MULTIPLEXED ASSAY

Abstract

A microfluidic device is provided for conducting multiplex immunoassays to detect multiple target analytes in a liquid sample using a set of tags and anti-tags. The device comprises an array of different anti-tags immobilised on a surface of a sensor for measuring a signal. Each anti-tag is arranged to immobilise a tagged antibody or tagged antigen thereby in use separating tagged immune complexes from the liquid phase affecting the signal measured. The device comprises a sensor chamber for tagged immune complexes to form in the liquid phase and one or more detection zones each comprising the array of different anti-tags for immobilising tagged immune complexes. In use, liquid comprising the tagged immune complexes flows or is fluidically driven from the sensor chamber to the detection zone(s).

Claims

1: A microfluidic device for conducting multiplex immunoassays to detect multiple target analytes in a liquid sample using a set of tags and anti-tags: wherein the device comprises an array of different anti-tags immobilised on a surface of a sensor for measuring a signal, wherein each anti-tag is arranged to immobilise a tagged antibody or tagged antigen thereby in use separating tagged immune complexes from the liquid phase affecting the signal measured; wherein the device comprises a sensor chamber for tagged immune complexes to form in the liquid phase, wherein the device comprises one or more detection zones comprising the array of different anti-tags for immobilising tagged immune complexes, and wherein, in use, liquid comprising the tagged immune complexes flows or is fluidically driven from the sensor chamber to the detection zone(s).

2: The device according to claim 1, wherein the detection/signal measurement comprises colorimetric detection, fluorometric detection, faradaic or non-faradaic impedance spectroscopy, or redox active precipitate detection.

3: The device according to claim 1, wherein either: i) a portion of the surface of the sensor comprises a conductive or semi conductive polymer: or ii) the surface of the sensor comprises the surface of one or more electrodes.

4: The device according to claim 1, wherein the device further comprises an array of electrodes with a different anti-tag on each electrode.

5: The device according to claim 1, wherein the device further comprises a microfluidic delay channel between the sensor chamber and the detection zone(s) to increase the liquid phase incubation time of reagents that react to form tagged immune complex before reaching the anti-tags.

6: The device according to claim 1, wherein the target analytes comprise antibodies and/or antigens.

7-9. (canceled)

10: The device according to claim 1, wherein the device further comprises an array of 6 different anti-tags, and wherein the 6 different anti-tags are antibodies against: FITC or biotin, Flag, His-6, c-Myc, V5 and GST.

11. (canceled)

12: The device according to preceding to claim 1, wherein the multiplex assay comprises one or more of the following immunoassay formats: a. two site immunometric or sandwich assay with anti-tag separation; b. competitive immunoassay with anti-tag separation; c. competitive immunoassay (inverted) with anti-tag separation; d. direct immunoassay for detecting antibodies with anti-tag separation; and e. indirect immunoassay for detecting antibodies with anti-tag separation.

13: The device according to preceding to claim 1, wherein the array of different anti-tags is arranged to conduct immunoassays of at least two different formats on a single device.

14-15. (canceled)

16: A method for detecting multiple target analytes in a liquid sample, using the device according to claim 1 wherein at least one assay is a two site immunometric sandwich assay with tag/anti-tag for detecting target analyte X, wherein a capture antibody carries the tag and is specific for the target analyte X, wherein a signal antibody is also specific for the target analyte X, wherein both antibodies bind the target analyte X in the liquid phase as it migrates through the device, wherein an immobilised anti-tag binds to the tag and thereby removes the complex from the liquid phase as it comes into contact with it, and wherein unreacted signal antibody flows away from the sensor.

17: A method for detecting multiple target analytes in a liquid sample, using the device according to claim 1, wherein at least one assay is a competitive immunoassay with anti-tag separation for detecting target analyte X, wherein target analyte X and analyte labelled with a tag compete for binding on the antibody which carries a signal, and wherein, on reaching the anti-tag, tagged analyte X becomes immobilised, such that in the absence of target analyte the signal will be maximal, whereas high target analyte concentrations will yield minimal signal.

18: A method for detecting multiple target analytes in a liquid sample, using the device according to claim 1, wherein at least one assay is a competitive inverted immunoassay with anti-tag separation for detecting target analyte X, wherein target analyte X and analyte labelled with signal compete for binding on an antibody that carries a tag, and wherein, on reaching the anti-tag, the tagged antibody becomes immobilised, such that in the absence of target analyte the signal will be maximal and high target analyte concentrations will yield minimal signal.

19: A method for detecting multiple target analytes in a liquid sample, using the device according to claim 1, wherein at least one assay is a direct immunoassay with anti-tag separation for detecting target analyte X, wherein target analyte X is an antibody, wherein, in the liquid phase, the target analyte X binds a signal labelled antigen and the target analyte X is captured by a capture antibody with a tag, and wherein the complex is separated when the immobilised anti-tag binds the tag on the capture antibody and excess signal labelled antigen migrates away from the sensor.

20-21. (canceled)

22: A method for detecting multiple target analytes in a liquid sample using the device according to claim 1, wherein at least one assay is an indirect immunoassay with anti-tag separation for detecting target analyte X, wherein target analyte X is an antibody, wherein, in the liquid phase, signal antibody binds the target analyte X, which binds tagged antigen, wherein tagged antigen becomes immobilised when bound to the anti-tag, wherein the signal generated is proportional to the amount of target analyte X in the sample, and no target analyte X results in no detectable signal, and wherein excess unbound signal antibody is removed before signal readings are taken.

23. (canceled)

24: The method according to claim 16 wherein the device comprises a microfluidic delay channel configured to increase the liquid phase incubation time of reagents that react to form a tagged immune complex before they reach the anti-tag.

25: The method according to claim 16, wherein the complimentary anti-tags/tags have medium to high binding affinity to allow for relatively short incubation times and rapid immobilisation of tagged immune complex.

26: A method for detecting multiple target analytes in a liquid sample, using the device according to claim 1, wherein the multiplex assay comprises one or more of the following immunoassay formats: a. two site immunometric or sandwich assay with anti-tag separation; b. competitive immunoassay with anti-tag separation; c. competitive immunoassay (inverted) with anti-tag separation; d. direct immunoassay for detecting antibodies with anti-tag separation; and e. indirect immunoassay for detecting antibodies with anti-tag separation, and wherein the detection/signal measurement comprises colorimetric detection, fluorometric detection, faradaic or non-faradaic impedance spectroscopy, or redox active precipitate detection.

27: A kit comprising: a. a device according to claim 1; and b. reagents, wherein the reagents comprise tag reagents.

28: A method for detecting multiple target analytes in a test liquid sample using a set of tags and anti-tags, the method comprising the steps of: a. providing a microfluidic device comprising an array of different anti-tags immobilised on a surface of a sensor for measuring a signal, wherein each anti-tag is arranged to immobilise a tagged antibody or tagged antigen; and b. adding a test liquid sample to the sensor chamber in the presence of reagents, comprising tag reagents, and optionally signal reagents e.g. signal antibody(s) or signal analyte(s)/antigen(s), so that a tagged immune complex can form in the liquid phase, wherein the array of different anti-tags are immobilised in a detection zone, and the tagged immune complex forms in the sensor chamber upstream of the detection zone and flows or is fluidically driven to the detection zone, and wherein, as a result of the anti-tags and tags binding, tagged immune complexes are separated from the liquid phase, affecting the signal measured by the sensor, and determining the presence or amount of each target analyte.

29-30. (canceled)

31: The method according to claim 28, wherein the device comprises multiple electrodes and there is a different anti-tag on each electrode.

32. (canceled)

Description

FIGURES

[0079] FIG. 1aBasic two site sandwich assay

[0080] FIG. 1bBasis two site sandwich assay with tag/anti-tag

[0081] FIG. 2aCompetitive immunoassay

[0082] FIG. 2bCompetitive immunoassay with anti-tag separation

[0083] FIG. 3aCompetitive (inverted) immunoassay

[0084] FIG. 3bCompetitive (inverted) immunoassay with anti-tag separation

[0085] FIG. 4aDirect immunoassay for patient antibody detection

[0086] FIG. 4bDirect immunoassay with anti-tag separation

[0087] FIG. 5aIndirect immunoassay for patient antibody detection

[0088] FIG. 5bIndirect immunoassay for patient antibody detection with anti-tag separation

[0089] FIG. 6Tag/anti-tag multiplexing: Different immunoassay formats This Figure provides an example of how different immunoassay formats can be combined in a single device/method using an array of different anti-tags

[0090] FIG. 7Device comprising a multiplex sensor

[0091] Brief description: A device 700 comprising a multiplex sensor 720. The multiplex sensor including a sensor chamber 702 for incubation of a sample with tag reagents which can then flow or be fluidically driven to a detection zone 704 comprising an array of different anti-tags (706, 708, 710, 712).

[0092] FIG. 8Device comprising an alternative multiplex sensor.

[0093] Brief description: A device 800 comprising a multiplex sensor 820. The multiplex sensor including a microfluidic delay channel 814 between the sensor chamber 802 and the detection zone 804.

DETAILED DESCRIPTION OF THE INVENTION

[0094] Immunoassays

[0095] An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody or an antigen. Antibodies have the potential of binding to an antigen, which itself could also be an antibody and newer developments have seen the deployment of other binding reagents such as Molecular Imprinted Polymers, Affimers, Aptamers and antibody fragments. From here on in, these are all referred to as antibody or antibodies. The molecule detected by the immunoassay is often referred to as an analyte and is in many cases a protein, although it may be other kinds of molecules, of different size and types, as long as the proper antibodies that have the adequate properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.

[0096] Immunoassays are often used for the detection of a specific analyte within a sample. There are a number of immunoassay formats. For example, five different common immunoassay formats are shown in FIGS. 1-5 (parts A) which all rely on the separation of antibody-bound analyte and free (non-bound) antibody. Variations of these five immunoassay formats with anti-tag separation of a tagged immune complex are shown in FIGS. 1-5 (parts B).

[0097] Basic two site sandwich assay (FIG. 1A): This typical sandwich assay uses a primary immobilised capture antibody and a secondary signal antibody to bind to different sites of an analyte. These sandwich assays rely on the immobilisation of the capture antibody and removal of excess signal antibody before readings are taken. Signal generated is proportional to the amount of analyte sandwiched. No target analyte results in signal noise only. Sandwich assays are used for example in pregnancy test devices.

[0098] Competitive immunoassay (FIG. 2A): These assays rely on the immobilisation of an analyte competing for binding with the target analyte on an antibody that carries a signal. Removal of excess signal antibody is needed before a reading is taken. Signal generated is inversely proportional to the amount of target analyte in the sample. No target analyte results in maximum signal. Competitive immunoassays are used for example in drugs of abuse assays.

[0099] Competitive (inverted) immunoassay (FIG. 3A): These assays rely on the immobilisation of an antibody with competition between the target analyte and analyte carrying the signal reagent. Removal of excess unbound signal analyte is needed before readings are taken. Signal generated is inversely proportional to the amount of target analyte in the sample. Competitive (inverted) immunoassays are used for example in therapeutic drug assays.

[0100] Direct immunoassay for detecting patient antibodies (FIG. 4A): These assays rely on an immobilisation of an antibody that binds the target analyte (antibody) which in turn binds an antigen that carries the signal. Removal of excess signal antigen is needed before readings are taken. Signal generated is proportional to the amount of target analyte (antibody) in sample. No target analyte results in no detectable signal. Direct immunoassays for detecting patient antibodies are used for example in detecting antibodies to Hepatitis B.

[0101] Indirect immunoassay for detecting patient antibodies (FIG. 5A): These assays rely on an immobilisation of an antigen being recognised by the target analyte (antibody) which in turn is recognised by a signal antibody. Removal of excess signal antibody is needed before readings are taken. Signal generated is proportional to the amount of target analyte (antibody) in sample. Indirect immunoassays for detecting patient antibodies are used for example in detecting antibodies to HIV1/2.

[0102] These standard assays are shown in FIGS. 1 to 5 (parts A). Variations of each of these assay formats with anti-tag separation (comprising tag/anti-tags) are shown in FIGS. 1 to 5 (parts B). The present inventors have developed a tag/anti-tag system for multiplex assays involving anti-tag separation methods as illustrated in FIGS. 1 to 5 (part B) and described below.

[0103] Basic two site sandwich assay with tag/anti-tag (FIG. 1B): Capture antibody carries a tag and is specific for the target analyte. Signal antibody is also specific for the target analyte and both antibodies bind the target as it migrates through the device. An immobilised anti-tag that binds to the tag effectively removes the tagged complex from the liquid phase as it comes into contact with it. Unreacted signal antibody will flow away from the sensor or can be washed away.

[0104] Competitive immunoassay with anti-tag separation (FIG. 2B): Target analyte and analyte labelled with a Tag compete for binding on the antibody which also carries the signal. On reaching the anti-tag, tagged analyte becomes immobilised. In the absence of target analyte the signal will be maximal, whereas high target analyte concentrations will yield minimal signal.

[0105] Competitive (inverted) immunoassay with anti-tag separation (FIG. 3B): Target analyte and analyte labelled with signal compete for binding on the antibody which also carries the tag. On reaching the anti-tag, tagged antibody becomes immobilised. In the absence of target analyte the signal will be maximal, whereas high target analyte concentrations will yield minimal signal.

[0106] Direct immunoassay with anti-tag separation (FIG. 4B): Target analyte (antibody) is bound by an antibody labelled with a tag. Target antibody also binds labelled antigen (analyte with signal). All of this occurs in the liquid phase. The complex is separated using an anti-tag which binds the tag on the capture antibody. Excess antigen signal migrates out of the sensor area.

[0107] Indirect immunoassay for patient antibody detection with anti-tag separation (FIG. 5B): Signal antibody binds the target analyte (antibody) which binds the tagged antigen in liquid phase. Tagged antigen becomes immobilised when bound to the fixed anti-tag.

[0108] Capture antibodies and signal antibodies for use in the immunoassays described herein are well-known in the art. As are the labelling techniques used in the immunoassay formats to provide signal antigen or signal analyte.

[0109] Multiplex Assay

[0110] A multiplex assay is a type of assay used to simultaneously measure multiple analytes in a single run/cycle of the assay. The conventional multiplex approach requires the deposition of individual binding reagents (antibodies or antigens) in a fixed array to make a test panel. This requires complex equipment & processes to deliver either on continuous reel systems or discreet dispensing into a device array. Increasing the number of analytes in a panel increases the risk of batch failure since only one assay has to fall out of specification for the entire panel to fail.

[0111] A multiplex device and assay methods provided herein use multiple tags/anti-tags. In particular, they use an array of different anti-tags which are immobilised to a solid support e.g. surface of a sensor. Each anti-tag can bind a complimentary tag and so is able to immobilise a complimentarily tagged antibody or complimentarily tagged antigen, thereby separating tagged immune complexes, that have formed in the liquid phase, from the liquid phase.

[0112] In some embodiments, there is a sensor chamber for incubation of a sample with tag reagents so that tagged immune complexes form in the liquid phase which can then flow or be fluidically driven to a detection zone comprising an array of different anti-tags. The sample may also interact with signal reagents (e.g. labelled antibodies or antigens) in the sensor chamber to facilitate detection.

[0113] In some embodiments, the flow of a fluid through a microfluidic device may be controlled and/or manipulated by an external means such as applying pressure to a fluid channel using an actuator such as a flow pump. The pump used to drive a fluid through the microfluidic device may be an electrically-driven pump, a thermally-driven pump or a pneumatically-driven pump. The use of a pump may be advantageous as it enable the precise control of a flow rate through the microfluidic device.

[0114] Additionally or alternatively, the flow of a fluid through a microfluidic device may be provided via capillary action. Capillary action or motion can be advantageous as it may enable a fluid to flow through the microfluidic device without the requirement of providing any external forces, such as pressure provided by a pump.

[0115] In some embodiments, a valve may be provided within the channels of the microfluidic device to control the flow rate of a fluid flow through the microfluidic device. In some embodiments, the channels of the may be provided with one or more resistors, which may be useful in controlling the flow rates of a fluid through the microfluidic device.

[0116] There may be multiple reaction zones for tagged immune complexes to form in the liquid phase, wherein reaction zone has different anti-tag.

[0117] Example 4 describes several different experimental setups for multiplexed assays in a cardiac panel which use basic two site sandwich assays e.g. with different detection methods and/or devices. The same array of anti-tags can be used to detect other target analytes (i.e. other than cardiac markers), and with other immunoassay formats. Other sets of anti-tags can be devised and tailored to the intended use. Though the invention advantageously enables the provision of an array of anti-tags that can be used for multiple tests.

[0118] In one embodiment, described in example 4E, there is a microfluidic device and method for detecting multiple target analytes in a test liquid sample using a set of complimentary tags and anti-tags and impedance spectroscopy. The detection zone of the device comprises an array of electrodes (e.g. 6), wherein there is a different anti-tag (e.g. antibodies raised against FITC, Flag, His-6, c-Myc, V5 and GST) on each electrode, and wherein each anti-tag is adsorbed or chemically linked to the electrode (e.g. wherein the electrodes are gold electrodes). The test liquid sample (e.g. serum or plasma) is added to a sensor chamber upstream of the electrodes that already contains the tag and signal reagents (e.g. tagged primary and secondary antibodies conjugated to HRP). After sufficient incubation time allowing for the formation of specific sandwich complexes with each target analyte, the mixture is fluidically driven to the anti-tag coated electrodes of the sensor and tagged immune complexes bind to their respective anti-tags. After sufficient incubation time, a wash step is introduced to remove any unbound excess material (antibody or antigens). Precipitating solution (e.g. DAB) is added to the sensor for a controlled period of time and reacts with signal reagent present in the immune complex (e.g. HRP conjugated to secondary antibody) to produce an insulating precipitate on the electrode surface. The quantity of the precipitate (which is proportional to the amount of HRP-tagged secondary and thus proportional to the amount of antigen) can be measured electrochemically using standard faradaic impedance spectroscopy using a redox probe solution. In this, the magnitude of the charge transfer resistance, Rct, at the half-wave potential of a dissolved reversible redox probe is measured by fitting an impedance spectrum to a suitable Equivalent circuit. Rct will be proportional to the amount of the insulating precipitate created on the surface. Alternatively, other parameters derived from the impedance response may be used to transduce the signal.

[0119] Target Analytes

[0120] The target analytes comprise antibodies and/or antigens.

[0121] Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in what might be a complex mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope.

[0122] In some cases, an immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an antibody rather than an antigen.

[0123] Sample

[0124] The sample is a liquid sample and can be any test solution. For example, blood, serum, plasma, cell and tissue lysates, or urine. The sample can be from a human, mammal or other animal, or the sample can be of a non-mammalian nature e.g. bacterial proteins. The sample is suitably chosen for human clinical testing or veterinary testing. However, the devices and methods provided herein can also be applied to other samples for other applications of immunoassay e.g. in the Agri-chemical business (e.g. testing for fungal residues and fungicides in fruit, pesticides in water or mycotoxins in grains/coffee as well as the testing of hormones and antibiotics in milk are typical examples).

[0125] Solid Surface

[0126] The solid surface on which the multiple different anti-tags are immobilised, is suitably the surface of a sensor. The sensor surface can comprise a metallic surface or metallic film, polymeric surface and in general conductive or non-conductive materials. However, the surface of the sensor comprises either i) the surface of an electrode/conductive structure itself or ii) the conductive or semi-conductive coating of a non-conductive material (to function as an electrode).

[0127] The anti-tags can be adsorbed or chemically linked the surface of the sensor e.g. a metal surface or an electrode surface. In some embodiments, the sensor comprises multiple electrodes and there is a different anti-tag on each electrode. In one example, the sensor may be composed of one or more electrodes such as gold electrodes or silver/silver chloride electrodes or BST electrodes. By providing a sensor with one or more electrodes on its surface, the sensor can be suitably modified for electrochemical impedance measurements. In some embodiments, the sensor may comprise a reference electrode, a working electrode and a counter electrode, whereby a current flowing between the counter electrode and the working electrode can be continuously monitored.

[0128] The substrate (surface) of the electrode may comprise any electrically conducting material. The electrode surface may comprise a metal or carbon. The metal may be a metal in elemental form or an alloy of a metal. Optionally, the whole of the electrode surface comprises a metal or carbon. The electrode surface may comprise a transition metal. The electrode surface may comprise a transition metal selected from any of groups 9 to 11 of the Periodic Table. The surface may comprise a metal selected from, but not limited to, rhenium, iridium, palladium, platinum, copper, indium, rubidium, silver and gold. The surface may comprise a metal selected from gold, silver and platinum. The surface may comprise a carbon-containing material, which may be selected from edge plane pyrolytic graphite, basal plane pyrolytic graphite, glassy carbon, boron doped diamond, highly ordered pyrolytic graphite, carbon powder and carbon nanotubes.

[0129] Alternatively, the anti-tags can be adsorbed or chemically linked to the surface of the sensor comprising a non-conductive plastic surface, if this surface is coated with a conductive or semi-conductive polymeric material. In some embodiments the sensor surface comprises a conductive polymer, e.g. PEDOTpolystyrene sulfonate or poly(3,4-ethylenedioxythiophene), PANI (polyanilines), and/or PPY (poly(pyrrole)s). It is known in the art to coat plastic surfaces with conductive polymers such as PEDOT to function as electrodes.

[0130] Anti-Tags/Tags

[0131] Provided herein is an array of different anti-tags immobilised to a solid surface. Each anti-tag is able to bind a complimentary tag. The anti-tag may be a group (e.g. antibody fragments) or species.

[0132] Epitope tags are used extensively in proteomics & protein chemistry and can be an amino acid sequence or molecule attached to or assembled into a target analyte (protein or nucleic acid). The matching binding reagentthe anti-tag is typically an antibody but could also be an aptamer, affimer, molecularly imprinted polymer (MIP) or other binding reagent, or nucleic acid(s) or antibody fragments. Other suitable binding reagents include: Nucleic acids (oligonucleotides), peptides (e.g. flag tags), enzymes and chemicals (e.g. FITC). The most common tag/anti-tag system is biotin-avidin but there are many options available (e.g. see Table 1). Typically, anti-tags are antibodies with good affinity.

TABLE-US-00001 TABLE 1 Example tag/anti-tag pairs Tag Anti-Tag antibody Primary ab e.g. mouse Sheep anti-mouse ab Biotin Anti-biotin (or avidin) FITC Anti-FITC HRP Anti-HRP R Maltose Binding Protein Anti-MPB Streptavidin Anti-Streptavidin (or biotin) Flag Anti-Flag His-6 Anti-His-6 c-Myc Anti-c-Myc biotin acceptor peptide Anti-biotin acceptor peptide V5 Anti-V5 glutathione S-transferase (GST) Anti- glutathione S-transferase (GST)

[0133] In a device or method provided herein, any of the anti-tags shown in Table 1, or other tag/anti-tag pairs, may be combined to form a panel/array of different anti-tags. The set of tags/anti-tags are tailored to the signal measurement method used e.g. anti-FITC/FITC would not be selected as an anti-tag/tag pair for use with fluorometric detection, and anti-HRP/HRP would not be selected as an anti-tag/tag pair for use with colorimetric detection.

[0134] In some embodiments the multiplex assay comprises 3-10 different tag/anti-tag pairs. In some embodiments, the panel of anti-tags comprises 6 different anti-tags, e.g. antibodies against FITC, Flag, His-6, c-Myc, V5 and GST; or e.g. antibodies against biotin, Flag, His-6, c-Myc, V5 and GST.

[0135] The immunoassay formats with tag/anti-tag separation as shown in FIGS. 1-5 part B) provided herein, use tagged antibody or tagged competing analyte and immobilised anti-tag.

[0136] It would be understood that each tag/anti-tag binding pair could be used in any suitable configuration. For example, the tag could be immobilised and the anti-tag chemically attached to the antibody or antigen as well as the configuration of immobilised anti-tag that is described herein. Any combination of the anti-tag/tag binding pairs can be deployed to create a suitable configuration for multiplexed assays.

[0137] Signal/Signal Measurement Technique

[0138] In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding. Most, though not all, immunoassays involve chemically linking antibodies or antigens with some kind of detectable label. A large number of labels exist in modern immunoassays, and they allow for detection through different means. Many labels are detectable because they either emit radiation, produce a color change in a solution, fluoresce under light, or can be induced to emit light.

[0139] In some embodiments, the multiplex assay comprises a signal antibody or signal antigen/analyte. For examples, FIGS. 1, 2 and 5 show use of a signal antibody (antibody carrying a signal/label) and FIGS. 3 and 4 show use of a signal antigen (antigen carrying a signal/label). Some embodiments utilise colorimetric detection or fluorometric detection, e.g. see examples 4A and 4B respectively. Some embodiments utilise FRET (Fluorescence resonance energy transfer), e.g. see example 4G.

[0140] Some embodiments utilise impedance measurement or redox active precipitate for detection. See examples 4C and 4D. In these examples, secondary antibodies (that can bind the target analytes) are conjugated with HRP and a precipitating solution (e.g. DAB, TMB, AEC) which reacts with HRP is added to the sensor to produce a precipitate.

[0141] In some embodiments, the assay methods provided herein utilise electrical impedance spectroscopy methods (e.g. faradaic or non-faradaic impedance spectroscopy) to determine the presence and/or amount of analytes of interest. In this case, the anti-tags are immobilised at fixed points on the sensor surface suitable for impedance measurement (e.g. electrode surface). In these embodiments, a precipitating solution is added to produce an insulting precipitate on an electrode surface. The quantity of the precipitate (which is proportional to the amount of target analyte) can be measured electrochemically using standard faradaic impedance spectroscopy. In this, the magnitude of the charge transfer resistance, Rct, at the half-wave potential of a dissolved reversible redox probe is measured by fitting an impedance spectrum to a suitable Equivalent circuit. Rct will be proportional to the amount of the insulating precipitate created on the surface. Alternatively, other parameters derived from the impedance response may be used to transduce the signal. See Example 4C.

[0142] In some embodiments, the quantity of the precipitate (which may be proportional to the amount of HRP-tagged secondary and thus proportional to the amount of antigen) can be measured electrochemically using any range of electrochemical techniques which include chronoamperometry, cyclic voltammetry, pulsed voltammetric methods such as Different Pulsed Voltammetry (DPV) and

[0143] Square Wave Voltammetry (SWV), and potentiometric methods such as Open Circuit Potential (OCP) or chronopotentiometry. For example, the amount of the precipitate can be measured using DPV by scanning from low to high potential in an inert electrolyte and measuring the peak oxidative current generated as the precipitate (initially in reduced form at low potentials) is re-oxidised.

[0144] Precipitating substrates that may be used in the assay methods described herein are set out in table 2 below. Precipitating substrates are typically used for western blot and immunohistochemistry detection and are, therefore, well known in the literature and available for use in the present assay methods.

TABLE-US-00002 TABLE 2 Precipitating chromogenic substrates for HRP and AP. Relative Substrate Format Enzyme Features sensitivity Signal colour DAB Dry HRP Can be formulated Medium Brown powder Metal- 2- HRP 50X more sensitive Highest Brown to enhanced component than DAB alone black DAB reagent kit BCIP Dry AP Can be formulated Medium Blue to powder purple NBT Dry AP and Non-carcinogenic Medium Blue to powder glucose purple oxidase 1-step Single AP Low background High Black to NBT/BCIP step, for high purple ready-to- sensitivity use 1-step Single AP May or may not High Black to NBT/BCIP + step, contain levamisole purple suppressor ready-to- for endogenous use alkaline phosphatase inhibition TMB Single HRP Very high high Blue step, sensitivity and ready-to- stability use AEC Single HRP Ready to use low Orange- step, moderate stability brown ready-to- use Vector 3 AP Very high high Black Black component sensitivity reagent Vector 4 HRP Very high high red NovaRED component sensitivity reagent

EXAMPLES

Example 1Universal Platform

[0145] Consider a multiplex panel for infectious disease testing looking to detect human IgG antibodies to toxoplasma, CMV, HSV1, HSV1, measles, mumps & rubella. This could use a direct immunoassay format and each chemistry would require the immobilisation of its respective antibody onto a surface in a fixed array suitable for reading the signal. This process is typically done as a large batch on a dedicated pipetting/dispensing system.

[0146] Large scale manufacturing a diverse range of multiplex tests by depositing specific antigens or antibodies that could also include allergy testing, therapeutic drugs, cardiac markers, blood borne viruses and hormones would create either a bottleneck in production or the need for a many dispensing systems.

[0147] By using multiple tag/anti-tags, a single universal platform could be utilised for all the different multi-panel tests. This would require just one sensor platform with 6 different anti-tags attached in an array. Selectively adding different tags to the selected antibody or antigen for a given panel and adding these as a separate downstream reagent would complete the device.

[0148] As an example, Table 3 below shows the same universal platform with anti-tags (1-6) fixed above the sensor area. For Rubella within the infectious disease panel using an indirect format, each rubella antigen would be labelled with tag 1 and a common signal antibody of say anti-human IgG HRP conjugate added. Human IgG to rubella would bind to the antigen and also be bound by the signal antibody. The immobilised anti-tag 1 would extract the complex onto the sensor surface. This would be repeated using different tag/anti-tags (2-6) for the measles, mumps etc

[0149] Using the same universal platform with the same array of anti-tags, other test panels can be created by substituting in the relevant chemistries.

TABLE-US-00003 TABLE 3 Example panels comprising 6 immobilised anti-tags Panel Device Tag 1 Tag 2 Tag 3 Tag 4 Tag 4 Tag 5 Tag 6 Infection Rubella Measles Mumps HSV 1 HSV 2 CMV Toxo Disease Cardiac Troponin T Troponin I FABP BNP proBNP CKMB Myoglobin Drugs Amps Benzo THC Opiate Cocaine PCP Tramadol Immuno-status IgG1 IgG2 IgG3 IgG4 IgA IgM Light Chains

[0150] This is analogous to the manufacturing processes used in the car industry: The Volkswagen Group use a common platform (known as MQB) that covers over a dozen different cars (Audi TT, Seat Ateca, Skoda Octavia and VW Tiguan to name a few).

[0151] Within many areas of testing there could easily be anywhere from 10 to 100 different target analytes of interest depending on the market and this often requires subtle changes to the panels offered. For example there are at least 14 different drugs of abuse that could be tested for but typically this is restricted to 2 or 3 variations of 6-panel tests: they tend to be created if substantial market demand and volume dictates. The number of 6-panel permutations from 14 drugs could yield over 3000 different 6-panel tests which would not be economically viable. Smaller volume requests for different 6-panel tests could be achieved using the greater flexibility, choice and speed to market of the universal platform.

[0152] Allergy testing is another example where specific combinations of tests could be required from the many allergens within a class.

Example 2Faster Kinetics

[0153] Antibody and antigens bind quickly in liquid phase, the strength of the binding is called the affinity. Affinities can be described as weak, medium or strong. Immobilising one or other of these on a solid surface reduces the opportunity of antibody-antigen interaction and as such reduces the binding kinetics. To compensate for this incubation times are increased when one moiety is immobilised on a solid phase.

[0154] Using the tag/anti-tag format, the primary incubation between target specific antibodies and antigens happens in liquid phase. This benefits weak to medium affinity reactions as well as detecting low concentrations (free movement of molecules allows greater opportunity of meeting up). Detecting low concentration analytes can be improved by lengthening the liquid phase incubation, this can be achieved in the sensor by moving these key reagents further away from the sensor. For example, including a microfluidic delay channel into the device can increase incubation time.

[0155] Separation of the liquid phase immune complex using tag/anti-tag also relies on the binding affinity of the tag/anti-tag. These can be used in high concentration and be selected to have medium to high binding affinity. This allows for relatively short incubation times.

[0156] Primary incubation in the liquid phase allows for incubation time to be varied, and allows for a more automated approach to multiplexed assays.

Example 3Mixed Mode Immunoassay

[0157] One of the reasons different immunoassay formats have evolved has been driven by the need to measure at greater analytical sensitivity. As a general rule sandwich assays tend to have greater sensitivity than competitive assays. Sandwich assays are not typically used to detect small molecules such as drugs and steroid hormones (though a sandwich format can be potentially be used by either using other species (aptamers) or indirectly by targeting metabolic products of drugs/hormones).

[0158] In some circumstances a multiplex panel needs to include analytes that span a wide range in analytical concentration and include small and large molecules. For example sports medicine testing could involve simultaneous monitoring of low levels of cortisol (0.00003 to 0.0003 g/L) whilst also quantitating higher levels of Immunoglobulin G (8 to 16 g/L) and Immunoglobulin A (1 to 4 g/L).

[0159] Testosterone and Immunoglobulin M might also be included in such a panel. The ability to simultaneously run different formats on the same panel, without significantly altering the dilution of the sample, is a good benefit.

[0160] Other scenarios might involve the detection of an anti-cancer drug whilst measuring various tumour markers.

Example 4Experimental Set-Ups

[0161] A) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays in a Cardiac Panel Using Colorimetric Detection

[0162] In this setup, a panel of 6 antibodies raised against FITC, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a plastic or metal surface. These are part of an array (sensor) that is composed of 6 electrodes. Capture (primary) antibodies specific to several different markers e.g. CRP, TnI, TnT, D-Dimer, BNP and proBNP, each are conjugated with one of the above tags using available coupling chemistry conjugation kits (EDC/NHS). A set of secondary antibodies are selected against the targets mentioned above and are conjugated to HRP. Sample, either serum or plasma is being added to a sensor chamber that is before the electrodes and already contains the tagged primary and secondary antibodies. Sufficient incubation time allows for the formation of specific sandwich complexes with each target analyte. Afterwards the mixture is being fluidically driven to the anti-tag coated electrodes of the sensor. Another binding event takes place, with each of the capture antibody, containing a specific tag e.g. His-6, binding to the respective anti-his-6 antibody on the surface of the electrode. After sufficient incubation time, a wash step in introduced to remove any unbound excess material (antibody or antigens). A chromogenic solution, (e.g. TMB) is added to the sensor and reacts with the HRP to produce a characteristic blue colour. The intensity of the colour can be measured colorimetrically at 605 nm after a specific reaction time. The signal intensity is proportional the target analyte.

[0163] B) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays in a Cardiac Panel Using Fluorometric Detection

[0164] In this setup, a panel of 6 antibodies raised against biotin, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a plastic or metal surface. These are part of an array (sensor) that is composed of 6 detection zones. Capture (primary) antibodies specific to several different markers e.g. CRP, TnI, TnT, D-Dimer, BNP and proBNP, each are conjugated with one of the above tags using available coupling chemistry conjugation kits (EDC/NHS). A set of secondary antibodies are selected against the targets mentioned above and are conjugated with different fluorogenic molecules. The secondary anti-CRP and anti-TnI antibodies can be conjugated with FITC, the secondary anti-TnT and anti D-Dimer with Alexa Fluor 750 and the secondary anti-BNP and anti-proBNP antibodies with Cy7.5. The selection of these dyes also offers greater flexibility as they have distinct excitation/emission wavelengths. Sample, either serum or plasma is being added to a sensor chamber that is before the detection zones and already contains the tagged primary and secondary antibodies. Sufficient incubation time allows for the formation of specific sandwich complexes with each target analyte. Afterwards the mixture is being fluidically driven to the anti-tag coated electrodes of the sensor. Another binding event takes place, with each of the capture antibody, containing a specific tag e.g. His-6, binding to the respective anti-his-6 antibody on the surface of the electrode. After sufficient incubation time, a wash step in introduced to remove any unbound excess material (antibody or antigens). Detection is performed by use of a fluorometer using the corresponding excitation/emission wavelengths. Signal intensity is proportional to the bound analyte.

[0165] C) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays in a Cardiac Panel Using Faradaic or Non-Faradaic Impedance Spectroscopy

[0166] In this setup, a panel of 6 antibodies raised against FITC, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a gold electrode. These are part of an array (sensor) that is composed of 6 electrodes. Capture (primary) antibodies specific to several different markers e.g. CRP, TnI, TnT, D-Dimer, BNP and proBNP, each are conjugated with one of the above tags using available coupling chemistry conjugation kits (EDC/NHS). A set of secondary antibodies are selected against the targets mentioned above and are conjugated with HRP. Sample, either serum or plasma is being added to a sensor chamber that is before the electrodes and already contains the tagged primary and secondary antibodies. Sufficient incubation time allows for the formation of specific sandwich complexes with each target analyte. Afterwards the mixture is being fluidically driven to the anti-tag coated electrodes of the sensor. Another binding event takes place, with each of the capture antibody, containing a specific tag e.g. His-6, binding to the respective anti-his-6 antibody on the surface of the electrode. After sufficient incubation time, a wash step in introduced to remove any unbound excess material (antibody or antigens). Precipitating solution (e.g. DAB) is added to the sensor for a controlled period of time and reacts with the HRP to produce an insulating precipitate on the electrode surface. The quantity of the precipitate (which is proportional to the amount of HRP-tagged secondary and thus proportional to the amount of antigen) can be measured electrochemically using standard faradaic impedance spectroscopy. In this, the magnitude of the charge transfer resistance, Rct, at the half-wave potential of a dissolved reversible redox probe is measured by fitting an impedance spectrum to a suitable Equivalent circuit. Rct will be proportional to the amount of the insulating precipitate created on the surface. Alternatively, other parameters derived from the impedance response may be used to transduce the signal.

[0167] D) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays in a Cardiac Panel Using Redox Active Precipitate

[0168] In this setup, a panel of 6 antibodies raised against FITC, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a gold electrode. These are part of an array (sensor) that is composed of 6 electrodes. Capture (primary) antibodies specific to several different markers e.g. CRP, TnI, TnT, D-Dimer, BNP and proBNP, each are conjugated with one of the above tags using available coupling chemistry conjugation kits (EDC/NHS). A set of secondary antibodies are selected against the targets mentioned above and are conjugated with HRP. Sample, either serum or plasma is being added to a sensor chamber that is before the electrodes and already contains the tagged primary and secondary antibodies. Sufficient incubation time allows for the formation of specific sandwich complexes with each target analyte. Afterwards the mixture is being fluidically driven to the anti-tag coated electrodes of the sensor. Another binding event takes place, with each of the capture antibody, containing a specific tag e.g. His-6, binding to the respective anti-his-6 antibody on the surface of the electrode. After sufficient incubation time, a wash step in introduced to remove any unbound excess material (antibody or antigens). A solution of precipitating solution capable of producing a redox active precipitate (e.g TMB, DAB, AEC) is added to the sensor and reacts with the HRP to produce the redox active precipitate on the electrode surface. The quantity of the precipitate (which is proportional to the amount of HRP-tagged secondary and thus proportional to the amount of antigen) can be measured electrochemically by several conventional techniques, including amperometry (measuring the charge transferred to the precipitate), voltammetry or impedance (by measuring the ability of the precipitate to capacitate charge at its half-wave potential).

[0169] E) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays for a Cardiac Panel in a Microfluidic Device.

[0170] In this setup, a panel of 6 antibodies raised against FITC, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a gold electrode for 1 hour in a concentration of 1 ug/ul. These are part of an array (sensor) that is composed of 6 electrodes. Capture (primary) antibodies specific to several different markers e.g. CRP, TnI, TnT, D-Dimer, BNP and proBNP, each are conjugated with one of the above tags using available coupling chemistry conjugation kits (EDC/NHS). A set of secondary antibodies are selected against the targets mentioned above and are conjugated with HRP. 100 ul of whole blood are added in the slot of a microfluidic cartridge and around 50 ul of plasma are separated using a plasma separation membrane. Then the plasma is diluted to an appropriate concentration using a sample diluent. The diluted samples is then microfluidically driven into a chamber containing the capture and detection antibody either in a dried format or in liquid form in concentrations of 0.5 ng/ml. After 5 min of incubation the mixture containing the sandwich complexes of the antibodies with the antigens is further incubated for 4 min on the 6 electrodes of the sensor. Each electrode depending on the tag it contains will bind to the respective sandwich complex from the previous incubation. After the incubation is completed, the sensor chamber is flushed with 200 ul wash buffer and incubated for further 2 min. Finally, 100 ul of precipitating chromogenic solution (DAB, TMB) are added into the sensor chamber and incubated for 3 min. After the incubation, the reaction is terminated by flushing the chamber with 300 ul of wash buffer. The quantity of the precipitate (which is proportional to the amount of HRP-tagged secondary and thus proportional to the amount of antigen) can be measured electrochemically using standard faradaic impedance spectroscopy using 100 ul of redox probe solution. In this, the magnitude of the charge transfer resistance, Rct, at the half-wave potential of a dissolved reversible redox probe is measured by fitting an impedance spectrum to a suitable Equivalent circuit. Rct will be proportional to the amount of the insulating precipitate created on the surface. Alternatively, other parameters derived from the impedance response may be used to transduce the signal.

[0171] F) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays for a Cardiac Panel in a Lab Setup

[0172] In a high binding 96 well ELISA plate 100 ul of each of 6 antibodies raised against FITC, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a gold electrode for 1 hour in a concentration of 1 ug/ul. After incubation, the wells are washed 3 times with PBS-T and then blocked for 30 min using a 5% BSA solution in PBS-T. In separate vials 100 ul of plasma diluted 1/1000 is being added in a vial that contains capture and HRP-labelled detection antibodies in a concentration of 0.5 ng/ml. Samples are incubated for 5 min in RT and afterwards 100 ul of the mixture is added into each of the 96 well plates. Then the samples are incubated for a further 3 min in RT. After the incubation, the wells are emptied and washed 3 times using PBS-Tween. After the wash steps, 100 ul of TMB solution is added into each well and the reaction is further incubated for 3 min in RT. The reaction is being terminated by addition of 100 ul of 2M sulfuric acid and the absorbance of each well is read at 450 nm, also using a reference wavelength at 650 nm. The change in absorbance is correlated with the concentration of each different analyte in each of the wells.

[0173] G) Experimental Setup Using the Tag/Anti-Tag System for Multiplexed Assays in a Cardiac Panel Using FRET

[0174] In this setup, a panel of 6 antibodies raised against biotin, Flag, His-6, c-Myc, V5 and GST tags are adsorbed or chemically linked to a plastic or metal surface. These are part of an array (sensor) that is composed of 6 detection zones. Capture (primary) antibodies specific to several different markers e.g. CRP, TnI, TnT, D-Dimer, BNP and proBNP, each are conjugated with one of the above tags using available coupling chemistry conjugation kits (EDC/NHS). A set of secondary antibodies are selected against the targets mentioned above and are conjugated with different fluorogenic molecules. The secondary anti-CRP can be conjugated to RPE-APC, the anti-TnI antibodies can be conjugated with RPE-Cy5, the secondary anti-TnT can be conjugated with RPE-Cy7, the anti D-Dimer with RPE-DyLight 650, the anti-BNP with APCDyLight755 and the anti-proBNP antibodies with FluoresceinRPE the selection of these dyes also offers greater flexibility as they have distinct excitation/emission wavelengths and can yield distinct FRET signals. Sample, either serum or plasma is being added to a sensor chamber that is before the detection zones and already contains the tagged primary and secondary antibodies. Sufficient incubation time allows for the formation of specific sandwich complexes with each target analyte. Afterwards the mixture is being fluidically driven to the anti-tag coated electrodes of the sensor. Another binding event takes place, with each of the capture antibody, containing a specific tag e.g. His-6, binding to the respective anti-his-6 antibody on the surface of the electrode. After sufficient incubation time, a wash step in introduced to remove any unbound excess material (antibody or antigens). Detection is performed by use of a fluorometer using excitation wavelengths of the donor molecule and emission wavelengths of the donor molecule. Signal intensity is proportional to the bound analyte.

[0175] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

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

[0176] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0177] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.