ASSAY

Abstract

The present invention relates to methods for detecting an analyte present in a fluid sample using a microfluidic device comprising a detection zone characterized by an optically transmissible portion and reagent(s) associated with a porous matrix, wherein the analyte is detected with an optical detector. The present invention also provides a microfluidic channel and a microfluidic cartridge for use in such a method.

Claims

1.-24. (canceled)

25. A kinetic assay method for use in detecting an analyte within a sample, the method comprising: a) providing a sample to a detection zone of a microfluidic channel, the detection zone comprising an optically transmissible portion and a porous matrix comprising one or more reagent(s) localised to an inner luminal surface of the optically transmissible portion of the microfluidic channel, wherein the reagent(s) is/are capable of reacting with the analyte or a reaction product thereof to form a reagent reaction product, the reagent reaction product capable of being detected, using an optical detector which is extra luminal to the optically transmissible portion of the microfluidic channel; b) taking at least one optical measurement of the reagent reaction product through the optically transmissible portion; and detecting any analyte, based upon the at least one optical measurement of the reagent reaction product.

26. The assay method according to claim 25, wherein the optically transmissible portion of the channel is a top portion of the channel.

27. The assay method according to claim 25, wherein the microfluidic channel comprises at least one additional matrix and/or one or more assay reagents deposited outside the matrix.

28. The assay method according to claim 27, wherein said at least one additional matrix and/or one or more assay reagents is localized to a section of the microfluidic channel which is not the detection zone.

29. The assay method according to claim 25, wherein the matrix/matrices comprises at least one carrier molecule.

30. The assay method according to claim 29 wherein the carrier molecule is generally insoluble in the sample fluid.

31. The assay method according to claim 29 wherein the at least one carrier molecule comprises at least one polymer.

32. The assay method according to claim 31 wherein the at least one polymer comprises at least one disaccharide and/or polysaccharide.

33. The assay method according to claim 32 wherein the at least one disaccharide is selected from the group consisting of sucrose, lactose, maltose, trehalose, cellobiose and chitobiose and the at least one polysaccharide is selected from the group consisting of amylose, amylopectin, cellulose, cellulose derivative, chitin, callose, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan and galactomannan.

34. The assay method according to claim 29 wherein the at least one carrier molecule comprises trehalose and at least one cellulose derivative, selected from the group consisting of carboxymethylcellulose (CMC), cellulose ethyl sulfonate (CES), hydroxyethylcellulose (HEC), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), microcrystalline cellulose (MCC), methylcellulose and salts thereof.

35. The assay method according to claim 25, wherein the sample is provided to the detection zone by an active fill mechanism.

36. The assay method according to claim 25, wherein the sample is a sample of blood or other bodily fluid.

37. The assay method according to claim 25 wherein the analyte is an enzyme, lipid, lipoprotein, cytokine, hormone or endotoxin.

38. The assay method according to claim 37 wherein the analyte is an enzyme.

39. The assay method according to claim 38 wherein the enzyme is thrombin or a protease.

40. The assay method according to claim 37 wherein the enzyme is thrombin and the method is used to determine a prothrombin time (PT) or international normalised ratio (INR) value.

41. The assay method according to claim 38 wherein the reagent comprises a thrombin cleavable substrate reagent, which is capable of being cleaved by thrombin to form a reagent reaction product which generates an optical signal.

42. The assay method according to claim 41 wherein the thrombin cleavable substrate reagent comprises a peptide sequence which is recognisable and cleavable by thrombin and an associated fluorescent molecule, the associated fluorescent molecule forming the reagent reaction product which is capable of detection following cleavage of the peptide sequence.

43. The assay method according to claim 37 wherein the analyte comprises a lipid or lipoprotein.

44. The assay method according to claim 43 wherein the analyte comprises cholesterol or a cholesteryl ester.

45. A microfluidic channel for use in a method according to claim 25, the microfluidic channel comprising: a detection zone for receiving at least a portion of a sample provided to the microfluidic channel, the detection zone comprising an optically transmissible portion and a porous matrix comprising one or more reagent(s) localised to an inner luminal surface of the optically transmissible portion of the microfluidic channel, wherein the reagent(s) is/are capable of reacting with the analyte or an analyte reaction product thereof to form a reagent reaction product, the reagent reaction product being capable of being optically detected; and optionally wherein the optically transmissible portion is a top portion of the channel.

46. A microfluidic cartridge for use in conducting an assay according to claim 25, the microfluidic cartridge comprising: at least one microfluidic channel, wherein each/said microfluidic channel(s) comprises a detection zone, the detection zone comprising an optically transmissible portion and a porous matrix comprising one or more reagent(s) localized to an inner luminal surface of the optically transmissible portion, wherein the reagent(s) is/are capable of reacting with the analyte or an analyte reaction product thereof to form a reagent reaction product, the reagent reaction product being capable of being optically detected; and optionally wherein the optically transmissible portion is a top portion of the channel.

47. An assay method for use with a sample, the method comprising; inserting a cartridge into a reader device, the cartridge comprising a microfluidic channel comprising: a detection zone for receiving at least a portion of a sample provided to the microfluidic channel, the detection zone comprising a optically transmissible portion and a porous matrix comprising one or more reagent(s) localized to an inner luminal surface of the optically transmissible portion, wherein the reagent(s) is/are is/are capable of reacting with the analyte or an analyte reaction product thereof to form a reagent reaction product; the reagent reaction product capable of being optically detected, optionally expelling air from the microfluidic channel using force application means within the reader device; introducing the sample to a first end of the microfluidic channel; drawing the sample along the microfluidic channel to the detection zone using means within the reader device; permitting said analyte or analyte reaction product in the sample to react with the reagent(s) to form the reagent reaction product; taking at least one optical measurement of the reagent reaction product using an optical detection device within the reader, the optical detection device being extra luminal to the optically transmissible portion; and detecting any analyte or analyte reaction product based upon the at least one optical measurement of the reagent reaction product.

48. The assay method according to claim 47, wherein the optically transmissible portion is a top portion of the channel.

Description

DETAILED DESCRIPTION

[0220] The present invention will now be further described by way of example and with reference to the following figures which show:

[0221] FIG. 1 (A and B) show a schematic representation of the coagulation pathway (or clotting cascade) and potential analyte targets of this pathway for measurement by a kinetic assay in accordance with the present invention;

[0222] FIG. 2 shows a thrombin cleavable agent comprising Rhodamine 110, which may be used in an embodiment of the present invention;

[0223] FIG. 3 is a schematic of an assay in accordance with an embodiment of the invention;

[0224] FIG. 4 is a schematic of another assay in accordance with an embodiment of the invention;

[0225] FIG. 5 (A) is a diagram of a microfluidic cartridge in accordance with an embodiment of the present invention. (B) is a photograph showing signal development at the optically transmissible portion during the assay. (C) shows the different signal intensity which can be obtained depending on where the porous matrix is positioned in relation to the optical detector. (D) shows signal development at different timepoints when using a porous matrix which was spread when deposited onto the luminal surface and (E) shows signal development when using a thick versus a thin porous matrix;

[0226] FIG. 6 shows blood entering and filling the portion of the cartridge shown in FIG. 5A;

[0227] FIG. 7 is a diagram of microfluidic architecture in a cartridge in accordance with an embodiment of the invention;

[0228] FIG. 8 is a diagram of microfluidic architecture in a cartridge in accordance with an embodiment of the invention;

[0229] FIG. 9 (A and B) show the effect of HEC (hydroxyethylcellulose) as a component of the porous matrix on the optical measurement. (C and D) show the effect the molecular weight of HEC in the porous matrix has upon clot time;

[0230] FIG. 10 shows the effect of other components of the porous matrix on optical measurement. (A) Clot time versus ACL INR on a sample of whole blood, the porous matrix having a concentration of 10% PEG (polyethylene glycol). (B) Effect of Avicel on assay rate;

[0231] FIG. 11 shows the effect of CMC (sodium carboxymethylcellulose) in the porous matrix on the assay. (A and B) Effect of concentration of CMC in the porous matrix upon clot time. (C) Effect of new forms of CMC (DS (degree of substitution) CMC) upon the assay;

[0232] FIG. 12 shows images of signal development in the porous matrix for optical detection using different concentrations of CMC and PEG in the matrix;

[0233] FIGS. 13 A and B show a modelled schematic of thrombin conversion as a measure of INR in accordance with an embodiment of the present invention;

[0234] FIG. 14 shows assay results using different samples;

[0235] FIG. 15 shows the extended assay range of the present invention;

[0236] FIG. 16 shows a graph which demonstrates how clot time (and thus INR) may be calculated in an embodiment of the invention. PT is measured at clot onset, which is calculated as being 6×SD (standard deviation) over background;

[0237] FIG. 17 shows the stability profile of the porous matrix after up to 13 weeks storage at 45° C. (A) and after up to 120 days at temperatures of between 5 and 70° C. (B);

[0238] FIG. 18 is a schematic representation of the cholesterol pathway and potential reagents for use in detecting cholesterol in accordance with the present invention;

[0239] FIG. 19 is a diagram of dihydrorhodamine 123; and

[0240] FIGS. 20 A and B show cholesterol assay results in accordance with an embodiment of the invention.

EXAMPLE 1

[0241] FIG. 1 shows the analytes involved in the coagulation pathway, which is a series of enzyme assisted biochemical reactions that produce a blot clot. For many individuals, for example, patients taking anti-coagulant therapy such as warfarin or coumarin, the coagulation status must be regularly monitored. This enables the dose of therapy to be regularly adjusted to be within the therapeutic range while avoiding severe side effects such as increased bleeding.

[0242] As FIG. 1A shows, the cascade has two pathways, an intrinsic or contact activation pathway and an extrinsic or tissue factor pathway. The cascade is self-propagating once initiated, yet also exhibits self-regulation, since there are a number of inhibitors in the blood system which impede pro-coagulant steps and break down formed clots. The extrinsic and intrinsic pathways merge into the common pathway, at the centre of which is thrombin. This molecule has proteolytic activity, cleaving soluble fibrinogen and releasing insoluble fibrin which cross-links clots.

[0243] To monitor the coagulation status in known assays, a component is added to the patient sample which initiates clotting, for example, tissue factor. Tissue factor is a lipoprotein found only in tissue material and so is normally absent from the cardiovascular system. When a blood vessel is ruptured and no longer provides an effective barrier against tissue material, tissue factor enters the blood stream and the extrinsic cascade is initiated. One or more downstream analytes can then be measured. In the following embodiments, thrombin is the analyte which is detected by the assay of the present invention. However, because of the self-propagating nature of the cascade, numerous analytes of the pathway may be considered as potential biomarkers for coagulation status. As FIG. 1B shows, other factors, such as Russell's Viper Venom, Ecarin, Textarin, Taipan venom, Reptilase or Protac can be added to the patient sample to initiate clotting at different points of the pathway. There is accordingly the potential to monitor other components of the cascade, for example Factor X, Factor Xa, Factor II, Fibrin monomer or protein c. Thus, it is readily possible to envisage methods which are capable of detecting and/or determining the effect of other members of the extrinsic or intrinsic pathway, or other unrelated analytes.

[0244] Reagents Capable of Generating a Photometric Change

[0245] The present inventors found that a quenched reagent could be cleaved by thrombin, thus emitting fluorescence. FIG. 2 shows the thrombin cleavable agent, a bisamide derivative of Rhodamine 110 (Rhodamine 110, bis-(p-Tosyl-L-Glycyl-L-Prolyl-L-Arginine Amide, catalogue number R22124, Molecular Probes, Thermo Fisher Scientific). This reagent contains a fibrinogen peptide unit (peptide arm) covalently linked to each of Rhodamine 110's amino groups.

[0246] As shown in the Figure, in the cascade thrombin cleaves fibrinogen peptide units A and B, converting fibrinogen to insoluble fibrin monomers. The peptide arms attached to Rhodamine 110 replicate the fibrinogen unit peptides recognised naturally by thrombin. Thus, thrombin can cleave the peptide arms of the bisamide derivative of Rhodamine 110.

[0247] In an embodiment of the invention, the thrombin cleavable bisamide derivative of Rhodamine 110 is present within the porous matrix. The porous matrix is localised to an inner luminal surface of the optically transmissible portion of the microfluidic channel. It will, however, be appreciated that in other embodiments a porous matrix is not necessary. During the assay, the thrombin cleavable bisamide derivative of Rhodamine 110 comes into contact with a portion of the sample, for example blood. When thrombin is present in the sample of blood, thrombin cleaves the peptide arms of the bisamide derivative of Rhodamine 110. The cleavage of the peptide arms activates the bisamide derivative of Rhodamine 110 so that it emits an optical signal.

[0248] Describing the optical signal in more detail, as a result of cleavage of the peptide arms (Arg-Pro-Gly sequence), the non-fluorescent bisamide derivative Rhodamine 110 is converted first to a fluorescent monoamide and then to Rhodamine 110. Rhodamine 110 emits a fluorescent signal measurable on the wavelength of the optical detection device (excitation at 498-505 nm, emission at 521-525 nm). As the clotting cascade progresses downstream the conversion of prothrombin to thrombin increases and more Rhodamine 110 is released, therefore increasing the magnitude of the fluorescent signal. The conversion of prothrombin to thrombin is proportional to the fluorescent signal generated in a given time. The value for determining thrombin synthesis is known as prothrombin time (PT), or its derived measure, international normalized ratio (INR).

[0249] Hence, a high rate of fluorescent signal formation is indicative of a fast rate of prothrombin to thrombin conversion (low INR), while a low rate of fluorescent signal formation is indicative of a slow rate of prothrombin to thrombin conversion (high INR).

[0250] In a sample from a patient who is not taking anticoagulant therapy (non-therapeutic sample), the conversion of prothrombin to thrombin is very fast so a fluorescent signal is produced rapidly. In a therapeutic sample (a sample from a patient taking anticoagulant therapy), the cascade of reactions is slowed down, for example, in the case of Warfarin, due to inhibition of Vitamin K factors. Thus, PT is prolonged in patients on anti-coagulant therapy.

[0251] An example of such an assay is shown schematically in FIG. 3. In this Figure the porous matrix for generating an optical change/signal 21 is shown immobilised to the upper inner luminal surface 5 of a detection zone 26 of a microfluidic channel 4. A blood sample 7 enters detection zone 26, in which the porous matrix 21 is immobilised, permitting the blood sample 7 to come into contact with components including reagents within the porous matrix 21. Prothrombin within the blood sample 7 initially reacts with thromboplastin (TP) within the porous matrix 21, which initiates the clotting cascade and activation of clotting enzymes as shown. This results in the ultimate generation of thrombin which in turn is able to react with the bisamide derivative of Rhodamine 110 (R110) as discussed above, cleaving the peptide arm and generating an optically detectable signal within the porous matrix 21. The optical signal is detected using an optical detector present within an associated reader device, through an optically transmissible portion 9 of the detection zone 26. In this embodiment, the upper inner luminal surface 5 and the optically transmissible portion 9 are formed of the same optically transmissible material; however, in other embodiments it will be appreciated that the optically transmissible portion 9 may be formed of a different material to the rest of the upper inner luminal surface 5.

[0252] In the above example all the necessary components are present within a single porous matrix. FIG. 4 shows an alternative embodiment where some of the reaction components are separated into two porous matrices (121, 221). In this example, the first porous matrix 221 comprises thromboplastin (TP) for use in initiating the clotting cascade. The blood sample 7 initially comes into contact with the first porous matrix 221 in order to permit prothrombin within the blood sample 7 to come into contact with the thromboplastin within the first porous matrix 221 and the clotting cascade initiated. Thereafter the blood sample 7 is transported downstream to the second porous matrix 121 (the porous matrix for generating an optical change/signal) which comprises the reagent bisamide derivative of R110. Thrombin within the blood sample 7 cleaves the bisamide derivative of R110, thereby generating the reagent reaction product and an optically detectable signal, which in this embodiment, can be detected through the optically transmissible portion 9 of the detection zone 26 of the microfluidic channel 4.

[0253] It will be appreciated that although the embodiment of FIG. 4 shows only the second porous matrix 121 in the detection zone 26, in other embodiments the first porous matrix 221 can also be in the detection zone 26.

[0254] Separating or de-coupling the components/reagents such as exemplified schematically in FIG. 4 may be of use when the various components/reagents may potentially interfere with one another, or where it may be desired to alter the physical and/or chemical nature of each porous matrix. For example, it is possible to alter the porosity, wettability, pH etc. for each matrix and hence tailor this to the specific reactive components within each porous matrix. As made clear previously, it is readily possible for some reagents simply to be outside the porous matrix for optical detection. Thus, for example, a reagent could simply be dried down directly on to a surface of the microfluidic channel, or coupled with a carrier, but need not be present within a further porous matrix. Tests have shown this de-coupled embodiment to produce very reproducible results.

[0255] The close proximity of the optically transmissible portion 9/matrix 21 and the optical detector minimises any effect components which may be present in the sample may have in interfering with and/or obscuring optical detection. Without wishing to be bound by theory, the inventors also believe that the porous matrix is capable of excluding material of a particular size, such as red blood cells. This may reduce interference from such material during optical detection.

[0256] The assay performance of the free thrombin cleavable bisamide derivative of Rhodamine 110 (R110) (i.e. by “free” it will be appreciated that the bisamide derivative of Rhodamine 110 is not attached to another particle) when localised to the inner luminal surface of the optically transmissible portion was compared to magnetic or latex particles functionalised with the thrombin cleavable bisamide derivative of Rhodamine 110. Performance (measured as INR values), was comparable between the free thrombin cleavable bisamide derivative of R110 and latex particles functionalised with the thrombin cleavable bisamide derivative of R110. The performance of magnetic particles functionalised with the thrombin cleavable bisamide derivative of R110 was reduced compared to the other two groups.

[0257] It was decided to use the free thrombin cleavable bisamide derivative of R110 in further experiments since this removed the requirement of a solid phase, therefore reducing the complexity and cost of the porous matrix.

[0258] For the purposes of brevity, R22124 (catalogue number) as used herein refers to the free thrombin cleavable bisamide derivative of Rhodamine 110.

[0259] The effect of the concentration of R22124 within the porous matrix on the performance of the assay was also assessed. Concentrations (i.e. as provided in a liquid form, which is then allowed to dry by evaporation or other means in the matrix) of 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1 mM R22124 were tested on contrived plasma samples and the results compared. A similar performance (as measured by clot time) was achieved from 0.25 to 0.75 mM; concentrations at 0.1 and 1 mM led to the highest clot times. Results are shown in Table 1.

TABLE-US-00001 TABLE 1 Effect of the concentration of R22124 (shown as [Rhod]) within the porous matrix on the performance of the assay. ACL INR [Rhod] mM Mean clot time SD 2.008 0.1 90.8 7.4 2.008 0.25 47.1 5.8 2.008 0.5 44.4 6.5 2.008 0.75 54.2 1.6 2.008 1 66.4 11.0

[0260] Microfluidic Channel/Cartridge

[0261] A sample microfluidic cartridge for use in the present invention is shown in FIG. 5a. In FIG. 5a, the cartridge 1 comprises a sample input port 3 connected to a microfluidic channel 4. The channel 4 extends within the cartridge 1 and branches to form a plurality of microfluidic channels 11, each channel 11 being fluidly connected to an independent gas filled chamber 10. In other embodiments the cartridge may comprise one, two or three channels 4.

[0262] The cartridge is formed from three separate planar layers, a first, second and third layer, in this embodiment a top and a bottom layer with a middle layer disposed between the top and bottom layers, which are sandwiched together to define the microfluidic channel 4 and the gas filled chamber 10. The middle layer is in the form of an adhesive layer which adheres the top and bottom layers. In the present embodiment, the channels of the cartridge are disposed within the middle layer. Hence, the channel walls are formed by the middle layer, and the base of the channels is formed by the bottom layer.

[0263] The cartridge of the embodiment of FIG. 5a is to be considered as self-contained; in that prior to application of a sample it is substantially liquid free and is considered as dry. The only fluid prior to application of the liquid sample which is present in this cartridge is a gas, in this embodiment air.

[0264] In use, a fluid sample (in the present embodiment, a blood sample) fills the channel 4 and this can be detected by electrodes which are in electrical contact with corresponding electric 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.

[0265] Describing each channel 11 in more detail, there are printed features (20, 22, 24) which are designed to limit movement of any reagent/porous matrix which is positioned within each channel 11 during the manufacturing process. The printed features (20, 22, 24) also function to limit further movement of the sample once it reaches the region of the printed features. In the present embodiment, the printed features are formed of a carbon-based hydrophobic ink and function as electrodes. In other embodiments, the printed features are formed of silver and can also function as electrodes. It will be appreciated that in other embodiments, the number of printed features may differ. For example, the printed features may comprise a printed feature bordering the detection zone (i.e. printed feature 20). Conveniently, the porous matrix is initially deposited within the area defined by the printed features and mechanically spread within. This limits and defines the area where the porous matrix is located The printed features may have a different shape to the shape shown in FIG. 5A. Printed feature 20 may be a different shape to the feature shown in FIG. 5A. For example, printed feature 20 may be a u-shape, i.e., open at one end. The printed feature 20 defines an area of 2×1.5 mm.

[0266] In use, an increasing force is applied to the chambers 10 of the cartridge 1, expelling gas from the chambers 10. Upon application of a blood sample to the cartridge, the sample is then drawn into the cartridge by air returning to the gas chambers 10 following a release in pressure being applied to the chambers (as will be described later). Once the sample reaches the printed feature 24 downstream of the detection zone, an increase in pressure is applied to the gas chambers to prevent further downstream movement of the sample.

[0267] In other embodiments, initial sample flow into and along the microfluidic channel take place by capillary action alone, to a first stop feature. Thereafter and in order to pass the stop feature, further sample flow is effected by an active force, as described above and below.

[0268] Located within the boundary of the printed feature 20 is a detection zone 26 of each assay channel 11 into which has been deposited a porous matrix (not shown) comprising a reagent designed to react with a particular analyte or reaction product thereof which may be present in a sample to be assayed. The porous matrix is optically transmissible and is localised to the inner luminal surface of an optically transmissible portion of the top layer. Multiple assays may be carried out on one microfluidic cartridge; the number of assays depends upon the number of separate channels and detection zones. In the present Figure four different channels 11 and detection zones 26 are shown.

[0269] Located further downstream to the detection zones 26 and printed features (20, 22, 24) 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 11. A decrease in the force applied to the chambers 10 causes air to be drawn back into the chambers 10 from the assay channels 11.

[0270] Once the blood sample reaches the printed feature 24 which is located downstream of the detection zone 26, further movement of the blood sample is restricted. In addition, the printed feature 24 triggers the taking of at least one optical measurement by an optical detection device and thus the start of the assay.

[0271] Various reagents may be suitable for localisation within the porous matrix of the detection zone 26. For example localised in the porous matrix of the detection zones 26 may be free fluorogenic particles or fluorescently labelled latex particles functionalised with a further antibody designed to specifically bind a different epitope of analyte to be detected. In the present embodiment R22124, as shown in FIG. 2, is a reagent in the detection zone 26.

[0272] FIG. 5B shows a detection zone 26 once the assay of the present invention has started and detection of signal is ongoing. As shown in this Figure, a portion of a blood sample has reached the printed feature 24 which is located downstream of the detection zone 26, and so further movement of the blood sample is restricted. In this way, the portion of the blood sample is primarily localised to the detection zone 26 thus ensuring optimal detection. Optical measurements are taken using the LED (not shown), which is positioned above the detection zone 26. The matrix 21 remains localised to the inner luminal surface of the optically transmissible portion following the blood sample filling the detection zone 26. During the assay, no or very few red blood cells enter the matrix, as shown by the primarily clear matrix 21 in FIG. 5B. There is thus minimal interference with the optical detection of signal.

[0273] Although the above embodiment is shown with the porous matrix being immobilised on the upper intraluminal surface of the microfluidic channel and the optical detection taking place from above, it is possible for the porous matrix to be immobilised to the lower intraluminal surface of the microfluidic channel and detection taking place from below. In accordance with the invention detection should take place through the same surface to which the porous matrix is immobilised. Thus, if the porous matrix is immobilised to an upper intraluminal surface of the microfluidic channel, the optical detection system should not be arranged to detect any optical change through the lower intraluminal surface of the microfluidic channel and vice versa. That is, the bulk blood sample should not be positioned between the porous solid matrix and the optical detection system.

[0274] FIG. 5C shows the difference in signal intensity which can be obtained depending on where the porous matrix is positioned. The two top images show two repeats of the fluorescent intensity in the detection zone when a porous matrix according to an embodiment of the invention is retained on an inner luminal surface (in this case the upper surface) of the microfluidic channel, which is immediately adjacent to the optical detector present in the reader. The two bottom images show two repeats of the significant reduction in intensity displayed when the same porous matrix is positioned on the lower surface of the channel, when the optical detector is immediately adjacent to the upper surface. Hence the sample of blood within the channel is between the porous matrix and the optical detector. All images show the fluorescent intensity 30 seconds after a blood sample entered the detection zone. The same formulation was used for each porous matrix in each Figure. As can be seen, there is a distinct advantage in not having blood positioned between the porous matrix and the optical detector, in order to ensure efficient signal detection.

[0275] The porous matrix is initially spotted and contained within the boundary of printed feature 20 as shown in FIG. 5A. If simply spotted, the resulting dried spot will be generally round in nature and when dried will have an uneven thickness in cross section. That is the spot will be thinner at the edges of the spot, as compared to the center of the spot. The inventors have observed that by spreading the liquid spot, prior to it drying down, a more uniform thickness can be achieved, leading to a very good reproducible signal generation being achieved. FIG. 5D shows examples of different formulations of porous matrices in accordance with the present invention, in which each liquid spot has been spread and thinned prior to drying onto the surface. As can be seen good signal generation (from the reagent reaction product), over the three time points (5, 15 and 30 seconds after a blood sample entered the detection zone) across the entire spot is observed for each formulation. In this Figure, two porous matrices; a first porous matrix outside of the detection zone, and a second porous matrix for generating an optical change/signal in the detection zone, were utilised. In the top two rows, the porous matrix for generating an optical change/signal (the second porous matrix) comprised a formulation containing readiplastin, CMC, trehalose and rhodamine, amongst other components (formulation 1). In the bottom two rows of the Figure, the porous matrix for generating an optical change/signal (the second porous matrix) comprised a formulation which did not contain readiplastin (formulation 2). Both embodiments contained a further porous matrix (the first porous matrix) outside of the detection zone comprising trehalose and readiplastin. It will be appreciated that these formulations are exemplary and that other formulations can be used.

[0276] Signal development from a thin porous matrix as compared to a thicker porous matrix is shown in FIG. 5E. As is clear from this Figure, use of a thin layer (a) resulted in a fast and even reaction (see fluorescence), as compared to a thicker layer (b), where the reaction was slower and more patchy.

[0277] FIG. 6 shows a cartridge according to an embodiment of the present invention in use in the assay. A blood sample is contacted with and introduced into the cartridge 1 by way of the input port 3, as shown in step I of FIG. 6. The blood sample fills the channel 11 by the active fill mechanism, wherein following gas being expelled from the chamber 10 by pressure applied by the piezoelectric bender (not shown), the blood sample is drawn into the cartridge 1 and channel 11 by air returning to the gas chambers following a release in pressure being applied to the chambers 10 (step II and III of FIG. 6). The careful control of the reduction in force applied to the chambers 10 controls how far the blood sample is drawn into the detection zones 26, which are bounded by the printed features 20, and minimises sample variation effects on filling speed. This can also be controlled via electrode sensed feedback.

[0278] Further downstream of the detection zones 26 are the printed features 24, which are electrodes. The blood sample fills over the printed feature 20, and continues further downstream to meet the printed feature 24 (step IV of FIG. 6). Once the blood sample meets the printed feature 24, this printed feature 24 sends a signal to turn on the LED. At least one optical measurement is then taken by the LED (detector) in the reader. Further movement of the blood sample is restricted by the printed feature 24 and the stop flow mechanism. By waiting until the blood sample meets the printed feature 24 before starting to take optical measurements, this provides a start time 0 to the assay which is independent of operator error. It also confirms that the blood sample has successfully filled the detection zone 26.

[0279] FIGS. 7 and 8 show schematically some of the microfluidic architecture of two embodiments which employ an initial capillary fill, followed by an active fill. Each embodiment shows an input port 303 connected to a first portion of microfluidic channel 304. At the end of each first portion there is an enlarged region 314, where a haematocrit value of the blood sample can be obtained. Suitable methods for obtaining haematocrit values are known, for example, from US006064474A and Van Kempen E J and Zijlstra W. G et al. 1983 (Spectrophotometry of hemoglobin and hemoglobin derivatives. Advances in Clinical Chemistry, Vol 23, 199-257.) Extending downstream from the enlarged region 314 is a second portion of the microfluidic channel 316, which contains a small vent arm 318. This type of vent arm is described in more detail in WO2018/002668 to which the skilled reader is directed.

[0280] In use, once a sample of blood is introduced to the input port 303, the blood sample will fill the first portion 304, enlarged portion 314 and second portion 316 by capillary action, until encountering the vent arm 318, at which point capillary flow of the blood will stop. In order for further blood flow to occur, an active fill mechanism is utilised, as described above. Briefly, the active fill involves depression of the air/gas filled chamber 310. This causes the blood sample to flow past the vent arm 318 and into the detection zone 326 comprising one or more porous matrices (not shown), as previously described herein. The necessary reactions take place in the detection zone 326 and any optical change is detected using an optical detector as previously described.

[0281] The embodiment depicted in FIG. 8 is shown as having a longer second portion 316 before the detection zone 326. This readily accommodates the split or de-coupled method as described herein. For example, a first porous matrix, or one or more reagents may be deposited with the second portion 316 and further reagents or a second porous matrix comprising the reagents necessary for generating the optical change/signal, may be deposited in the detection zone 326. For example, the first porous matrix, or one or more reagents may be deposited in the section of the second portion 316 between the vent arm 318 and the detection zone 326. As previously discussed, in such a split matrices embodiment, the compositions of each matrix may be different and tailored to the requirements of the particular assay and reagents which are necessary. For example, for a PT/INR assay as discussed herein, the thromboplastin present in the first spot, may not need to be tightly bound within the spot, such that upon blood sample contact, it may be free to migrate from the porous matrix. However, the reagent which reacts to form the reagent reaction product (for example the bisamide derivative of Rhodamine 110), should be contained within a porous matrix, which substantially prevents or minimizes the reagent and the resulting reagent reaction product, from being washed out of the porous matrix. The inventors have observed that the use of CMC is particularly efficacious in this regard.

[0282] The above provides a description of one specific embodiment of the present invention, but the present invention is designed to be in the form of a platform assay which can easily be adapted. For example, the sample may be moved in the cartridge from capillary action alone, for example due to the addition of vents to the cartridge.

[0283] Moreover, multiple test formats may be assayed; cartridges with 2, 4 or 10 channel formats, for example, may be used.

[0284] Although the primary measurement technology is fluorescence the assay also incorporates electrochemical measurement and other components can easily be incorporated.

[0285] Various matrix formulations have been developed by the inventors for localisation to the optically transmissible portion to deliver accurate optical measurement of a sample. These are described in more detail below.

[0286] Other Components of the Matrix

[0287] Initial assessment of the matrix considered a simple thrombin-R110 method. In the assessment, R22124 was dissolved in a solution of ethanol (i.e. a wet assay). Lyophilised thrombin was solubilised in buffer and serial dilutions made. A volume of thrombin was reacted with the R22124 in ethanol in a cartridge, and after a period of incubation optical measurements were taken using a reader such as a reader described in WO2018/002668. A thrombin dose response curve was thus obtained. This confirmed that thrombin activity (and thus coagulation status) could be measured by a wet assay.

[0288] A dry matrix localised to the optically transmissible portion was then investigated. Free R110 was solubilised in DMSO or ethanol and deposited on the inner luminal surface of the optically transmissible portion of the microfluidic channel using trehalose as a carrier molecule for binding and to provide viscosity and stability. Trehalose is a non-reducing homodisaccharide in which two glucose units are linked together in an .-1,1-glycosidic linkage. Trehalose is classified as a kosmotrope or water-structure marker. It was found that this dry matrix returned a fluorescent signal when tested against lyophilised thrombin or plasma samples, although signal was low.

[0289] The effect of the concentration of trehalose in the matrix upon assay performance was assessed. The signals obtained from a sample with a known INR value of 3.5 using a matrix having a trehalose concentration of 20, 30, 40 or 50% (w/v) were observed. It was found that an increased trehalose concentration enhanced the signal. Later studies found that trehalose concentrations of 10 and 20% (w/v) were comparable.

[0290] Additional components of a dry matrix were then investigated. The effect of tissue factor upon assay performance was assessed. As previously noted, tissue factor is added to the patient sample to initiate clotting and thus to enable the determination of the coagulation status. Tissue factor was thus added to the formulation (which was then allowed to dry) at a concentration of between 1× and 8×.

[0291] To explain the 1×-8× nomenclature, RecombiPlastin 2G (Werfen) was used as the tissue factor. Product instructions recommend that the lyophilised RecombiPlastin 2G is dissolved in 20 mL of diluent. Werfen does not disclose the actual concentration of tissue factor in this product and so the initial concentration used in the formulation was not known.

[0292] Hence, to investigate the effect of tissue factor concentration, the lyophilised material was dissolved in lower volumes than recommended. Accordingly:

[0293] 1×=20 mL diluent addition as recommended by the manufacturer;

[0294] 2×=10 mL diluent;

[0295] 4×=5 mL diluent;

[0296] 8×=2.5 mL diluent.

[0297] 16×=1.25 mL diluent

[0298] It was calculated that the concentration of tissue factor in the 16× concentration was 2189 pm.

[0299] Assessing a sample with a known INR of 7, it was observed that increased tissue factor in the matrix increased the rate of fluorescence generation of the assay.

[0300] In later studies the inventors switched from Recombiplastin 2G to Readiplastin. Clinical performance of Readiplastin was comparable to Recombiplastin 2G. Readiplastin is a concentrated liquid form of Recombiplastin 2G in which calcium has been removed

[0301] The effect of clotting factors upon assay performance was then assessed by measuring end-point fluorescence. Factor V/Va is a cofactor which forms the prothrombinase complex with Factor Xa and calcium. This complex cleaves prothrombin converting it to the active form, thrombin. Factor V is modulated by thrombin being directly activated by thrombin on a positive feedback and indirectly inactivated by thrombin through the thrombomodulin-protein C complex (with the cofactor protein S) pathway.

[0302] Factor Va was added to the sample at concentrations between 0 and 100 units/ml, and the effect upon the signal obtained from clinical plasma samples with different known INR values (5.0, 2.8 and 3.8) was observed. Adding factor Va primarily increased the signal observed. However, it slightly decreased the signal (fluorescence as measured at an end point) obtained in some samples at the higher concentrations (50 or 100 units/ml).

[0303] Factor X/Xa is another clotting factor which is activated by factor VIIa/tissue factor complex. Factor Xa binds to Factor Va and calcium to form the prothrombinase complex and converts prothrombin to thrombin. Factor Xa was added to the sample at concentrations between 0 and 15 μg/ml, and the effect upon the signal obtained from clinical plasma samples with different known INR values (7.8, 2.5 and 3.8) was observed. Adding factor Xa increased the signal observed, with a slight reduction of signal at 15 μg/ml only.

[0304] Prothrombin is the inactive zymogen of thrombin. The rate of conversion to thrombin is what assays measure to monitor clot time. Prothrombin was added to the sample at concentrations between 0 and 0.2 mg/mL, and the effect upon the signal obtained from clinical plasma samples with different known INR values (2.1, 3.9, and 6.5) was observed. An increasing concentration of prothrombin increases the signal observed.

[0305] Calcium, another clotting factor, was also investigated. Ca.sup.2+ was added to the sample at a concentration of between 0 and 25 mM, and the effect upon the signal obtained from plain whole blood samples was observed. Similarly to many other clotting factors, calcium had an initial enhancing effect upon signal followed by an inhibitory effect when concentration was too high (above 12 mM).

[0306] Other clotting factors investigated were Factors Vila, IXa and XIa.

[0307] One or more of these clotting factors could optionally be used in a quality control channel in a cartridge of the assay.

[0308] Studies were undertaken to assess the effect of different carrier molecules in the matrix on assay rate. Without wishing to be bound by theory, the inventors believe that the carrier molecule acts as a scaffold to hold R22124. The inventors also believe that the carrier molecule acts as a selective filter, allowing thrombin (or other analytes) to enter the formulation yet preventing red blood cells, or other particulate material/components from entering the matrix. Therefore, the interaction of R22124, thrombin and optionally tissue factor is favoured.

[0309] The effect of incorporating the polymer carrier molecule Hydroxyethylcellulose (HEC) into the matrix was investigated. HEC is a non-ionic water-soluble polymer made by reacting ethylene oxide with alkali-cellulose. Solutions of HEC are pseudo-plastic or shear-thinning. HEC is easily dissolved in cold or hot water to give crystal-clear solutions of varying viscosities. Furthermore, low to medium molecular weight types are fully soluble in glycerol and have good solubility in hydro-alcoholic systems containing up to 60% ethanol. HEC is generally insoluble in organic solvents. HEC can be found as a polymer of different lengths and molecular weights. In the present examples, HEC had an average molecular weight of 720,000 g/mol, but as the skilled person will appreciate, there are other commercially available molecular weights, for example 90,000, 250,000 or 1,300,000 g/mol.

[0310] FIG. 9 shows the effect of adding HEC as a carrier molecule of the matrix. FIG. 9A shows the end-point signal generated from a blood sample when either 35% trehalose or a concentration of 1%, 0.5% or 0.25% HEC was included in the matrix. As FIG. 9A shows, replacing trehalose with HEC increases the signal (i.e. end point fluorescence signal) generated. FIG. 9B shows the improved signal when HEC replaces trehalose in the matrix. The same concentrations were used in B as in A. End-point signals were obtained for FIGS. 9A and 9B using a reader as described in WO2018/002668

[0311] The effect of the molecular weight of HEC upon clot time, when used in the matrix, was then investigated. FIG. 9C shows that for a sample with an INR value of 4.2, decreasing the molecular weight of the HEC in the matrix decreased the clot time. The effect of the molecular weight of HEC upon clot time was further investigated as shown in FIG. 9D. In this Figure, samples with known INRs of between 0 and 8 (as measured using a reference system, in this Figure the CoaguChek® XS system, Roche, see x axis) were used in an assay according to an embodiment of the invention, wherein the formulation of the matrix contained either 1% HEC with a molecular weight of 750 kDa, or 1% HEC with a molecular weight of 90 kDa. In accordance with FIG. 9C, decreasing the molecular weight reduced clot times.

[0312] The inventors observed that in the absence of trehalose, HEC was very viscous. To aid coverage of the optically transmissible portion, it was therefore decided to add HEC and trehalose to the matrix to provide stability and aid deposition of the free R110. When in combination with HEC, Trehalose was included at lower concentrations than those previously studied, for example, 10%.

[0313] Other carrier molecules were also assessed. The assay was tested upon a whole blood sample using a matrix containing 0.3% HEC, 5% trehalose and 20% PEG (polyethylene glycol) (as well as Triton X 100, free R110 and tissue factor); clot times generated from this assay are shown in FIG. 10A. As this Figure shows, using the matrix described above, the clot time as determined by a method according to the present invention and the INR are linearly related.

[0314] An INR of 1 as measured by a reference system (ACL elite—plasma based lab analyser, Werfen, UK.) was detected in a reader according to the present invention in 10-12 seconds, whereas an INR of 10 was detected at between 70 and 90 seconds. The reference INR values were provided by reference plasma samples and appropriately diluted reference plasma samples in accordance with the Hart Biologicals INR correction kit (Hartlepool, UK). This shows that high INRs can be measured in less than 100 seconds using a method according to the present invention.

[0315] Avicel was also studied as a potential carrier molecule for the matrix. Avicel RC-591 is a spray-dried blend of microcrystalline cellulose (MCC) and sodium carboxymethylcellulose (CMC), which acts as a water dispersible organic hydrocolloid. In the presence of water and mild shear, the Avicel RC-591 powder particles swell and are then peptized, forming a dispersion of cellulose microcrystals. These microcrystals create a stable lattice structure.

[0316] Due to the small size of the microcrystals (approximately 60% of the crystallites in dispersion are ⋅0.2 μm), there are a large number of microcrystals packed in each powder particle. The large number of small microcrystal particles helps to promote a slower and more uniform settling rate, suspension stability and the absence of hard packing. It also provides dispersion and stability.

[0317] The effect on the assay rate of replacing HEC and/or trehalose with Avicel was studied. Contrived blood samples with known INR values were tested in an assay wherein the carrier molecule in the matrix localised to the inner luminal surface of the optically transmissible portion consisted of HEC and trehalose, HEC only, Avicel and trehalose or Avicel alone. As can be observed in FIG. 10B, the use of Avicel in the matrix reduced the assay times. Hence, an INR of 8 is detected approximately 140 seconds faster in an Avicel and trehalose matrix as compared to a matrix containing HEC.

[0318] It was then decided to study the effect of the polymer sodium carboxymethylcellulose (CMC) as a carrier molecule of the matrix when localised to an inner luminal surface of the optically transmissible portion, since CMC is a component of Avicel RC-591. FIG. 11A shows the effect of different concentrations of CMC (0.1%, 0.2% or 0.5%) in the matrix upon the clot time of the assay (see y axis) when tested upon samples with identical known INR values (the INR values previously determined using a CoaguChek® XS reference system, see x axis).

[0319] Samples tested were reference plasma samples with known INRs and appropriately diluted reference plasma samples in accordance with the Hart Biologicals INR correction kit (Hartlepool, UK). In this Figure CMC was used instead of Avicel RC-591. Assay times were found to be comparable to those achieved using Avicel RC-591; assay times were still within 10 to 60 seconds for INR values 1-5.

[0320] Further investigations were carried out regarding the effect of the concentration of CMC in the matrix. 0.4% CMC or 0.2% CMC was incorporated into the matrix, and assays carried out on samples with identical known INR values (as previously measured using a ACL INR reference system). A CMC concentration of 0.4% resulted in longer clot times (shown plotted as seconds against the y axis) than those observed from a CMC concentration of 0.2% (FIG. 11B). For both CMC concentrations the assay was able to measure a wide range of INRs (INRs ranging from 1 to 10.3 are shown in FIG. 11B).

[0321] The CMC used in FIGS. 11A and B was a blend of polymers with unknown weight and purity. Following a further search, some pure forms of CMC were identified with differing degrees of substitution. The degree of substitution (DS) of a polymer is the average number of substituent groups attached per base unit (in the case of condensation polymers) or per monomeric unit (in the case of addition polymers). Some of the commercial CMCs used have a DS of 0.7 (i.e. an average of 7 carboxymethyl groups per 10 anhydroglucose units). Assays were performed on samples with identical INR values using CMC at differing DS' (0.7 DS, 0.9DS or 1.2 DS) in the formulation. FIG. 11C shows the clot times from these assays (see y axis), as plotted against the known INR values (as previously determined using an ACL reference system). All three CMC forms provided similar assay rates, but it was observed that increasing the degree of substitution prolonged clot times.

[0322] Combinations of carrier molecules for the porous matrix for optical detection were further considered. The effect of differing concentrations of CMC and PEG, and the ratio of CMC to PEG in the porous matrix for optical detection was assessed. In particular, the solubility of the matrix and hence the distribution of the reagent for detection was considered.

[0323] Formulations tested comprised PEG at a concentration by weight of between 0.83% and 3.33% and CMC at a concentration by weight of between 0.08% and 0.12% The formulations were used in a porous matrix for optical detection. The porous matrix, in addition to other components, comprised the fluorescent reagent R22124. Samples of blood having a contrived INR of 2.7 were used to test the different ratios of CMC to PEG in the matrix.

[0324] It was observed that increased fluorescence, and distribution of fluorescence occurred when the ratio of PEG to CMC was higher, i.e. when the porous matrix comprised a low concentration of CMC and a higher concentration of PEG. These results (0.08% CMC and 0.83% PEG or 0.08% CMC and 1.67% PEG) are shown in bottom two rows of images in FIG. 12.

[0325] The images in FIG. 12 show fluorescence detected in the porous matrix in the detection zone 20 seconds, 30 seconds and 40 seconds (from left to right, consecutively) after the blood sample entered the detection zone. In this embodiment, only one porous matrix, a porous matrix for optical detection in the detection zone, was used.

[0326] The top row of images show the fluorescence observed when the porous matrix comprised 0.08% CMC and 0.83% PEG. The middle row of images show the fluorescence observed when the porous matrix comprised 0.08% CMC and 1.67% PEG. The bottom row of images show the fluorescence observed when the porous matrix comprised 0.08% CMC and 3.33% PEG.

[0327] Without wishing to be bound by theory, the inventors believe that PEG may assist in the wettability of the porous matrix, while CMC may assist in the binding nature of the matrix, i.e. in localising the matrix to the surface of the microfluidic channel. By optimising the ratio of the two components, this may achieve a matrix with excellent localisation to the surface with improved wettability to allow the even distribution of the analyte and resulting reaction product. This may help to improve fluorescent signal.

[0328] Another molecule which the inventors decided to study was heparin. Heparin is an anticoagulant which prevents the formation of blood clots. Two types of heparin are currently available: unfractionated heparin (UFH) and low molecular weight heparin (LMWH). Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change which results in its activation through an increase in the flexibility of its reactive site loop. The activated AT then inactivates thrombin, factor Xa and other proteases. The rate of inactivation of these proteases by AT can increase by up to 1000-fold due to the binding of heparin.

[0329] A known inhibitor of heparin is hexadimethrine bromide (HMB or polybrene). It was decided to assess the effect of the heparin enoxaparin upon the clot time of samples with known INR values in the assay. Addition of the heparin enoxaparin at a concentration of 3 U to the matrix (when wet, which was then allowed to dry) increased clot times as compared to assays without enoxaparin (samples with known INR values were tested). For example, when testing an INR value of approximately 4.4, the clot time when the matrix included enoxaparin was approximately 70 seconds longer than the clot time when the matrix did not include enoxaparin. The addition of 0.25 mg/mL polybrene inhibited this effect, such that clot times obtained from assays where enoxaparin and polybrene were included in the matrix were comparable to clot times from control matrices.

[0330] The dry formulation of the matrix brings a number of important differentiating advantages. Firstly, the dry formulation is viscous and can withstand blood flow; as a result the matrix remains aligned with the optical detection device for optimal detection. Without being bound by theory, the present inventors believe that the viscous nature of the matrix acts as a polymer scaffold to hold the active components of the matrix.

[0331] The dry matrix is porous. The porous nature of the matrix allows the ingress of necessary analytes or analyte reaction products, in this embodiment thrombin, while preventing the entry of larger components in the sample, such as red blood cells. This leads to a fast and sensitive assay.

[0332] By using a dry matrix, the matrix can be localised to the inner luminal surface of the optically transmissible portion of the microfluidic channel; the matrix is thus in close proximity to the optical detection device, for example an LED. This prevents optical interference. Due to the close proximity of the matrix to the optical detection device, the assay has an improved sensitivity to known assays. Without wishing to be bound by theory, the present inventors believe that this allows a much faster assay than those previously known in the art; interference by other components in the sample is overcome since the assay can kinetically detect thrombin activity.

[0333] In addition, the matrix is very viscous. Hence, while porous, the matrix remains in position even when in contact with the sample, for example a liquid. This ensures that the reagent/reagent reaction product thereof is localised in close proximity to the sample, further facilitating a rapid and sensitive assay.

[0334] Real-Time Kinetic Measurement

[0335] The close proximity of the matrix, the sample and the optical detector means that the conversion of prothrombin to thrombin, as detected by fluorescent signal, can be detected kinetically.

[0336] In the present assay, the rate of conversion from prothrombin to thrombin is faster in samples with low INR values (e.g. a value of 1 INR: i.e. a higher level of coagulation) than for samples with high INR values (e.g. a value of 10 INR: i.e. a lower level of coagulation). Accordingly, the present assay does not require an end point (i.e. a single measurement is performed after a fixed incubation period—although this is envisaged within the scope of the present invention) and so the assay may instead be stopped after reaching a threshold value or a certain rate. This results in a fast and efficient assay.

[0337] Once the sample has successfully filled the detection zone, this provides a start time of 0, and the taking of a plurality of optical measurements by the reader is triggered. During the taking of the plurality of optical measurements, time is counted as clotting time. The increasing fluorescence due to the continuing conversion of prothrombin to thrombin can continue until a threshold value is reached. Once the threshold signal is reached, the taking of optical measurements stops.

[0338] FIG. 13A shows a schematic model based upon the assay of the present invention. This Figure demonstrates that high levels of thrombin conversion (i.e. from prothrombin to thrombin) equate to low INR values, while low levels of thrombin conversion relate to high INR values. FIG. 13B is a schematic to demonstrate the linear relationship between clot time (PT) and INR values; a low clot time indicates a high level of thrombin conversion and a low INR value, while a high clot time indicates a low level of thrombin conversion and a high INR value. The samples tested were reference plasma samples (and appropriately diluted reference plasma samples with known INR values as provided by the Hart Biologicals INR correction kit (Hartlepool, UK).

[0339] Effect of Sample on Optical Detection

[0340] Many known assays require the separation of plasma from a whole blood sample before detection can occur. This is costly and time consuming. FIG. 14 shows that the present assay can be used to test various different samples including citrated plasma, plasma calibrators (for example, as provided by the INR correction kit as described herein) and clinical whole blood. The Figure also compares values obtained using an assay in accordance with the present invention as compared to other known assays.

[0341] FIGS. 14A and B shows results when testing various different samples including citrated plasma, plasma calibrators and clinical whole bloods. The same samples were also tested on an ACL device as described above. For FIG. 14A, the y axis label “lumira PT (secs)” represents the uncalibrated PT in seconds obtained using a method according to the present invention. For FIG. 14B they axis shows calibrated INR values determined using a method according to the present invention. The x axis label “ACL INR” represents INR values calculated using the ACL elite reference system.

[0342] FIG. 14C shows the uncalibrated PT in seconds obtained from clinical whole blood and contrived blood samples from a method according to the present invention (y axis) as compared to the INR values obtained from the same samples using a CoaguChek® XS system.

[0343] As is apparent from this Figure, the assay is equally effective when testing blood or plasma samples, and citration does not affect the results. Advantageously, the assay maintains linearity across a broad range of INR values.

[0344] Increased Range of Assay

[0345] The fast rate of the assay enables the measurement of very high INR samples with very low thrombin activities. Accordingly, the assay in accordance with an embodiment of the invention can advantageously measure a very broad range of INR values from clinical samples, as shown in FIG. 15. As shown in this Figure, linearity is maintained even at high INR values.

[0346] Calculation of INR

[0347] FIG. 16A shows how clot time (in relation to thrombin levels) was calculated in an embodiment of the present assay. Time 0 represents the time point at which the sample fills the detection zone. As the sample fills the detection zone, the dry porous matrix is wetted and the clotting cascade is triggered. This triggers the conversion of pro-thrombin to thrombin, and the subsequent cleavage of R22124 in the matrix to generate a fluorescent signal, in this instance with emission at 525 nm. The pro-thrombin time (or INR) value was measured at clot onset (red dot), which was defined as being 6× standard deviations over the averaged baseline. FIG. 16B represents a magnified form of the section of 16A showing the clot onset.

[0348] An alternative INR method is based on determining a PT value in order to derive an INR result through an assay calibration scheme. The PT represents a time in seconds taken for a sample to respond to activation of the extrinsic clotting cascade which is initiated by the thromboplastin contained in the cartridge.

[0349] A sequence of calculations is applied to the fluorescent signal data which is captured by the Instrument as the cascade reaction progresses—this sequence is referred to as the PT Algorithm.

[0350] The PT algorithm may be based on the following parameters and steps:

[0351] Code Parameters

TABLE-US-00002 start_wl = 515 # lowest wavelength to include end_wl = 530 # highest wavelength to include threshold1 = −300 # threshold for determining blood filling threshold2 = 1000 # threshold for the initial estimate of clot time threshold3 = 200 # threshold for the final estimate of clot time loCut = 0.35 # low level of regression data cut (e.g. 0.35 is 35%) hiCut = 0.65 # high level of regression data cut (e.g. 0.65 is 65%) PT time = Tclot − T0

[0352] Ts Determination: Ts is the initial fall in signal indicating arrival of sample and is caused by sample starting to move into the measurement chamber.

[0353] T0 Determination: T0 indicates the time where the detection zone is almost full of sample. It represents the start of mixing of sample with the reagents in the chamber and is used as the start of reaction time to calculate PT time.

[0354] T100 Determination: T100 indicates the signal has risen far enough that the point of clot formation is sure to be contained within the collected data point set.

[0355] T17000 Determination: the instrument shall proceed to Clot time Extraction below if a datapoint is found that is >17,000 count above the T0 value.

[0356] T100+30 s Determination: the instrument shall proceed to Clot time Extraction below if more than 30 seconds has elapsed since the T100 was detected.

[0357] Clot Time Extraction:

[0358] The Instrument shall perform a linear regression on the datapoints between T35 (loCut) and T65 (hiCut) to determine a slope and intercept for baseline correction.

[0359] Note: T35 is located 35% of the time between T0 and T100. T65 is located 65% of the time between T0 and T100 the reader shall apply the offset and slope to all datapoints to form a set of baseline-corrected datapoints.

[0360] The reader shall find the first point after T50 in the baseline corrected data at which the count value rises above 200 units.

[0361] The reader shall record this time as Tclot.

[0362] The reader shall calculate a PT time by subtracting the time value recorded for T0 from the time value recorded for Tclot.

[0363] The reader shall apply a calibration given by the calibration table for INR in the Lot Calibration File to the measured PT Time to obtain the INR value.

[0364] Summary:

[0365] As an alternative to a SD method described above, this is a fixed threshold with a correction of the baseline to ensure it is always flat.

[0366] There are also features of the transient that can be detected (drop, slope) as well as error trapping mechanisms to ensure all the expected steps are occurring as expected and related to the strip/assay/meter workflow (e.g. sample arrival within a given time, no breaching of the sample, slope reaches a certain signal within a given time, time outs, etc.)

[0367] Stability of Formulation

[0368] FIG. 17A shows the stability profile of a dry porous matrix according to an embodiment of the present invention (comprising tissue factor, rhodamine 11—and trehalose, amongst other components) after up to 39 weeks storage at 45° C. Storage of the dry matrix for up to 39 weeks at this temperature did not affect the clot times calculated by the assay when using such matrices and samples with known INR values.

[0369] FIG. 17B shows the stability profile of this dry matrix after up to 12 days storage at temperatures between 5 and 70° C. Clot time remained consistent even after storage of up to 60° C. for 120 days.

[0370] In embodiments comprising a plurality of porous matrices, the formulation of each porous matrix may be the same, or may be different. For example, the porous matrix for generating an optical change/signal may have a more viscous formulation and/or be less soluble that the other porous matrix/matrices. An exemplary porous matrices arrangement may comprise a porous matrix for generating an optical change/signal (i.e. a second porous matrix), having a low solubility formulation and a first porous matrix positioned outside of the detection zone having a low solubility formulation.

[0371] An example formulation for the porous matrix for generating an optical change/signal may comprise 0.20% CMC, 5% Trehalose, 33.1% thromboplastin (in embodiments directed to the detection of thrombin) and 0.125 mM R22124. The second porous matrix may comprise 5% trehalose.

[0372] When a plurality of porous matrices is envisaged, in some embodiments the porous matrices may not comprise PEG.

[0373] Other formulations can be envisaged.

EXAMPLE 2

[0374] It is envisaged that the present invention may be used for other assays, for example kinetic assays such as the determination of cholesterol levels in a sample, for example whole blood.

[0375] FIGS. 18 and 19 provide an illustrative example of the pathway and components which may be utilised to detect cholesterol. The cholesterol for detection may comprise free cholesterol and/or cholesteryl esters.

[0376] If the analyte for detection is cholesteryl ester, the ester can be reacted with the enzyme cholesterol esterase to result in the generation of free cholesterol.

[0377] When cholesterol is in the presence of the enzyme cholesterol oxidase, the following chemical reaction occurs:

cholesterol+O.sub.2⇄cholest-4-en-3-one+H.sub.2O.sub.2

[0378] H.sub.2O.sub.2 generated from the above reaction can be converted to H.sub.2O by the enzyme Horse Radish Peroxidase (HRP), the by-product of which can be used to oxidise a reagent resulting in an oxidised reagent reaction product and the emission of an optical signal. By oxidising the reagent, this can result in the generation of an optical signal. In the present Example, the reagent is a fluorescent reagent, which emits fluorescence upon oxidation. In one embodiment, this fluorescent reagent is dihydrorhodamine 123, as shown in FIG. 19. Dihydrorhodamine 123 is an uncharged and nonfluorescent reactive oxygen species (ROS) indicator that can be oxidized to cationic rhodamine 123 which exhibits green fluorescence. Dihydrorhodamine 123 is commercially available from various suppliers, including, but not limited to, Thermo Fisher Scientific, Sigma-Aldrich and Abcam.

[0379] Hence, the porous matrix/matrices of the present invention can comprise the above described components in order to detect levels of cholesterol and/or cholesteryl esters. For example, for the detection of cholesterol, the porous matrix/matrices could comprise cholesterol oxidase, HRP and dihydrorhodamine 123. For the detection of cholesteryl esters, the porous matrix/matrices could additionally comprise the enzyme cholesterol esterase.

[0380] As described in Example 1, the components may be in one porous matrix, or may be separated or decoupled so as to reduce interference, or where it may be desired to alter the physical and/or chemical nature of each porous matrix. Suitable cartridge architecture for use in a cholesterol assay are shown in FIGS. 7 and 8, although it will be appreciated that other cartridge architecture can be envisaged.

[0381] An embodiment of a cholesterol assay is described below and in relation to FIG. 7.

[0382] A sample, for example blood is contacted with and introduced into the cartridge by way of the input port 303, which in this embodiment is circular (but it will be appreciated that other shapes can be envisaged).

[0383] Once a sample of blood is introduced to the input port 303, the blood sample fills the first portion 304, enlarged portion 314 and second portion 316 by capillary action, until encountering the vent arm 318, at which point capillary flow of the blood will stop. Further downstream movement of the sample of blood is controlled by an active fill mechanism, in which depression of the air/gas filled chamber 310 causes the blood sample to flow past the vent arm 318 and into the detection zone 326 comprising a porous matrix for generating an optical change/signal (not shown). In this embodiment, the porous matrix for generating an optical change/signal is immobilised to the upper luminal surface in the detection zone 326. It will be appreciated that in other embodiments, the porous matrix may be immobilised to the lower luminal surface.

[0384] The detection zone may be enclosed by a printed feature, which in this embodiment is used to detect the flow of the sample in order to determine a start time to the assay, as described above. In this embodiment, the printed feature is also used to control the active fill mechanism in order to stop, start, reduce or increase the active fill mechanism in order to change the flow rate and/or stop or start sample flow downstream.

[0385] Once the blood sample enters the detection zone 326, any cholesteryl esters present in the sample interact with the enzyme cholesterol esterase in the porous matrix to generate free cholesterol. The free cholesterol then interacts with the enzyme cholesterol oxidase in the porous matrix to generate cholest-4-en-3-one+H.sub.2O.sub.2. H.sub.2O.sub.2 is converted to H.sub.2O by the enzyme Horse Radish Peroxidase (HRP) in the porous matrix, the by-product of which is then used to oxidise dihydrorhodamine 123, dihydrorhodamine 123 being present in the porous matrix. Oxidised dihydrorhodamine 123 emits an optically detectable signal, in this example a fluorescent signal. Any optical signal is then detected using an optical detector as previously described. Detection by the optical detector is initiated by the printed feature, which once it detects the sample in the detection zone 326 starts the assay.

[0386] In other embodiments, more than one porous matrix may be utilised. For instance, the channel may have a first portion (zone A), a second portion downstream of the first portion (zone B) and a third portion downstream of the second portion (zone C). A porous matrix may be positioned in each zone. A printed feature may separate each zone. Zone C may comprise the detection zone 326 in which is located the porous matrix for generating an optical change/signal. In zone A, the porous matrix comprises the reagent cholesterol esterase. The porous matrix in zone B comprises the reagent cholesterol oxidase and the porous matrix in zone C comprises the reagents dihydrorhodamine 123 and HRP. In such embodiments, the sample will fill the first portion of the channel 304 and fill downstream to zone A. In zone A, any cholesteryl esters present in the sample will interact with the enzyme cholesterol esterase in the porous matrix to generate free cholesterol. The free cholesterol will flow with the sample downstream to fill zone B, in which the free cholesterol will interact with the enzyme cholesterol oxidase to generate cholest-4-en-3-one+H.sub.2O.sub.2

[0387] These reaction products will mix with the sample which will then fill the downstream zone C. In the porous matrix of zone C H.sub.2O.sub.2 is converted to H.sub.2O by the enzyme Horse Radish Peroxidase (HRP), the by-product of which is then used to oxidise dihydrorhodamine 123, which results in the generation of an optically detectable signal. Any optical signal can then be detected using an optical detector as previously described.

[0388] Alternatively, rather than having a zone A, B and C, the cartridge may have only a zone A and B and so only two porous matrices. In such embodiments the porous matrix in zone A may comprise cholesterol esterase and/or cholesterol oxidase. In embodiments comprising than one porous matrix, only one of the porous matrices may be deposited in the detection zone. However, in other embodiments more than one porous matrix may be deposited in the detection zone.

[0389] FIG. 20 shows the results of a cholesterol assay carried out in accordance with an embodiment of the invention. In this embodiment, a porous matrix was utilised, which was immobilised to an inner luminal surface of the detection zone. In this particular embodiment the porous matrix was immobilised to the lower inner luminal surface of the detection zone, but it will be appreciated that mobilisation to the upper inner luminal surface of the detection zone could instead be envisaged. The porous matrix comprised the components cholesterol esterase, cholesterol oxidase, HRP and dihydrorhodamine 123. The porous matrix contained trehalose as a carrier molecule.

[0390] A 10 μl sample containing a known concentration of cholesterol was added to the input port and the sample filled the channel to the detection zone. After 90 seconds of the sample being in the detection zone, an optical measurement was taken. Plots of the peak detected optical signal, in this embodiment a fluorescent signal (y axis), against total cholesterol concentration (x axis) are shown in FIG. 20A. FIG. 20B shows the lower cholesterol concentration values of FIG. 20B plotted as a smaller scale.

[0391] Exemplary Test Descriptions

[0392] Summary Test Sequence 1: [0393] 1. Cartridge Insertion into the reader. [0394] 2. Cartridge gas chamber compression by reader. [0395] 3. Sample application to the cartridge, filling by active fill (by partial chamber decompression) to the printed feature which is located downstream of the detection zone. [0396] 4. Wetting of the detection electrodes of the printed feature located downstream of the detection zone determines the test start timing and also initiates a stop-flow mechanism to prevent flow of the sample further downstream. [0397] 5. The sample wets the porous matrix localised to an inner luminal surface of an optically transmissible portion of the detection zone of the cartridge, the matrix containing a reagent capable of reacting with an analyte or analyte reaction product thereof of the sample. [0398] 6. The reagent reacts with the analyte/analyte reaction product in the sample to form a reagent reaction product which generates an optical signal. [0399] 7. A plurality of optical measurements is taken by an optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached. [0400] 8. The gas chamber is further decompressed completely by a removal in force applied to the cartridge gas chamber. This removal of force completely forces all of the sample into the cartridge for subsequent safe disposal.

[0401] Summary Test Sequence 2: [0402] 1. Cartridge Insertion into the reader. [0403] 2. Cartridge gas chamber compression by reader. [0404] 3. Sample application to the cartridge, filling by active fill (by partial chamber decompression) to the printed feature which is located downstream of the detection zone. [0405] 4. Wetting of the detection electrodes of the printed feature located downstream of the detection zone determines the test start timing and also initiates a stop-flow mechanism to prevent flow of the sample further downstream. [0406] 5. The sample wets the porous matrix localised to an inner luminal surface of an optically transmissible portion of the detection zone of the cartridge, the matrix containing a reagent reacting with an analyte or analyte reaction product thereof of the sample. [0407] 6. The reagent reacts with the analyte/analyte reaction product thereof in the sample, to form a reagent reaction product which generates an optical signal. [0408] 7. An optical measurement is taken by an optical detector positioned extraluminally to the optically transmissible portion after a fixed period of time. Each strip batch and analyte channel is calibrated separately so the signal is transformed into component concentration.

[0409] Summary Test Sequence 3: [0410] 1. Cartridge Insertion into the reader. [0411] 2. Sample application to the cartridge, initial filling of the microfluidic channel by passive capillary fill and then active fill to the detection zone. [0412] 3. During filling of the microfluidic channel, the sample wets a first porous matrix localised to an inner luminal surface of the microfluidic channel, an analyte in the sample reacting with a reagent in the first porous matrix. [0413] 4. The analyte reaction product of the reagent and the analyte mixes with the sample which fills downstream of the first porous matrix to the detection zone. [0414] 5. The sample wets the second porous matrix for generating an optical change/signal localised to an inner luminal surface of an optically transmissible portion of the detection zone, the matrix containing a further reagent capable of reacting with the analyte reaction product. [0415] 6. The further reagent reacts with the analyte reaction product in the sample to form a reagent reaction product which generates an optical signal. [0416] 7. A plurality of optical measurements is taken by an optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached.

[0417] Summary Test Sequence 4: [0418] 1. Cartridge Insertion into the reader. [0419] 2. Sample application to the cartridge, initial filling of the microfluidic channel by passive capillary fill and then active fill to the detection zone. [0420] 3. The sample wets the porous matrix for generating an optical change/signal localised to an inner luminal surface of an optically transmissible portion of the detection zone, the matrix containing a reagent capable of reacting with an analyte in the sample. [0421] 4. The reagent reacts with the analyte in the sample to form a reagent reaction product which generates an optical signal. [0422] 5. A plurality of optical measurements is taken by an optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached.

[0423] Summary Test Thrombin Sequence: [0424] 1. Cartridge Insertion into the reader. [0425] 2. Cartridge gas chamber compression by force application feature of reader expels gas from the cartridge. [0426] 3. Blood sample application to the cartridge. The force applied by the force application feature to the gas chamber is reduced. This results in partial decompression of the gas chamber and the ingress of air back into the chamber. This pulls a portion of the blood sample along the channel by an active fill mechanism. [0427] 4. The portion of the blood sample continues to move downstream until it reaches the printed feature which is located downstream of the detection zone. This acts as a boundary to prevent further downstream flow of the sample and also initiates a stop-flow mechanism to prevent flow of the sample further downstream. [0428] 5. Wetting of the detection electrodes of the printed feature located downstream of the detection zone determines the test start timing. [0429] 6. The blood sample wets the porous matrix for generating an optical change/signal localised to an inner luminal surface of a optically transmissible portion of the detection zone, the matrix containing R110. The fibrinogen peptide arms of R110 are cleaved by thrombin present in the sample, generating a fluorescent monoamide which is excited by the light emitted by the optical detector, the fluorescent monoamide emitting a fluorescent signal at 525 nm. [0430] 7. A plurality of optical measurements is taken by the optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached. The signal is transformed into component concentration.

[0431] Summary Test Thrombin Sequence 2: [0432] 1. Cartridge Insertion into the reader. [0433] 2. Blood sample application to the cartridge. The blood sample fills downstream into the microfluidic channel and detection zone due to a combination of passive capillary and active fill mechanisms. [0434] 3. The blood sample wets the porous matrix for generating an optical change/signal localised to an inner luminal surface of an optically transmissible portion of the detection zone, the matrix containing R110. The fibrinogen peptide arms of R110 are cleaved by thrombin present in the sample, generating a fluorescent monoamide which is excited by the light emitted by the optical detector, the fluorescent monoamide emitting a fluorescent signal at 525 nm. [0435] 4. A plurality of optical measurements is taken by the optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached. [0436] 5. The gas chamber is further decompressed completely by a removal in force applied to the cartridge gas chamber. This removal of force completely forces the all of the blood sample into the cartridge for subsequent safe disposal.

[0437] Summary Test Cholesterol Sequence 1: [0438] 1. Cartridge Insertion into the reader [0439] 2. Cartridge gas chamber compression by force application feature of reader expels gas from the cartridge. [0440] 3. Blood sample application to the cartridge. The force applied by the force application feature to the gas chamber is reduced. This results in partial decompression of the gas chamber and the ingress of air back into the chamber. This pulls a portion of the blood sample along the channel by an active fill mechanism. [0441] 4. The portion of the blood sample continues to move downstream until it reaches the printed feature which is located downstream of the detection zone. This acts as a boundary to prevent further downstream flow of the sample and also initiates a stop-flow mechanism to prevent flow of the sample further downstream. [0442] 5. Wetting of the detection electrodes of the printed feature located downstream of the detection zone determines the test start timing. [0443] 6. The blood sample wets the porous matrix for generating an optical change/signal localised to an inner luminal surface of an optically transmissible portion of the detection zone of the cartridge, the formulation containing dihydrorhodamine-123. The dihydrorhodamine-123 is oxidised by hydrogen peroxide present in the sample, the hydrogen peroxide formed as a oxidisation product of cholesterol when catalysed by the enzyme cholesterol oxidase [0444] 7. The oxidised dihydrorhodamine-123 forms the reagent reaction product fluorescent form cationic rhodamine 123 which is excited by the light emitted by the optical detection device, the rhodamine-123 emitting a fluorescent signal at 530 nm. [0445] 8. A plurality of optical measurements is taken by an optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached. The signal is transformed into analyte, in this embodiment cholesterol, concentration.

[0446] Summary Test Cholesterol Sequence 2: [0447] 1. Cartridge Insertion into the reader. [0448] 2. Sample application to the cartridge, initial filling of the microfluidic channel by passive capillary fill and then active fill to the detection zone. [0449] 3. During filling of the microfluidic channel, the sample wets a first porous matrix localised to an inner luminal surface of the microfluidic channel, cholesterol in the sample reacting with cholesterol oxidase in the second porous matrix. [0450] 4. The analyte reaction product of the cholesterol and the cholesterol oxidase mixes with the sample which fills downstream of the first porous matrix to the detection zone. [0451] 5. The sample wets the second porous matrix for generating an optical change/signal localised to an inner luminal surface of an optically transmissible portion of the detection zone, the matrix containing dihydrorhodamine 123, which is capable of reacting with the analyte reaction product. The second porous matrix also comprises HRP to facilitate this interaction. [0452] 6. Dihydrorhodamine 123 reacts with the analyte reaction product in the sample to form a reagent reaction product which generates an optical signal. [0453] 7. A plurality of optical measurements is taken by an optical detector positioned extraluminally to the optically transmissible portion until a threshold signal is reached.