DISPOSITION OF REAGENTS IN ASSAY DEVICE

Abstract

An assay cartridge for detecting a target component in a liquid sample is provided. The cartridge comprises: a sample collection unit configured to introduce the liquid sample into the cartridge; a fluid pathway commencing at its proximal end at the sample collection unit and extending distally through the cartridge including: one or more capture components immobilised within the fluid pathway; one or more detection reagents provided within the diffusion distance of the capture components.

Claims

1. An assay cartridge for detecting a target component in a liquid sample, the cartridge comprising: a sample collection unit configured to introduce the liquid sample into the cartridge; a fluid pathway commencing at its proximal end at the sample collection unit and extending distally through the cartridge including: one or more capture components immobilised within the fluid pathway; one or more detection reagents provided within the diffusion distance of the capture components.

2. The assay cartridge according to claim 1, wherein detection of the target component includes identifying the presence of the component.

3. The assay cartridge according to claim 1 or claim 2, wherein the fluid pathway commences at a location at which the sample is introduced into the cartridge.

4. The assay cartridge according to any one of claims 1 to 3, wherein the fluid pathway has a rectangular or square cross section comprising four substantially orthogonal walls.

5. The assay cartridge according to any one of claims 1 to 3, wherein the fluid pathway is elongate and cylindrical and includes a single annular wall.

6. The assay cartridge according to any one of claims 1 to 3, wherein the fluid pathway is a well.

7. The assay cartridge according to claim 4, wherein the detection reagents are placed on different walls from the capture components.

8. The assay cartridge according to any one of claims 1 to 7, wherein the detection reagents are substantially equidistant with the capture components.

9. The assay cartridge according to any one of the preceding claims, wherein at least one of the liquid droplets comprises an additive that minimises evaporation.

10. The assay cartridge according to any one of the preceding claims, wherein the fluid pathway includes one or more indentations.

11. The assay cartridge according to any one of the preceding claims, further comprising a flow controller configured to reduce the bulk movement of the sample in the vicinity of the capture components.

12. The assay cartridge according to claim 11, wherein the flow controller is provided distally of the capture components.

13. The assay cartridge according to claim 12, further comprising a porous-structure pump provided distally of the flow controller.

14. The assay cartridge according to any one of the preceding claims, wherein the detection reagent and the capture components comprise antibodies.

15. The assay cartridge according to any one of the preceding claims, wherein the detection reagent and capture components both comprise single-stranded oligo- or polynucleotides.

16. The assay cartridge according to any one of the preceding claims, further comprising a channel downstream of the capture components that contains a confirmation element configured to show when the liquid sample is present in the channel.

17. The assay cartridge according to any preceding claim, further comprising one or more target components immobilised within the fluid pathway.

18. The assay cartridge according to any one of the preceding claims, in which the liquid droplet within which the detection reagents are provided includes a degradable shell.

19. An apparatus for detecting the presence and/or the amount of a target component in a sample of biological fluid, the apparatus comprising: an assay cartridge according to any one of claims 1 to 18, and a detector detecting the presence and/or the amount of the emitted light to provide an indication of the presence and/or the amount of the target component within the sample.

20. The apparatus according to claim 19, wherein the apparatus further comprises an excitation source configured to enable TIR illumination.

21. The apparatus according to claim 19 or 20, further comprising a component for acoustic mixing.

22. The apparatus according to any one of claims 19 to 21, further comprising an optical readout component.

23. The apparatus according to claim 22, further comprising an optical mask in the optical path between the bound detection reagent and the optical readout component.

Description

[0060] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which:

[0061] FIGS. 1 to 4 show, schematically, various permutations and combinations of detection reagents and capture components;

[0062] FIG. 5 shows an embodiment that allows a check that detection reagents have reached the capture component locations;

[0063] FIG. 6 shows a multi-lane configuration of the fluid pathway;

[0064] FIGS. 7 to 9 show possible detection reagent and capture component locations in embodiments including an elongate flow pathway;

[0065] FIGS. 10 to 13 each comprise a top view and a side view of embodiments of a cartridge showing the locations of the detection reagents in various indentations;

[0066] FIG. 14 shows a graph of calculated diffusion length vs. time for IgG antibodies in water and in mucus;

[0067] FIGS. 15 and 16 show further embodiments of the cartridge focussing on the introduction of the liquid sample into the assay cartridge;

[0068] FIG. 17 shows a TIRF-based CRP sandwich immunoassay where detection reagents and capture antibodies were disposed on opposing walls and where the spots of detection antibody were dry until introduction of the sample; and

[0069] FIG. 18 shows a TIRF-based PIGF sandwich immunoassay where detection and capture antibodies are both disposed on the TIRF-illuminated wall and where the spots of detection antibody were liquid until introduction of the sample.

[0070] Referring to FIG. 1, there is provided an assay cartridge 10 for detecting a target component 20 in a liquid sample. The cartridge comprises a sample collection unit 12, in which the sample collection unit 12 has an inlet 18 which is configured to introduce the liquid sample 14 into the cartridge 10. The liquid sample 14 is able to flow through a fluid pathway 16 commencing at its proximal end at the sample collection unit 12 and extending distally through the cartridge 10 including one or more capture components 22 immobilised within the fluid pathway 16. Therefore, the terms proximal and distal are used to define positions relative to the sample collection unit 12 where the liquid sample 14 enters the cartridge 10. The fluid pathway 16 starts at the sample collection unit 12 and continues through the cartridge 10 until it reaches the capture components 22. The fluid pathway 16 then continues downstream of (in use), or distally from, the capture components 22. One or more detection reagents 24 provided proximally of or level with the capture components 22 each contained within a liquid droplet 15. The detection reagents 24 are level with the capture components 22 and therefore equidistant from the proximal end of the fluid pathway 16 SO that the liquid sample 14 will come into contact with the detection reagents 24 at the same time as it comes into contact with the capture components 22 because they are the same distance from the inlet 18.

[0071] In the example shown in FIG. 1, the fluid pathway 16 is substantially linear as the fluid sample 14 flows from left to right as illustrated. The left hand end of the fluid pathway 16 is therefore the proximal end, owing to its location adjacent to the sample collection unit 12. In FIG. 2, the inlet is central and therefore the flow is radially outwards in all directions and therefore the proximal end of the flow pathway 16 is the centre of the well and the distal end of the flow pathway 16 is adjacent to the circumferential wall of the well.

[0072] As shown in FIGS. 1 and 2, the detection reagents 24 are in the form of liquid droplets which enables them to be combined with the sample such that they undergo bulk movement with the liquid sample 14. As the liquid sample 14 moves over the detection reagents 24, they are dragged along with the bulk movement of the liquid sample 14 and, because the flow of the liquid sample 14 is substantially laminar, they create streaks of detection reagent 24. The detection reagent 24 is configured to bind to the target components and move to the capture components 22 along the fluid pathway 16.

[0073] In FIG. 2, the detection reagents 24 are transported through bulk movement of the sample until they reach the vicinity of the detection reagents and the capture components, at which point the bulk flow is slowed sufficiently for diffusion to predominate. In FIGS. 1 and 4, the detection reagents 24 are transported predominantly through diffusion.

[0074] As shown in FIGS. 1 to 4, the detection reagent 24 is contained within a liquid droplet 15. The liquid droplets 15 in the illustrated embodiments have a circular cross section and are part-spherical. Alternatively, in embodiments not shown in the accompanying drawings, the liquid droplets can be non-spherical such as a rectangular liquid droplet. Liquid droplets that have a non-circular cross section can be created through manipulation of the surface tension of the liquid droplet 15 when it is deposited at its intended location on the fluid pathway 16. At these locations, or reagent sites, the surface of the fluid pathway is provided with a surface coating (not shown) to help manipulate or mould the liquid droplet 15 into a suitable shape. For example, a hydrophilic area may be provided which encourages the liquid droplet to overcome surface tension and spread across the surface. This hydrophilic area may then be bounded by a hydrophobic barrier which halts the spread of the droplet. The droplet will tend to assume the shape of the hydrophilic area in this example. This area may be rectangular, square or an irregular shape dictated by the geometry of the fluid pathway 16.

[0075] Referring to FIG. 7, the capture component 22 is deposited onto an optical element 26. The capture component 22 is deposited onto the optical element 26 via by printing. The optical element is a prism, dove or a cuboid optical element. The capture component 22 can be an antibody or a fragment thereof, a peptide, or a nucleic acid.

[0076] Referring to FIGS. 1 to 4, there is shown a flow controller 19 to help control fluid flow through the fluid pathway 16. The flow controller 19 can include one or more of the following: a capillary channel; a narrow, a long and/or a tortuous path; a capillary stop; a capillary stop with a vent or gas buffer or a flow resistor. As shown in FIG. 1, the fluid controller 19 may comprise, but is not limited to, a vent 34, a chamber 36 containing gas buffer, a flow resistor 38 or a capillary stop 44. In some examples, the capillary stop 44, the vent 34 and the chamber 36 containing gas buffer are used in combination to control the flow of the liquid sample through the fluid pathway 16. The flow controller 19 is provided for each fluid pathway 16. For example, the number of fluid controller 19 will be T.sub.1, T.sub.2, T.sub.3 and corresponds to the number of N.sub.1, N.sub.2, N.sub.3 fluid pathway 16 provided on the cartridge 10. The flow controller 19 is provided distally of the capture components 22. By placing the flow controller distally, or downstream, of the capture components 22, as shown in FIG. 1, the flow of sample into the fluid pathway 16 is relatively unimpeded thereby enabling the sample to be quickly introduced into the cartridge 10. The flow controller 19 then acts to slow the flow of the sample once it has reached the capture components 22. The flow controller 19 is taken to be any form that is effective in slowing the flow. The flow controller 19 is required to slow the bulk movement of the sample sufficiently so that the detection reagents 24 can bind to the target components and move to the capture components 22 via diffusion.

[0077] Referring to FIGS. 2 to 4, there is shown an alternative embodiment of a fluid pathway 16, in which the fluid pathway is a well 28. As shown in FIGS. 2 to 4, there is shown the co-location of the capture components 22 and detection reagents 24 within the well 28. The capture components 22 can be equal to or more than the number of the detection reagent 24 located within the well 28. In another example, the number of capture components 22 can be less than the number of detection reagents 24 located within the well 28. The inlet 18 for receiving the liquid sample is located at the centre of the well, as shown in FIGS. 2 to 4, where the detection reagents 24 are positioned nearer to the inlet 18.

[0078] The flow controller 19 is provided by the geometry of the fluid pathway 16 in the case where the fluid pathway is a well 28, as shown in FIGS. 2 to 4. The sidewall or sidewalls of the well 28 provide the flow controller 19 as they prevent the sample from flowing further and cause the sample to stop in the vicinity of the capture components that are applied to the base of the well or to the wall or walls near the base of the well 28.

[0079] FIG. 6 shows multiple lanes with a physical barrier 32 provided between the lanes. Referring to FIG. 6, there is provided a cartridge 10 comprising a sample collection unit 12, an inlet 18 for receiving the liquid sample and multiple lanes 30. A physical barrier 32 such as a pair of walls is provided to separate the lanes as shown in FIG. 6. Without the walls 32, through diffusion, detection reagents 24 for one target component may cross-over to capture components for another target component; thus, cross-talk (unintended affinity) between the two might contribute to background signal and increase cross-talk between multiple assays on one device.

[0080] FIG. 6 shows an embodiment with multiple lanes 30, in which each of the lanes 30 can be used to test for a different capture component 22. Referring to FIG. 6, there is shown a physical barrier 32 i.e. a wall. The physical barrier 32 is configured to divide the fluid pathway 16 into a plurality of parallel flow lanes or channels 30. This configuration thereby enables a number of different detections reagents 24 to be deployed for the same liquid sample 14 and the same time without risk of cross-talk. One or more capture components 22 is deposited in each of the lanes 30 whereby the capture component is deposited onto the optical element 26. As the liquid sample 14 comes into contact with the detection reagents 24 and capture components, the bulk motion of the flow is slowed so that diffusion predominates and allows the target components to reach and react with the detection reagents and the capture components. A fluid controller 19 is provided downstream of the capture components 22 in order to control the flow of the liquid sample along each of the lanes 30 and to facilitate an initial, rapid fill of the fluid pathway and then a considerable slowing of the bulk flow to such a point where diffusion can predominate.

[0081] The physical barrier 32 i.e. the walls separating the well 28 into four quarters extend over the entire fluid pathway 16 from the sample collection unit 12 to the well base. However, for TIR methods in particular, such a separating wall 32 can cause unwanted reflections and scattering of the excitation and emission light.

[0082] In an alternative embodiment, there is provided a partial barrier or a soft texture not shown in the accompanied drawings that runs along the fluid pathway 16. The physical barrier 32 would be at a partial height and would not extend all the way to the top of the sample collection unit 12 but to provide a gap. This ensures that the flow channels or lanes 30 are partially separated. In one example, the partial barrier or the soft texture is a semi-permeable membrane that is designed to reduce cross talk between multiple parallel flow channels or lanes 30 without contacting the optical surface and thereby risking interference with the TIR measurements.

[0083] Channels are typically of rectangular cross-section due to two-dimensional nature of current mass fabrication processes. In many microfluidic devices, channel dimensions are limited by the amount of available sample and/or cost of reagent. However, in TIRF-based detection systems, channel height is less of an issue. For the case where reagents are disposed on the wall along which the TIRF evanescence is generated, as long as Reynolds numbers remain sufficiently low (<1e3), laminar flow will keep reagent concentrated near the wall. The fluid dynamics of the illustrated embodiments are such that the fluids all execute laminar flow throughout. Turbulence is minimised so that the dominant lateral motion arises from diffusion, not turbulence.

[0084] In some embodiments, down-stream passive pumping structures may be provided that create a second flow regime that is slower than the initial sample fill up to the test site The flow rate created by the pumping structures must be slow enough for target components to reach the capture components by diffusion, specifically, flow velocity over the test site must be less than 10 mm/min, less than 5 mm/minute, less than 2 mm/minute or even less than 1 mm/minute.

[0085] Besides capillary driven flow, low flow rates may also be achieved using evaporation. The chamber containing the gas buffer 36 after a capillary stop 44, may contain e.g. dry air or dry nitrogen gas (which requires device packaging to be sealed until use). Its initial humidity and volume may be designed such that humidity remains sufficiently low so that the evaporation rate does not drop significantly for the required duration of the assay. Alternatively, it may be designed such that it saturates with water vapour during the assay measurement, thus stopping the flow and preventing the test site from drying out.

[0086] In some embodiments, not illustrated, a vent 34 may be provided after the capillary stop 44. This demands less real estate on the cartridge, but it leads to a variable flow rate that is dependent on ambient humidity thus limiting the operating conditions.

[0087] In absence of second (slow) flow regime, a capillary stop 44 is required downstream of the test site. It must be no further from the capture components 22 in the test site than the length of streaks of detection reagent (for upstream or proximal deposition in channel geometries). For diffusion-based assays (co-located capture components and detection reagents), the capillary stop 44 must be as close as possible to the capture components 22 but far enough to not interfere with the assay (e.g. for optical detection, the meniscus may need to be outside the field of view of the detection element in order to avoid intense background light from reflection off the meniscus).

[0088] The combination of capillary stop 44 with evaporation into a chamber 36 containing the gas buffer or the vent 34 can also be used to concentrate the sample including capture components and detection reagents at the test site. This can be effective when flow velocity due to evaporation is higher than the velocity of diffusion of target components or detection reagents. Evaporation rate is determined by meniscus area and curvature and by humidity; diffusion distance vs. time x(t) is determined by diffusion constant D according to x≈2√{square root over (Dt)}

[0089] Referring FIG. 5, there is provided a check that detection reagents have reached the capture component locations. FIG. 5 shows an example of co-located capture component 22 (“Ab1”) and detection reagent (“Ab2”) 24 within a well 28. The central spot 46 is a spot of capture components 22 that are complementary to the detection reagents (“anti-Ab2”) 24.

[0090] Referring to FIGS. 7 to 9, there is shown possible detection reagent 24 and the capture component 22 locations in embodiments including an elongate flow pathway 48. There is also provided a capillary stop 44, a chamber 36 containing gas buffer and a vent 34 to control fluid flow, as shown in FIGS. 7 to 9.

[0091] In some examples, illustrated in FIGS. 10 to 13, a surface of the fluid pathway is provided with one or more indentation, such as a recess, a trough, a ditch, a trench, a groove, a gully, a via that is essentially perpendicular to the surface, or a porous structure in order to provide a location for the deposition of the liquid droplets. This can have several advantages, including precise location of the deposited droplets, reducing evaporation by reducing surface-to-volume ratio, having the option to deposit droplets from the opposite side of the pathway's wall (in the case of a via or porous structure), increasing the amount of detection reagent per unit length of the fluid pathway, and reducing the amount of detection reagent that is dragged by the sample flow. The latter advantage applies to assays in diffusion configuration. As an example only, the indentation is provided where the detection reagents 24 are positioned within fluid pathway 16. The liquid droplet is at a suitable configuration to enable it to position itself within the indentation on the surface of the fluid pathway. FIGS. 9 to 13 also show that the fluid pathway 16 includes the location of the capture component 22 or components and any intervening geometry. In some examples, an indentation on the surface of the optical element may be suitable to provide a location for deposition of the capture component. In some embodiments, where indentation is unsuitable on an optical element, a gasket can be provided between the optical element and the fluid pathway to help position the capture component.

[0092] FIG. 13 shows a configuration of the fluid pathway 16 that is particularly suited to liquid droplets of detection reagent. The fluid pathway 16 is provided with a series of indentations which are filled with the detection reagent in liquid form. The indentations in the fluid pathway 16 protect the liquid droplets of detection reagent during the initial, rapid, liquid fill so that they retain their position for the diffusion phase. Although in the illustrated embodiment, each of the detection reagents is housed in an indentation, other modifications to the fluid pathway 16 geometry may be selected to protect the liquid detection reagents. For example, a bump or protrusion may be provided at the upstream side of the detection reagent and this will provide a similar sheltering effect for the liquid detection reagent.

[0093] FIG. 14 shows a graph of calculated diffusion length vs. time for IgG antibodies in water and in mucus; diffusion length is an estimate for the distance that a molecule is able to travel when driven by Brownian motion. The calculations are based on measured diffusion constants as described above.

[0094] FIG. 15 shows an assay cartridge 10 in which the sample collection unit 12 is an opening 80 at the proximal end of the fluid pathway 16. The opening is provided in the upper surface of the cartridge 10 so that the introduction of the fluid sample 14 is aided by gravity. The liquid sample 14 is introduced into the cartridge 10 using a pipette 81. The opening that provides the sample collection unit 12 is sized to accommodate the tip of the pipette 81 so that the fluid sample 14 can be introduced into the cartridge 10 accurately.

[0095] FIG. 16 shows an assay cartridge 10 in which the sample collection unit 12 comprises a pad of porous material 82 such as a sponge, a filter 83 and a support mesh 84. The pad 82 soaks up the fluid sample 14 as it is introduced into the cartridge 10. The structure of the pad 82 holds the fluid sample 14 in place and prevents it from easily leaving the cartridge 10 once it has been introduced to the cartridge. When sufficient fluid sample 14 has been collected, the cartridge 10 is closed using a plunger 86 which forms a seal around the opening in the cartridge 10 using an O-ring 85. The plunger 86 compresses the pad 82 and forces the fluid sample 14 through the filter 83 and into the fluid pathway 16. The support mesh 84 is provided to hold the filter 83 in place and to prevent it from moving into the fluid pathway 16. The filter 83 is provided to remove particulate matter and/or biological matter such as mucins from the fluid sample 14 that is undesirable and could interfere with functioning of the assay cartridge 10.

[0096] Within the context of this invention, a capture component and a detection reagent can be a protein or peptide, including an antibody or enzyme; an oligo- or polynucleotide, such as DNA or RNA; a modified oligo- or polynucleotide, such as a locked nucleic acid (LNA); an aptamer; a morpholino; a small molecule that may be grafted via a spacer molecule; a cell; a cell membrane; a membrane receptor; a viral particle; a glycan; a solid particle or bead coated with a reagent or other type of molecule or material that can have a ligand receptor type of interaction with the target component of interest. For optical detection, detection reagents may be labelled with a luminophore such as a fluorophore or a phosphor or a chemiluminescent molecule, or an enzyme and its substrate that produces a colorimetric or luminescent signal.

[0097] The detection reagent can also be any reagent including a cofactor or any molecule used to process the sample (e.g. sodium dodecyl sulfate used for lysing cells).

EXAMPLE 1

[0098] FIG. 17 shows a TIRF-based CRP sandwich immunoassay where detection and capture antibodies were disposed on opposing walls and where the spots of detection antibody were dry until introduction of the sample. Shown are four incubation times after filling a channel with a saliva sample from a healthy subject. The saliva was pre-filtered (5-μm pore size). Detection reagent (Alexa-647-labelled monoclonal IgG targeting native human CRP) in SSC buffer was contact-printed onto the top surface of a channel of cross-section 10×0.09 mm, in two lines spaced 3 mm apart with a spot pitch of 0.6 mm within each line. The capture component (monoclonal IgG targeting native human CRP) was deposited in print buffer as well (3 rows with 1-mm pitch on the bottom surface of the channel), but rinsed with MilliQ water prior to chip assembly. During imaging, the bottom surface was illuminated with TIRF.

EXAMPLE 2

[0099] FIG. 18 shows a TIRF-based human epithelial growth factor sandwich immunoassay where detection and capture antibodies are both disposed on the TIRF-illuminated wall and where the spots of detection antibody were liquid until introduction of the sample. Shown are three different incubation times after filling a channel with a solution of recombinant human epithelial growth factor in PBSA (3×10{circumflex over ( )}9 PIGF/μL and 4% BSA in phosphate buffer).

[0100] Detection reagent (Alexa-647-labelled monoclonal IgG targeting human epithelial growth factor) in print buffer was contact-printed onto the bottom surface of a well of diameter 9 mm and depth of 0.6 mm, in a 2×2-mm square pattern. The capture component (monoclonal IgG targeting human epithelial growth factor) was deposited in print buffer as well, as a single droplet, but rinsed with MilliQ water prior to chip assembly.

[0101] Within the context of this invention, a target component can be a protein or peptide, including an antibody or enzyme or membrane receptor; an oligo- or polynucleotide, such as DNA or RNA; a cell; a small molecule; a viral particle; a glycan; a drug candidate, or other type of molecule or particle of interest.

[0102] By the term ‘liquid droplet’ used herein we mean a spot on the device comprising at least some liquid component e.g. that carries a reagent directly solubilised or suspended within it. For example, this includes liquids, gels, suspensions, or combinations thereof. The droplet may also include a degradable shell that releases its contents due to contact with the sample. A liquid can be a solution that includes a polymeric compound or compounds. The droplet may be a partial sphere formed when a liquid mass is deposited on to a surface. However, it should be understood that the term “droplet” also covers other shapes of fluid amalgam. For example, if the surface onto which the liquid is deposited is treated with one or more of a hydrophilic or hydrophobic layer, this may overcome the surface tension of the liquid and cause it to flow such that it has a non-circular footprint. Alternatively, adjacently placed and connecting liquid masses deposited in a pattern can maintain the pattern through contact line pinning. The footprint of the liquid droplet may be therefore, in addition to a circular footprint, rectangular, square or elliptical. It may even have an irregular shape which may be at least partially dictated by the packaging requirements of the fluid pathway. Within the context of this invention a liquid droplet ceases to exist when it is absorbed into a porous matrix, such as a nitrocellulose matrix or the liquid evaporates leaving behind a spot of dried matter that is no longer in solution.

[0103] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0104] “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.

[0105] Throughout the specification, unless context dictates to the contrary, the singular should be understood to encompass the plural. That is, “one” and “a” and “the” should be understood to encompass “at least one” or “one or more.”

[0106] 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.

[0107] 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.