitry present in the reader and connected to the piezobender.

23. The assay system according to claim 22 wherein the electrical circuitry is capable of causing the bending of the piezobender at a variable rate such that gas within the system can be drawn into and/or expelled from said/each gas filled chamber at different rates.

24. The assay system according to claim 16 further comprising heating and/or cooling means to allow assays to be conducted at a particular temperature, or plurality of temperatures.

25. A method of conducting an assay on a liquid sample, the method comprising: a) compressing a/said gas filled chamber(s) of the self-contained microfluidic system of the assay system according to claim 1, so as to expel gas from said/each gas filled chamber(s); b) introducing a liquid sample to the self-contained microfluidic system and allowing the sample to be drawn into said/each microfluidic channel(s) by capillary action, and/or partially decompressing said/each gas filled chamber(s) such that gas is drawn into said/each chamber(s) thereby causing the liquid sample to be drawn into said/each microfluidic channel(s); c) allowing one or more reagent(s) to react with any analyte present in the liquid sample; d) optionally partially further partially decompressing said/each gas filled chamber(s) of the microfluidic system, such that the liquid sample is drawn further along said/each microfluidic channel(s) towards said/each gas filled chamber(s) and optionally contacting the liquid sample with an analyte binding agent and/or one or more further reagent(s); e) optionally capturing any analyte or analyte reaction product and compressing said/each gas filled chamber(s), such that gas expelled from said/each chamber(s) causes the liquid sample and uncaptured material to be pushed away from any captured analyte or analyte reaction product; and f) detecting any analyte or analyte reaction product, or captured analyte or analyte reaction product.

26. The method according to claim 25 wherein the analyte/analyte binding agent complexes or analyte reaction product/analyte binding agent complexes to be formed comprise magnetic or paramagnetic particles.

27. The method according to claim 25 wherein the step e) is carried out as a single or multiple steps, whereby the sample is drawn to a further or a number of successive locations respectively within said/each microfluidic channel corresponding to the number of times a decrease in force is carried out.

28. The method according to claim 25 wherein the volume of gas which is expelled from the/said chamber(s) causing liquid to be expelled from at least a portion of the/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 or portion thereof, but not further along the microfluidic channel(s).

29. A multiplex assay platform for use in conducting multiple panels of assays, the multiplex assay platform comprising an assay system of claim 1, wherein the assay system comprises a plurality of the microfluidic systems, each system being capable of conducting a defined panel of assays on a sample and, wherein the reader is constructed to be capable of receiving and verifying each of said plurality of microfluidic systems, whereby the reader is configurable for detecting and/or determining levels of a panel of analytes which may be present in the sample.

30. The assay system according to claim 1 for use in detecting a heart condition and wherein the panel of separate assays is for detecting one or more of the following: lipid levels, apolipoprotein; homocysteine; C-reactive protein (CRP) troponin, BNP; and/or Cardiac enzymes.

Description

(1) The present invention will now be further described by way of example and with reference to the following figures which show:

(2) FIG. 1 shows a microfluidic cartridge in accordance with the present invention;

(3) FIG. 2 shows in detail portion A as identified in FIG. 1;

(4) FIG. 3 shows a reader in accordance with the present invention;

(5) FIG. 4 shows the internal mechanisms of the reader shown in FIG. 3;

(6) FIG. 5 shows in plan view an internal portion of a reader comprising a force control means of the invention;

(7) FIG. 6 shows a sectional view along line A-A of FIG. 5;

(8) FIG. 7: shows schematics of the exemplary cartridge formats which are capable of running different numbers of assays per cartridge;

(9) FIG. 8: shows a comparison plot detecting C-peptide in accordance with the present invention and Siemens Centaur C-peptide assay. N=350;

(10) FIG. 9: shows a bias plot comparison detecting C-peptide in accordance with the present invention vs. Siemens Centaur C-peptide assay. N=294;

(11) FIG. 10: shows a comparison plot detecting D-Dimer in accordance with the present invention and the HemolL D-Dimer HS 500 clinical analyser test;

(12) FIG. 11: shows a comparison plot detecting CRP in accordance with the present invention and the Siemens Dimension CRP clinical analyser test;

(13) FIG. 12: shows a comparison plot detecting hsCRP in accordance with the present invention and the Siemens Dimension hsCRP clinical analyser test;

(14) FIG. 13: shows a dose response curve of Plasmodium falciparum (P.f) HRP2 analyte spiked into blood and run according to the present invention;

(15) FIG. 14: shows a schematic of reagents used in a multi-step troponin I assay;

(16) FIG. 15: shows a schematic representation of the steps involved in a multi-step troponin I assay;

(17) FIGS. 16(a) and (b): show plots of Troponin I measured in healthy individuals using a multi-step assay according to the present invention as compared to the Siemens Centaur troponin Ultra test;

(18) FIG. 17 shows a comparison of a C-peptide assay response conducted in accordance with the present invention before and after buffer removal by air;

(19) FIG. 18 shows a comparison of a C-peptide assay response conducted in accordance with the present invention on a sample of blood, before and after removal of the liquid sample by air; and

(20) FIG. 19 shows a method comparison plot using a cartridge of the present invention which comprises a channel without a gas chamber to control liquid filling and/or removal, in order to carry out an INR test and the Roche CoaguCheck INR test.

(21) FIG. 1 shows a microfluidic cartridge (1) in accordance with the present invention, for carrying out 4 separate assays from a single sample. The cartridge (1) comprises a liquid sample input port (3) connected to a microfluidic channel (4) which splits into a plurality of separate channels (5 and 7). Each channel (5) extends within the cartridge (1) and is fluidly connected to gas filled chambers (10). The further channel (7) which is not connected to a gas filled chamber is a control channel for use in multiple control measurements. In use, a fluid sample fills the channels (5 and 7) and this can be detected by electrodes (not shown) which are in electrical contact with corresponding electrical contacts within the reader. Upon the reader detecting an appropriate signal that a sample has been loaded into the cartridge (1) the reader can start the assays. There is also provided a sink (13) for receiving liquid. Immediately upstream of the sink there is a liquid stop (15) which prevents liquid from entering the sink immediately (13) by capillary action alone. Thus, on initial sample application using a capillary application, the liquid sample does not pass the liquid stop (15)

(22) Describing each channel (5) in more detail, there are printed features (20, 22, 24, 26) which are designed to limit movement of any reagent which is deposited within each channel (5) during the manufacturing process. Adjacent the printed feature (20) and represented by section A, as shown in more detail in FIG. 2, is a smaller dimensioned channel (e.g. 0.1-0.2 mm) (50) extending perpendicularly away from assay channel (e.g. 0.75-1 mm) (5). Within each channel (50) is a one way valve (0.1 mm by 0.9 mm) (52) which is designed to permit gas or air present with each channel (5) to exit the cartridge (1) upon application of a liquid sample. Thus, upon application of a sample to the cartridge by a capillary application, the sample fills the channel (4) displacing air which is present in the channels (5) which exits 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 has been deposited one or more binding and/or reaction agents designed to react with and bind a particular analyte or reaction product thereof which may be present in a liquid sample to be assayed. For example deposited in the first zones (28) of said channels (5) may be magnetic particles functionalised with an antibody designed to specifically bind a first epitope of an analyte to be detected. Deposited with a second zone (30) of each channel may be fluorescently labelled latex particles functionalised with a further antibody designed to specifically bind a different epitope of analyte to be detected. Located distal/proximal to zones (28, 30) is a detection zone (32) where label/analyte/magnetic particle complexes can be detected.

(23) Located distal/proximal the detection zones (32) are the gas filled chambers (10), which are designed to collocate 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 assay channels (5). A decrease in the applied force to the chambers (10) causes air to be drawn back into the chambers (10) from the assay channels (5).

(24) In use, the cartridge (1) is inserted into a reader (100) as shown in FIG. 3. The reader has a closeable door (102), which may be opened in order to access a cartridge receiving port (103) of the reader. Once a cartridge has been inserted into the reader (100) and a sample applied to the cartridge (1), the door (102) may be closed. The reader houses a number of features which are designed to contact the cartridge (1) and/or facilitate with carrying out the assay of the present invention as will be described in more detail. The top surface of the reader (100) comprises a touch screen display (104) which allows a user to interact with the reader (100), as well as to receive information regarding the performance of any assays.

(25) FIG. 4 shows the internal features of the reader (100). The reader includes a rechargeable battery (110) for powering the reader and its various functions as will be described. Power to charge the battery (110) is provided via a DC jack (106). The reader (100) further includes a heater (111) for heating the cartridge (1) when required; optics block (112) containing the necessary optics for detecting a fluorescent signal from the cartridge (1); a movable magnet (113) which is designed to immobilise magnetic particles within the detection zone (32) of the cartridge; and a lever mechanism (114) which is designed to contact the chambers (10) of the cartridge (1) and apply a force so as to cause air to be expelled from the chambers (10).

(26) In use, a cartridge (1) is inserted into the reader (100) until the cartridge contacts an alignment feature (122) within the reader (100). Correct insertion of the cartridge (1) is detected by electrodes which are present on the cartridge with corresponding contacts which are present in the reader. This signals to the reader that a cartridge (1) has been correctly inserted and the start of an assay process may be commenced. A motor (120) is signaled to activate a rack and pinion mechanism. The gear (124) is turned in a clockwise direction so as to cause a rack mechanism (126) of a lever (128) to move vertically upwards. This movement causes the other end (132) of the lever (128), in the form of a finger, to move downwards and into contact with chambers (10) of the cartridge (1). Continued functioning of the motor causes the rack mechanism (126) upwards, with a corresponding downward movement of the other end (132) of the lever (128), such that an increasing 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). At this point the user will be advised by a message on the display (104) that a sample may now be applied to the cartridge (1).

(27) A sample is contacted with and introduced into the cartridge (1) by way of the input port (3). The sample fills the channels (4, 5, and 7) by capillary action, as previously described, with air being vented 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 turn the gear mechanism (124) in an anti-clockwise direction which in turn causes the rack mechanism (126) in a downwards direction and the other end (132) of the lever (128) upwards, such that the force as applied to the chambers (10) of the cartridge (1) is reduced. This reduction in force as applied to the chambers (10) causes air to be drawn back into the chambers (10), which in turn draws the sample into the first zones (28) of the channels (5). The motor (120) and associated lever movement are able to carefully control the reduction in force applied to the chambers (10) which controls 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 derivatised 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 which may be present in the liquid sample to bind to the functional analyte binding moieties on the surface of the magnetic particles in order to form analyte/magnetic particle complexes. After a defined 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 draws the sample and analyte/magnetic particle complexes into the second zone (30) of the channels (5). The second zone (30) of each channel (5) contains functionally derivatised fluorescently labelled latex particles which are capable of binding to the analyte/magnetic particle complexes in order to form a latex particle/analyte/magnetic particle complex sandwich. After a further period of time the force applied to the chambers (10) is further reduced and the liquid and associated complexes present therein, is drawn into a detection zone (32).

(28) 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) such that the magnet is brought into close proximity with the detection zones (32) of the cartridge, such that the magnetic complexes are attracted to the magnet and held in place within the detection zone (32) by the magnetic force of the magnet (113). Thereafter the motor (120) is reapplied so as 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) which results in the liquid sample and non-magnetically bound material which is present in the detection zone (32) to be pushed away from the detection zone (32) and along the channel (5) with a portion of the liquid exiting into the sink (13). It may not be necessary to expel all of the liquid into the sink (13) and in fact it may only be necessary to remove the liquid from the detection zone (32), such that the resulting magnetically bound complexes are present in an essentially air environment. This can be particularly advantageous in terms of not using extra sample volume to perform a wash as occurs in lateral flow products and no requirement for an on-strip buffer pouch or in-meter buffer delivery system.

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

(30) Following removal of the liquid from the detection zones (32), the captured complexes are present in an essentially liquid free environment and may be detected using a detector which 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 on the captured latex particle/analyte/magnetic particle complexes.

(31) In an alternative embodiment to that shown and described in relation to FIG. 4, piezoelectric benders may be employed to control force as applied to the gas filled chambers of the cartridge. FIG. 5 shows a force control means (200). The force control means (200) comprises a series of piezoelectric benders (202) which are fixed at a first end (201) by a fixing block (204). Each piezoelectric bender is also electrically coupled at the first end to electrical connections (206) which control the electrical signal provided to each bender (202). As can be seen each bender (202) is connected to its own set of electrical connections (206), such that each bender is independently controllable. As shown in FIG. 6, the other end (208) of each bender (202) rests upon the top surface (209) of a foot (210) which in use is designed to contact the external surface of a gas chamber of a microfluidic cartridge (220) of the present invention.

(32) FIG. 6 shows a sectional view along line A-A of FIG. 5 so the various parts of the force control means (200) and how they function can be better understood. In FIG. 6 the force control means (200) is shown together with a microfluidic cartridge (220) when correctly inserted within 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). The bottom 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 being applied to the foot (210) by the piezo bender (202), the foot (210) is capable of applying a variable force to the gas chamber of the microfluidic cartridge (220).

(33) As shown in FIG. 6, the piezo bender (202) is in its non-formed rigid state. In this embodiment, the force control means (200) is constructed such that the piezo bender (202) is able to extend maximum force upon the foot (210), such that the bottom surface (212) of the foot (210) pushes downwards and compresses the gas filled chamber, causing the gas within the chamber to be expelled from the chamber.

(34) Although not shown, applying an electrical charge to the piezo bender (202) will cause piezo bender (202) to bend and the end (208) of the piezo bender (202) to bend upwards. This upward bending of the piezo bender (202) reduces the force as applied to the foot (210), which in turn causes the foot (210) to reduce the force as applied to the gas filled chamber of the cartridge (220). Reduction of the force as applied to the gas filled chamber, provides a decompression to the gas filled chamber and a corresponding ingress of gas back into the chamber. Through appropriate electrical signaling it is possible to bend and relax the piezo bender (202) resulting in the gas filled chamber being decompressed or compressed accordingly and gas being expelled or drawn into the chamber.

(35) Many piezobenders are known in the art and may be suitable for use in the present invention. The skilled addressee will choose a bender which is suitable for a particular purpose. The present inventors have employed a variety of such piezobenders with displacement of up to several millimeters and response times in the millisecond range. A voltage programmable amplifier can be used to control each piezobender. Suitable amplifier include a 32-channel, 14-Bit DAC with Full Scale Output voltage programmable from 50V to 200V (AD5535) or High Voltage Quad-Channel 12-Bit voltage output DAC (AD5504) available from Analog Devices (Norwood, Mass. 02062, USA). Forces of 1N-2N may be attainable.

(36) The above provides a description of specific embodiments of the present invention, but the present invention is designed to be in the form of a platform which can easily be adapted. For example, the vent position can be changed to allow capillary fill to different positions within the channel (5), or a vent omitted altogether and sample filling taking place by an active fill following gas being expelled from the chamber (10) and the sample by drawn into the cartridge (1) and channels (5,7) by air returning to the gas chambers following a release in pressure being applied to the chambers (10)

(37) Moreover, the reader may be designed to utilise multiple test formats with a family of strip sizes defined by the product requirements. The strip may be designed to be manufactured in 2, 4, and 10 channel formats, for example, for specific product configurations and panel tests (see FIG. 5 showing different strip sizes). The availability of different strip sizes allows the present system to deliver multiplexed tests across mixed technologies to meet the specific user requirements of the Point of Care market at an increased performance and reduced cost structure as compared to established products in targeted markets.

(38) With reference to FIG. 7 showing different size strips, the 2 channel cartridge is designed for single assays with controls, the 4 channel cartridge for panels of 2-3 analytes with controls and the 10 channel cartridge allows complex assays of mixed technologies and products that require high multiplexing capability (e.g. Drugs of Abuse) to be performed. The described platform has a highly flexible sample and assay architecture and reader control and measurement capability allowing forward compatibility for new opportunities to be exploited as new test panels or test types are identified or move to Point of Care.

(39) Although the primary measurement technology is fluorescence the platform also incorporates electrochemical measurement and other methodologies can easily be incorporated. This is discussed in further detail below.

(40) To deliver multiple test types and formats on a single platform, a set of flexible core technology capabilities and controls have been developed which can be used as required and in sequences that deliver the different assay format steps. The system architecture design principles are: Magnetic Particle Capture Phase Liquid movement control Liquid removal from the detection zone Label Detection in air Multi-Channel Multiplexing Intra-channel Multiplexing Dynamic Range On-board Controls Electrochemical Measurements Heating and Temperature Control Sample Pre-treatment

(41) This platform architecture allows the many different test types and technologies to be formatted on the system. Each technology core principle is discussed below.

(42) Magnetic Particle Capture and Liquid Control

(43) 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 optimise performance of each test type. Paramagnetic particles ranging from 100 to 1000 nm have been utilised 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 the assay sensitivity and range requirements. Typical sizes of the fluorescent particles may be in the range 40 nm-4000 nm

(44) Some assays, such as C-reactive protein (CRP), require relatively high concentrations of analyte to be measured and utilise direct fluorophore labelled antibody conjugate in combination with magnetic particles, whilst high sensitivity assays generally utilise fluorescent particle labels in combination with magnetic particles. Importantly, both capture and label phases are mobile in the sample to drive capture events. This is further helped by the fact that unwanted flow within the strip is minimised. During channel filling, the sample flows over the dried test reagents. The reagent dissolution and therefore flow front effects are minimised by using formulations that allow good channel filling but result in controlled slower dissolution. After the initial sample fill event, the flow is stopped such 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 rate errors affecting the interrogated sample volume or binding kinetics.

(45) Performing the reagent dissolution and analyte capture in a optionally mixed, static, fixed volume as opposed to a variable flowing system (e.g. Lateral flow, Triage) significantly improves assay precision and accuracy.

(46) For more complex assays, such as Troponin (as described elsewhere), the assay is more efficiently performed as a multistep procedure using multiple reagent zones. In this case, the meter functionality of being able to compress the gas chambers (10) to expel gas and perform the liquid removal from the detection zone is also used to effect fine liquid movement control within the cartridge (1) and associated channels. 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 assay channels. The chambers (10) remain compressed by the meter during sample application and sample filling is by capillary action or entirely under gas driven fluidic control. The high-resolution motor or piezobender within the meter allows very controlled incremental release or increase of pressure on the gas chambers (10) with the rate and amount of pressure change specific to a particular test. This feature provides a number of important advantages including the ability to of mixing using fine positive and negative bending of any piezobenders.

(47) Sample fill time can have a significant effect on performance of a product by introducing variability of reagent dissolution, fluid front effects and the volume of sample interrogated. The fluidic control reduces the variation in fill time by directly controlling the sample fill rate. Fluidic control allows the sample to be moved in a controlled time to different zones within each channel, allowing sample pre-treatments and multi-step assays to be performed (described herein). Fluidic control and isolation is also a necessity for closed systems as required for NAT assays (see below).

(48) Liquid Removal from the Detection Zone

(49) Liquid movement and control is achieved by compressing or releasing the gas chambers (10) on the test cartridge using a motor and force applicator, or piezoelectric bender mechanism which contacts the fluid chambers (10). The resulting gas movement from each chamber (10) allows fine control of movement of sample and reagents including removal of unbound label from the detection zone (32) of the test channel and optionally into the sink area (13).

(50) The embedded fluid control function within each cartridge brings a number of important differentiating advantages.

(51) Firstly, the described system provides a very effective separation of bound and unbound assay components using gas control of liquid movement. This is important because it completely avoids the complexity and cost of an on-strip liquid reagent pouch or in-meter replaceable liquid wash reagent packs.

(52) Secondly, the present invention further enables the use of laminate manufacturing technology with very low cartridge costs and manufacturability using high throughput, high control web production systems.

(53) Thirdly, the removal of the sample and the unbound label from the detection zone (32) by the use of gas means the measurement of fluorescent labels can be made in an essentially liquid free, gas environment.

(54) Label Detection in Air

(55) Label measurement in gas results in several significant technical advantages for making fluorescent measurements compared to standard assay protocols of prior art products.

(56) Use of an essentially gas environment significantly decreases the quenching effect of a liquid sample thereby removing a primary source of assay variation and matrix effect. For example, the presence of blood cells and plasma proteins quenches the fluorescence signal reducing the sensitivity and increasing the variability of the fluorescence measurement. The measurement of fluorophores in gas or air environment enables the use of fluorophores that would not necessarily have been chosen due to sample quenching. This allows simpler optical designs, optimisation of fluorophores for each assay and multiplexing within a single channel. As described by example below and with reference to FIGS. 15 and 16, detection in air provides a significant improvement in sensitivity as compared to detection in buffer or whole blood.

(57) In summary, the use of gas to remove the sample and unbound label approach reduces assay variation by decreasing sample matrix quenching effects and gives access to a greater range of fluorophores for assay optimisation. This translates into assay design flexibility, speed of assay and unrivalled performance.

(58) Multi-Channel Multiplexing

(59) The platform of the present invention has multi-channel and intra-channel multiplexing capability. Panel tests may be delivered via multiple channels within a single strip combined with a scanning optical head to measure the label, e.g. fluorescence intensity in each channel. The number of channels can be varied depending on the product requirements.

(60) This allows the development of panel tests with each channel containing a different assay e.g. cardiac panel, metabolic panel, etc. As individual assays are spatially distinct within separate channels, each assay can be configured with unique reagents within the multi-channel strip. This brings a number of key advantages:

(61) Firstly, each assay can use an optimal formulation including reagents, buffers, pH etc. for: dissolution of reagents, anti-coagulation, neutralisation of matrix effects (HAMA etc.), optimum sensitivity, linearity, range and stability of the assay. It is not necessary to find a compatible optimisation for multiple assay reagent sets or compromise assay performance in order to develop panel products. Each assay can exist within its own optimum formulation within an individual channel and maintain its respective high assay performance.

(62) By contrast, multiplexing tests within a single channel inherently compromises performance of the individual tests as the reagent formulation has to be compatible with all assays. Individual assay requirements often conflict, for example something as fundamental as pH will significantly affect assay performance.

(63) Multi-channel multiplexing translates into panel test design flexibility, simplicity and speed of panel assay development and maintenance of single assay performance across panels.

(64) Secondly, the multi-channel approach allows the present platform to realise novel panel products that combine different assay technologies and different transduction methods on a single strip.

(65) There is increasing evidence that measurement of molecule families may be advantageous over measurement of a single molecule of that family. For example, the natriuretic peptides used in congestive heart failure stratification are generally separated into BNP and NT-proBNP tests. Multi-channel multiplexing allows measurement of proBNP, BNP, NT-proBNP and other natriuretic peptide forms on one strip and avoiding antibody epitope crossover within the peptide family. By contrast, intra-channel multiplexing leads to increased non-specificity of molecule family measurements. The presently described multi-channel approach is applicable to the troponin test market whereby different troponin isoforms can be measured in separate channels to improve diagnosis of myocardial infarctions.

(66) Intra-Channel Multiplexing

(67) Where ratio-metric measurements are required, for example HbA1c and blood ion measurements, intra-channel multiplexing is necessary in order to achieve the most accurate assay performance. The present platform achieves this by measuring more than one fluorophore in a single channel.

(68) The combination of multi- and intra-channel multiplexing allows for flexible and powerful product combinations with on-board controls that will improve accuracy and confidence.

(69) Dynamic Range

(70) 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 has to be capable of measuring high concentrations in order to monitor the changes observed in myocardial infarction patients. Dynamic range often leads to non-linearity across the required measurable range, which impacts precision and accuracy.

(71) The multi-channel design allows challenging tests with large dynamic ranges to be split into multiple channels on the strip covering high sensitivity and high concentrations of the required measurable range in a linear manner.

(72) For troponin (I and/or T forms), one channel could contain reagents optimised for measurement of 0-100 pg/ml whilst another channel could contain reagents optimised to measure 50-1000 pg/ml and a further channel optimised for 500-50000 pg/ml. The sensitivity and range each have their own calibration parameters with the sample concentration assigned from the confidence interval of the two results.

(73) On-Board Controls

(74) The present platform incorporates on-board control features to verify the validity of the test results obtained. Each test type requires unique on-board assay controls as well as several generic features. All tests can have fill-detect to ensure adequate sample application and used cartridges cannot be re-tested. Where required, the cartridge incorporates a hematocrit measurement to adjust 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.

(75) A microprocessor and associated software can control the timings, temperature, fluid control etc. for each particular assay, as these may have different requirements within a single cartridge.

(76) Electrochemical Measurements

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

(78) Heating and Temperature Control

(79) Temperature is a significant variable in most tests. For some assays, temperature effects can be compensated using a temperature correction algorithm. However, this is often problematic to determine for individual cartridge batches and fixed compensation can become a source of error in itself. Characterisation of temperature profiles across all process and matrix variables can significantly impact the development cycle of the product. In some products such as PT/INR and molecular tests, adequate temperature control is critical for functionality and performance of the test. The present platform allows incorporation of an integrated heating capability that provides the optimum temperature requirements for each test type. Typical operating temperatures are used for immunoassays (34° C.), PT/INR (37° C.) and nucleic acid detection (>37° C.) etc. The heating capability can be optimised to deliver a range of strip and pre-treatment controlled temperatures for maximum flexibility in test protocol.

(80) Sample Pre-Treatment

(81) Control of on-strip sample movement allows sample pre-treatment before the sample is presented to the assay specific reagents. This approach can be applied to immunoassays, for example, to remove interferents such as HAMA species or lipid panels to remove unwanted fractions for specific lipid measurements (e.g. HDL). The on-strip fluidic steps mimic the capabilities used by clinical analysers for optimising product performance allowing sample matrix and interferences to be rapidly resolved during product development.

(82) Exemplary Test Descriptions and Test Data

(83) One Step Immunoassays

(84) Summary Test Sequence: 1. Cartridge Insertion into the reader 2. Cartridge gas chamber compression by reader 3. Sample application to the cartridge, filling by capillary action or by reader controlled filling. 4. Wetting of the cartridge fill detect electrodes determines the test start timing 5. The sample rehydrates the dried reagents which contain: a. anti-analyte antibody (epitope 1) functionalised paramagnetic particle phase b. anti-analyte antibody (epitope 2) functionalised fluorescent label/particle phase 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 time (typically 2 minutes). 8. A magnetic field is applied to the strip localised to the optical detection zone accumulating the paramagnetic particles to this location forming a particle-analyte-label complex band 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 cartridge gas chambers. This compression force expels the gas from the gas chambers via the test channel resulting in the sample liquid and unbound fluorescent label/particles being expelled from the detection zone and optionally the channel and into the sample waste sink. The magnetic field is applied for the entirety of this step holding the paramagnetic particle-analyte-label complexes in the detection zone location by the magnetic field whilst the sample is expelled from this area. 10. The meter optical head scans 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 the fluorescence intensity is transformed into analyte concentration.

(85) Example Performance data sets for one step immunoassays are shown in FIGS. 8-13:

(86) C-Peptide

(87) C-peptide is a short 31-amino-acid polypeptide that connects insulin's A-chain to its B-chain in the pro-insulin molecule. Pro-insulin is cleaved into insulin and C-peptide in equimolar concentrations. In the context of diagnosis 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 assay against the commercially available ADVIA Siemens Centaur benchtop system (see FIG. 8).

(88) Table 1 below shows the percentage of results which are within a given bias of the reference system for the C-Peptide range as indicated. This shows that the present assay achieves typically around 95% of results within 20% of the reference system.

(89) TABLE-US-00001 TABLE 1 Accuracy of C-Peptide Assay within 10% within 15% within 20% above 0.5 ng/ml 73.1 89.2 95.9 above 0.25 ng/ml 70.9 87.6 94.7 above 0.1 ng/ml 69.7 86.2 93.4

(90) A bias 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 (FIG. 9) in comparison to an established commercially available clinical analyser. The percent bias of each point to the reference system value is plotted vs. the reference value. The plot shows the present assay system has comparable clinical accuracy to an established lab system.

(91) D-Dimer

(92) 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 the fibrin protein.

(93) D-dimer concentration is used to help diagnose thrombosis. It is an important test performed in patients with suspected thrombotic disorders. While a negative result practically rules out thrombosis, a positive result can indicate thrombosis but does not rule out other potential causes. Its main use, therefore, is to exclude thromboembolic disease where the probability is low.

(94) The inventors carried out a dose response analysis using the presently described methodology and compared results with those from a HemolL D-Dimer HS 500 (a commercially available clinical analyser) (see FIG. 10)

(95) C-Reactive Protein (CRP)

(96) 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 interleukin-6 secretion by macrophages and T cells.

(97) CRP has diagnostic utility for a number of disease types which can be summarised as follows: 1. Inflammation status in type 1 diabetic patients 2. Antibiotic stewardship for infection control and general infection status 3. Cardiovascular disease 4. Certain cancers

(98) A method comparison plot is shown in FIG. 11. The reportable range required is 5-200 μg/ml.

(99) High Sensitivity CRP (hs-CRP)

(100) High sensitivity CRP (hs-CRP) is used in assessing the risk of developing cardiovascular disease. General guidelines are as follows:

(101) 1. Low: hs-CRP level under 1.0 mg/L

(102) 2. Average: between 1.0 and 3.0 mg/L

(103) 3. High: above 3.0 mg/L

(104) A method comparison plot is shown in FIG. 12. The data demonstrates the present platform is well capable of measuring the required concentrations of hs-CRP.

(105) Malaria Plasmodium falciparum HRP2

(106) 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 has been used to demonstrate the measurement of HRP2 in blood samples. HRP2 protein was spiked into bloods and measured on the present platform and on Standard Diagnostics (SD) malaria Pf rapid test.

(107) The lowest HRP2 concentration measured on the present platform was 0.25 ng/ml. In comparison, using the SD test a very faint band was observed for 5 ng/ml. Lower concentrations could not be measured. The 0.25 ng/ml present platform test result took 7 minutes verses the recommended 30 minute test time required for SD test to measure the 5 ng/ml concentration. The 30 min assay time is necessary for the competitor tests to wash out the unbound gold sol label 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 wash step.

(108) The data was analysed and results are summarised in FIG. 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.

(109) Multi-Step Immunoassay—e.g. Troponin

(110) The present platform is configurable to carry out multi-step assays allowing step-wise binding reactions to occur to optimise binding kinetics, test time and sensitivity.

(111) In the high sensitivity Troponin assay, the antibody paramagnetic particle binding steps and label/particle binding steps are dissociated to significantly improve binding rate and capture efficiency of the analyte-antibody paramagnetic particle binding step for very low concentrations of Troponin. Subsequent stepwise binding of the label particle and the paramagnetic particle using high affinity anti-fluorescein isothiocyanate and Biotin-Streptavidin functionalized particles, respectively, enable higher capture and transduction of the bound Troponin complex.

(112) Summary Test Sequence: 1. Cartridge insertion into the reader 2. Gas chamber compression by the reader 3. Sample application to the cartridge, filling by capillary action to first vent-stop feature where first reagents are located (labelled antibodies) 4. Reagent re-solubilisation and antibody-analyte incubation and binding time. 5. A small chamber decompression results in the liquid sample being drawn further along the channel locating the sample reagent mix over a secondary reagent. 6. Reagent re-solubilisation and antibody-analyte-particle label incubation and binding time. 7. A second small chamber decompression results in the sample being moved further along the channel locating the sample reagent mix over a third reagent 8. Reagent re-solubilisation and antibody-analyte-particle label-paramagnetic particle incubation and binding time. 9. A magnetic field is applied to the cartridge localised to the optical detection zone accumulating the paramagnetic particles to this location forming an antibody-analyte-particle label-paramagnetic particle complex band in each channel. 10. Sample liquid and unbound label is removed from the detection zone by recompression of the chambers expelling the sample and unbound label from the optical detection zone 11. The reader's optical head scans across the strip and the fluorescence intensity for each channel is measured. The fluorescence intensity is proportional to the Troponin analyte concentration. Troponin I (TnI) assay—reagents are identified in FIG. 14 Step 1: This is a passive capillary fill. The TnI assay uses two capture antibodies each of which is tagged with a biotin group. The label antibody is tagged with a Fluorescein isothlocyanate (FITC) group. The biotin groups and FITC molecule serves as immunogenic tags for the second and third step. Step 2: The sample from step one is moved to a secondary reagent deposition area by fluidic reader control (chamber decompression). This deposition contains anti-FITC antibody coated latex particles. The anti-FITC latex particles will bind the FTIC tagged antibody (which is bound to the TnI complex). This reaction is rapid. Step 3: The sample is moved to the third deposition zone by fluidic reader control. The third deposition area contains streptavidin coated magnetic particles. The streptavidin paramagnetic particles will rapidly bind the biotin labelled antibodies which are bound to the TnI complex. Paramagnetic particle accumulation is followed by the sample/unbound label removal. The fluorescent optical scan is then performed. The fluorescence intensity is proportional to the TnI concentration. A schematic of the above method is shown in FIG. 15 Step 1 is capillary fill, step 2 & 3 are under reader fluid control. This approach is very attractive since it has generic application and greatly simplifies the assay reagents plus very importantly leads to excellent assay performance (see exemplary results shown in FIGS. 16(a) and (b)), which the sensitivity of the present method over a wide concentration range. For example, the anti-FITC latex is a generic label for other assays (e.g. BNP), likewise the streptavidin paramagnetic particles are also generic between assays. Batch to batch production of reagents will become much easier for the challenging assays such as TnI.

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

(114) It is possible for the present cartridges to also run assays which 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. The PT and INR are assays evaluating the extrinsic pathway of coagulation (PT/INR). They are used to determine the clotting tendency of blood, in the measure of warfarin dosage, liver damage, and vitamin K status.

(115) A method comparison plot of the PT/INR measurement is shown in FIG. 19 which was generated using a channel which does not have any fluid control provided by 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 allowing normalisation of fill rates. In comparison to previously described immunoassay examples, the channel is widened in the detection area of the strip in order to permit an increased volume of sample to be assayed. In addition, the INR/PT specific reagents are deposited in this area. The reagents contain all the required components to initiate the extrinsic clotting cascade and a specific thrombin fluorophore substrate which is converted from a non-fluorescent form to a fluorescent form by thrombin. The capillary filling resuspends the reagents and permits detection of thrombin activity. The measured thrombin activity (fluorescent intensity) is used to determine the PT/INR result.