Biosensor apparatus and methods

Abstract

The invention relates to apparatus for detecting analyte in sample liquid (eg. interrogation of sample liquid to detect analyte), methods of manufacture of apparatus, and methods and processes and a kit for detecting analyte in sample liquid using absorbent sensing material comprising super absorbent polymer. The invention provides an apparatus (10) for interrogation of sample liquid to detect analyte, comprising: at least one main chamber (20) of predetermined volume; and, within the main reaction chamber (20), a predetermined amount of dry, absorbent, sensing material comprising (e.g. comprising substantially) dry superabsorbent polymer (SAP); and, within the dry superabsorbent polymer, at least one dry reaction component for detecting and for signalling the presence of analyte (e.g. within the SAP); wherein, upon insult of sample liquid, the dry superabsorbent polymer absorbs sample liquid, the at least one dry reaction component dissolves and, if analyte is present in the sample liquid, its presence is detected and signalled.

Description

[0001] Biosensor Apparatus and Methods

[0002] Field of the Invention

[0003] The invention relates to apparatus for detecting analyte in sample liquid (e.g. interrogation of sample liquid to detect analyte), methods of manufacture of apparatus, and methods and processes and a kit for detecting analyte in sample liquid using absorbent sensing material comprising super absorbent polymer.

[0004] Background

[0005] Hydrogel-based Biosensors and Sensing Devices for Drug Delivery by Nicholas A. Peppas and Diana Snelling Van Blarcom, The Americas Special Issue of the Journal of Controlled Release 2016 describes glucose oxidase immobilized in a water swollen hydrogel for sensing glucose and swelling or de-swelling to provide insulin delivery in vivo. Stability of hydrogel sensing systems remains a problem.

[0006] In Hydrogel Based Sensors for Biomedical Applications: An Updated Review, Polymers 2017, 9, 364, Javad Tavakoli and Youhong Tang comment on stability problems: ‘Some limitations, including life time, storage, and adaptation with transducers for rapid quantitative analysis reveal that there is still a long way for hydrogel-based biosensors to go before they are used in commercialized health management systems. ’

[0007] Problems remain using hydrogel in quantitative and qualitative analysis. Cost and simplicity are also challenges in real life, commercial applications, particularly consumer applications.

[0008] In Superabsorbent Polymer Materials: A Review, Iranian Polymer Journal Vol 17 (6), 2008, 451-477, Mohammad J. Zohuriaan-Mehr and Kourosh Kabiri, describe superabsorbent polymers and their preparation which may be used in one or more embodiments of the present invention.

[0009] Biomedical applications of hydrogels: A review of patents and commercial products, Enrica Calo, Vitaliy V. Khutoryanskiy, in European Polymer Journal 65 (2015) 252-267 describes various applications including contact lenses, wound dressings, drug delivery, tissue engineering and hygiene products. The authors describe ‘swelling-controlled release devices’, seemingly for in vivo use.

[0010] Enzyme-Responsive Polymer Hydrogel Particles for Controlled Release, Thornton et al, 2007 - Advanced Materials 19, Issue 9 describes a swelling response in hydrogel particles releasing physically entrapped macro-molecules for drug delivery in vivo. Absorbent articles such as diapers comprising biosensing capabilities are known. For example, KR20180085577 LEE describes an attachment-type sensor in which the flow of the sample does not directly affect the test strip, so the reaction agent is less likely to be washed off. WOOO / 65348 ROE (Proctor and Gamble) describes disposable articles including a discrete detection device. LIS2015025347 SONG describes a separate lateral flow device attached to an absorbent article. WO2015085024 NELSON describes a disposable hygienic article with means of a separate test strip visible through a port. At paragraph 37 it states, ‘while some literature indicates that the presence of SAP in a diaper used for urine testing is considered benign, manufacturing diapers without SAP for the benefit of accuracy is contemplated by the present invention and may be considered beneficial’. Thus, the use of SAP is not considered beneficial when detecting disease. Further, such discrete detection devices or test strips may break or detach and are costly and complex to manufacture in addition to the disposable article.

[0011] US5468236 EVERHART describes a disposable product incorporating a chemically reactive substance which appears complex and costly to manufacture.

[0012] WO2015 / 127528 JOLLEZ describes a chromogenic absorbent material for animal litter comprising a water-absorbing first polysaccharide, and a structural second polysaccharide. Similarly, WO2017 / 165953 JOLLEZ describes a water absorbing material and uses thereof.

[0013] US5200321 KIDWELL describes a micro assay on a card with various layers. JPH11295306 BISEIKEN describes test materials for urine inspection using cellulose.

[0014] JP2017187336A KOGA SHIN describes a urinary ketone body detecting paper diaper with an indicator embedded between and absorbent body and a transparent window.

[0015] W02018 / 141017 RAJAPAKSA describes an absorbent article with indicator which does not appear easy to manufacture or cost-effective route nor provide long term stability.

[0016] US2006 / 0253047 FOX describes a device for non-invasively monitoring a medical condition.

[0017] WO2016191372 MEEK describes an indicator panel which may be in contact with an absorbent core. The problem of highly concentrated urine affecting perceived indicator colour is noted. W02008 / 072116 LONG is similar to MEEK describing absorbent articles that can detect medical conditions. LIS2015260658 SONG describes multi-layered devices for analyte collection using a control layer to inhibit the reaction at a sensing layer and acknowledges problems of repeated insults of bodily fluid. A separate absorption layer is required.

[0018] LIS2017 / 0172933 HARDER describes loadable polymeric particles for therapeutic and diagnostic applications.

[0019] Diaper Detective System 2014 is a diaper with an insert lateral flow channels for two biosensing tests. This is complex to manufacture.

[0020] W02007073139 and MXNL05000103 GONZALEZ describe a diagnostic disposable diaper including: a plurality of urine-sensitive indicator reagents which are fixed to the internal absorbent lining of the diaper.

[0021] SHIBATA et. Al. 17894-17898 I PNAS I October 19, 2010 describes injectable hydrogel microbeads for fluorescence based in vivo continuous glucose monitoring. WO2012043177 (EP2623530) SHIBATA describes injectable hydrogel particles.

[0022] US6203496 GAEL describes providing one or more chemical reagents near an absorbent region of a diaperand examples of various detection regimes, reagents and colour changes that may be useful in embodiments of the present invention.

[0023] In Simultaneous detection of pH value and glucose concentrations for wound monitoring applications, JANKOWSKA et al. describe a wound pad for non-invasive monitoring using sensing molecules in a biocompatible hydrogel matrix.

[0024] WO2019245468 SISMAN describes a method of manufacture to introduce chemicals to non-woven bulk product for diagnosing a disease.

[0025] Various non-biosensing applications are known. Non-biosensing applications include treatment. No signalling component is required or provided, or in several examples, possible.

[0026] US6051749(A) SCHULZ describes a fabric incorporating organophilic clay, preferably dispersed in a matrix of a superabsorbent polymer for diapers that can prevent skin irritation.

[0027] US2012 / 0058074 BRAIG describes dry odour-inhibiting compositions comprising water absorbing polymer particles and at least one oxidase and a substrate of the oxidase e.g. P-D-Glucose for glucose oxidase. KR930002270(B1) IM describes disposable absorbents with anti-bacterial enzyme.

[0028] US 2013 / 0345655 WU describes a dried polymeric matrix with embedded enzyme for deodour applications.

[0029] WO2019 / 077335 HALL describes antimicrobial super-absorption compositions comprising an enzyme, a substrate for the enzyme, and a superabsorbent component, such as a superabsorbent polymer, in the form of a powder.

[0030] WO2013116358 STAMPER describes insecticidal hydrogel feeding spheres.

[0031] US2017348162 ARIZTI describes an absorbent device and a detector device to indicate the presence or absence of bodily oxidates in an absorbent system but does not discuss disease detection.

[0032] US5226902 BAE describes a device for dispensing biologically active material into the surrounding environment in which hydrogel deswells or shrinks in response to contact by external physical or chemical stimuli.

[0033] WO2017178417A1 DONG describes swelling of polymer beads to close an inlet to a sample chamber.

[0034] Su, Xin et al describe Hydrophilic / hydrophobic heterogeneity anti-biofouling hydrogels with well-regulated rehydration in: ACS Applied Materials and Interfaces. 2020; Vol. 12, No. 22. Pp. 25316-25323.

[0035] Ye, B.-F.et al describe a Colorimetric photonic hydrogel aptasensor for the screening of heavy metal ions in Nanoscale, 4(19), 5998 (2012).

[0036] Liu, H. et al describe Enzyme encapsulation in freeze-dried bionanocomposites prepared from chitosan and xanthan gum blend. Materials Chemistry and Physics, 129(1-2), 488- 494 (2011).

[0037] Zhang, Z. et al describe Encapsulation of lactase (^-galactosidase) into K-carrageenan- based hydrogel beads: Impact of environmental conditions on enzyme activity. Food Chemistry, 200, 69-75 (2016).

[0038] Wolf, M. et al describe Stability of p-D-galactosidase immobilized in polysaccharide-based hydrogels. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 609, 125679 (2021). Ghanem, A., & Ghaly, A. describe Immobilization of glucose oxidase in chitosan gel beads.

[0039] Journal of Applied Polymer Science, 91(2), 861-866 (2003).

[0040] Problems remain with respect to the art: the concentration of analyte in a sample liquid, e.g. body liquid such as urine, may vary with time, and this can affect the measured level; there may be variable contact time, and uncontrolled, variable exposure and / or development time; additional insults of sample liquid may wash away and / or dilute reaction components; sample fluid discharged into an absorbent matrix may increase in concentration as water is selectively absorbed, affecting the remaining liquid presented to any test element; test sensors e.g. test strip suffer from other issues, such as cost and complexity of manufacture and use, particularly when multiple tests are desired (e.g. lab on a chip); test strips or other biosensor components may also be expensive to produce; hydrogel-sensing systems degrade relatively quickly and are unstable over the time periods for long term storage required for commercial products; defining a volume for liquid sample can be a challenge in manufacturing; complex structures with layers are difficult to manufacture and control, and may require pre-processing of analyte before a conjugate or the like is received in an absorbent layer.

[0041] Co-pending applications GB2204051.3 and PCT / GB2023 / 05063 HOWARTH seek to address many of these issues; however, problems remain and / or further problems may arise. For example, these do not provide sensor structures for multi-test arrangements nor for multi-analyte detection.

[0042] The present invention seeks to alleviate one or more of the above problems, and / or other problems of the prior art.

[0043] Statements of the Invention

[0044] In a first aspect of the invention there is provided, an apparatus for interrogation of sample liquid to detect analyte (e.g. the presence and / or concentration and / or amount and / or level of analyte e.g. of at least one or only analyte, optionally of two or more analytes) comprising: at least one main sample chamber of predetermined (e.g. of fixed and / or of known) volume (e.g. of fixed volumetric capacity); and, within the at least one main sample chamber, a predetermined amount of dry (e.g. at least initially dry), absorbent sensing material (e.g. preferably) comprising (or more preferably comprising substantially, or more preferably substantially consisting of, or more preferably essentially consisting of, or more preferably consisting of, or more preferably only consisting of):

[0045] (e.g. modified) dry superabsorbent polymer (SAP) (e.g. dry super absorbent polymer material which may be known as modified, e.g. pre-loaded, dry superabsorbent polymer) and within the (modified e.g. pre-loaded) dry superabsorbent polymer, (e.g. dried within superabsorbent polymer to form modified, e.g. pre-loaded, dry superabsorbent polymer material), at least one dry reaction component for (e.g. configured for) detecting and for signalling the presence of analyte (e.g. within the SAP or from within the SAP, e.g. in vitro).

[0046] Thus, in one or more embodiments, upon insult of sample liquid, the dry (modified) superabsorbent polymer absorbs sample liquid, the at least one dry reaction component dissolves and, if analyte is present in the sample liquid, its presence is detected and signalled (e.g. within the SAP, from within the SAP).

[0047] Where a quantity is described as predetermined in this document, it may be fixed (e.g. so as not to vary) and / or known (e.g. relatively - so each of a plurality are the same but may be unknown, or absolutely - so the absolute quantity is known) and / or predefined (e.g. of a known in advance, fixed amount).

[0048] Preferably the predetermined volume of the main sample chamber is fixed.

[0049] In one or more embodiments, preferably the predetermined amount of absorbent sensing material comprises a predetermined amount of (modified) super absorbent polymer (SAP) (e.g. with at least one reaction component within it). Alternatively, or in addition, in one or more embodiments, preferably the predetermined amount of absorbent sensing material comprises a predetermined amount of at least one reaction component.

[0050] In one or more embodiments, a or the predetermined amount of modified superabsorbent polymer, with at least on dry reaction component within it, comprises a predetermined amount of superabsorbent polymer (e.g. prior to modification by the addition of one or more reaction components) and / or a predetermined amount of one or more reaction components. Where the absorbent sensing material is provided as particles, particularly particles of the same size, e.g. within the tolerances described herein), providing a predetermined amount of absorbent sensing material in which one or both of a predetermined amount of SAP and / or a predetermined amount of at least one reaction component are provided, manufacturing is greatly simplified. For example, a predetermined number of such SAP particles can simply be added to one or more main sample chamber(s) to form a sensor apparatus of desired construction. Thus, this could be provided in kit form for easy transport, for example the SAP particles in one (e.g. desiccated) container such as a pouch, the main sample chamber and, optionally, an associated measurement module in another container, for transport.

[0051] In one or more embodiments, a plurality of main sample chambers (also known as cells) are provided.

[0052] In one or more embodiments, the plurality of main sample chambers have (e.g. each have) the same volume (e.g. are capable of receiving the same, within expected tolerances, sample volume e.g. have the same volumetric capacity). They may have different volumes.

[0053] In one or more embodiments, the plurality of main sample chambers have the same size and / or shape and / or configuration (e.g. with respect to the apparatus) as each other. They may have different sizes and shapes.

[0054] In one or more embodiments, the same predetermined amount of the dry, absorbent, sensing material is provided in each main sample chamber, e.g. the same amount of the same type of absorbent sensing material comprising preloaded SAP, e.g. the same number of preloaded SAP particles of the same size and / or type (e.g. having the same type and amount of one or more reaction component), where the SAP is provided as particles.

[0055] In one or more embodiments, modified SAP particles having the same reaction component but different quantities of this per particle may be provided in the same or different main sample chambers.

[0056] In one or more embodiments, the preloaded, dry, superabsorbent polymer is provided with the same (e.g. the same type and / or quantity) of at least one dry reaction component, throughout at least one, or more than one, or all main sample chambers.

[0057] In some embodiments, the preloaded, dry, superabsorbent polymer is provided with one or more different dry reaction component(s) (e.g. for different assays / analytes, and / or amounts of the same dry reaction component(s)). For example, a first at least one dry reaction component may be provided for detecting one analyte in a first SAP (e.g. in one or more first preloaded SAP particle(s)), or a first and a second at least one dry reaction component (e.g. for detecting multiple analytes) may be provided for detecting one analyte in a first SAP (e.g. in one or more first preloaded SAP particle(s)) or in respective first and second SAPs (e.g. in respective preloaded SAP particle(s)).

[0058] For example, ketone and glucose assays may be provided in one or more preloaded SAP particle(s), in a first main sample chamber, and a more sensitive ketone-only assay may be provided in one or more preloaded SAP particles in a second main sample chamber. Other combinations and variations will be apparent to those skilled in the art from this disclosure.

[0059] In one or more embodiments, one or more or all of the at least one main sample chamber is or are provided with one or more or all rigid walls (e.g. defining its internal sample receiving volume e.g. side walls, roof, floor etc.).

[0060] It will be understood that, in some circumstances, rigid walls such as a roof may not be rigid or may be absent. In other circumstances a capillary stop arrangement may be provided e.g. using hydrophilic coating on one or more internal surfaces of the walls of the one or more main sample chambers.

[0061] In one or more embodiments, the apparatus comprises at least one first measurement module for measuring a signal from the at least one main reaction chamber. One or more measurement modules may be provided, e.g. for different assays, which may be used for measuring with respect to one or more main sample chambers.

[0062] In one or more embodiments, the apparatus comprises a first and a second measurement module for measuring a first and / or second signal from at least one main reaction chamber.

[0063] Thus, one or more methods (and modules) of measurement (interrogation) may be used on the same and / or on different main sample chambers.

[0064] In at least one embodiment, the dry superabsorbent polymer (SAP) (which may be known as modified, e.g. pre-loaded, dry superabsorbent polymer) changes colour and / or fluoresces in order to indicate presence and / or amount and / or concentration of an analyte. In some embodiments, the change in size (volume and / or mass) of SAP, e.g. SAP particles, is used as a method of indicating that it is time to commence a step of measuring. Thus, if a swelling of 5x or 10x or 100x is noted, the step of measuring may commence. The absorbent sensing material preferably comprises at least 50%, more preferably at least 75%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% by weight of dry superabsorbent polymer comprising the at least one dry reaction component. Indeed, preferably it comprises at least 99% or even 100% by weight of dry superabsorbent polymer comprising the at least one dry reaction component.

[0065] The (modified) dry superabsorbent polymer material preferably comprises at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% by weight of dry superabsorbent polymer and may comprise up to 5%, more preferably up to 3%, more preferably up to 2%, or more preferably up to 1% by weight of one or more dry reaction component(s).

[0066] In one or more embodiments, at least one dry first reaction component is provided for detecting analyte and at least one dry second reaction component is provided for signalling the presence of the detected analyte.

[0067] In one or more embodiments, the (modified) dry superabsorbent polymer (SAP) is configured to receive a discrete (e.g. limited) amount of a sample liquid (e.g. in vitro) (e.g. in other words it provides a subsidiary sample chamber, which can be used in conjunction with the at least one main sample chamber to carry out more complex biosensing measurements and / or more complex biosensor structures, more easily).

[0068] In one or more embodiments, the (modified) dry superabsorbent polymer provides (e.g. defines) a subsidiary sample chamber. In one or more embodiments, the (modified) dry superabsorbent polymer provides (e.g. defines) a subsidiary sample chamber of limited mass. In one or more embodiments, the (modified) dry superabsorbent polymer provides (e.g. defines) a subsidiary sample chamber of limited volume (e.g. in a dry state and / or in a swollen hydrated state).

[0069] In one or more embodiments, the (modified) dry superabsorbent polymer is in the form of a continuous bulk medium, having a discrete (e.g. limited) absorption capacity per unit mass providing, in effect, subsidiary sample chamber of limited volume per unit mass.

[0070] In one or more embodiments, the (modified) dry superabsorbent polymer comprises particles, preferably dry, preferably discrete particles. In this way the SAP particles each provide a discrete quantity of SAP into which a restricted or limited, albeit large, volume of liquid can be absorbed. The SAP is preferably formed as particles (e.g. beads, pellets, flakes, grains of any shape e.g. spherical, cylindrical) of small mass and so (relatively) small swelling capability. SAP particles do tend to have very rapid and large swelling capability although this slows very quickly following after initial exposure to liquid. The SAP particles are preferably 3 dimensional (3D) although they may be generally 2 dimensional, and / or have limited size in one dimension e.g. flakes. They may be hollow. In one or more embodiments, it is the provision of discrete particles of dry SAP (in other words a known amount) with one or more dry detection and signalling components pre-loaded within the SAP in combination with one or more main sample chambers, preferably of predetermined and / or fixed volume, that provides one or more advantages of the invention. The (modified) dry SAP may be referred to as pre-loaded dry SAP.

[0071] Preferably, the dry SAP particles (of preloaded dry SAP) are of similar mass e.g. at least 90% by weight are within a range of < + / - 10% the average dry mass. Preferably, at least 90% by weight of the particles are within <±10% or <± 5% or <± 2% of the average dry weight of the particles. For example, so 1 mm size particles (e.g. beads) have approximately 200 particles (e.g. beads) per gram. 2.5 mm diameter particles (e.g. beads) may have approximately 65 particles per gram. 4 mm diameters particles (e.g. beads) may have approximately 20 particles per gram.

[0072] Preferably, the dry SAP particles (of preloaded dry SAP) are of similar or the same size e.g. within a range of < + / - 10% the average size (volume or other dimension). Preferably, at least 90% by weight of the particles are within <±10% or <± 5% or <± 2% of the average dimension (e.g. volume or length or diameter or radius). When dry, the particles may be of the order of tens or hundreds of nanometres e.g. submicron so for example < 100 nanometres, or 100 to 1000 nanometres, or may span the nanometre and micron range so for example 100 to 5000 nanometres, or may be of order of tens or hundreds of microns, so submillimetre e.g. from 5 to 1000 microns, or 5 to 500 microns, 5 to 250 microns, or < 500 microns, or above the half millimetre range so e.g. 0.5 to 5mm, or within the millimetre range from 1 mm to 4 mm or from 0.5mm to 3mm, or from 0.5mm to 2mm, or from 1mm to 2mm in main lateral dimension e.g. length or diameter (so, for example, 90% of particles may have these dimensions, e.g. within + / - 10% or + / - 0.1mm or + / - 0.2mm). Being of substantially or generally consistent density, each SAP particle may therefore have substantially or generally the same mass and so the same liquid absorption capacity. Thus, the absorptive capacity per unit mass, or per particle, defines a subsidiary ‘sample chamber’.

[0073] This may be particularly useful when the dimension is known. In one or more embodiments, for example, a main dimension of dry SAP particles (of preloaded dry SAP), such as length or diameter, an average main dimension over a known number of particles, is, preferably <100 nanometres, or 100 to 1000 nanometres, or 100 to 5000 nanometres, or 5 to 1000 microns, or 5 to 500 microns, or 5 to 250 microns, or 5 to 100 microns, or 5 to 50 microns, or < 500 microns, or <0.1 mm (100 micrometres), or < 0.05 mm (<50 micrometres), or <0.01 mm (<10 micrometres), or <0.005 mm (<5 micrometres), 0.5 to 5 mm, or from 1 mm to 4 mm, or from 0.5 mm to 3 mm, or from 0.5 mm to 2 mm, or from 1 mm to 2 mm. Preferably, for example, at least 50%, or at least 80%, at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% of particles have such a corresponding main dimension e.g. within a tolerance + / - 10%, or within + / - 5%, or within + / - 3%, or within + / - 1 %.

[0074] The purpose of selecting such a small main dimension for (for example the majority of SAP particles (of preloaded dry SAP) such as >50%, or >75%, or >90%, or >95%, or >98%, or >99%, in other words, preferably most) SAP particles is so that the volume of the subsidiary sample chamber provided by each particle is more easily delimited or defined to be of particular size or of a particular range of sizes as described elsewhere herein. The effective size of the subsidiary sample chamber from one particle to the next is preferably similar or the same within a similar range of tolerances.

[0075] Preferably, in use or, the limited volume (e.g. per particle) is less than an expected volume of sample liquid. Preferably, the limited volume (e.g. per particle) is at least an order of magnitude less than an expected volume of sample liquid.

[0076] In one or more embodiments, the at least one dry reaction component comprises: at least one dry transducing component for detecting an analyte; and, at least one dry signalling component responsive to the detection of analyte for signalling the presence of analyte.

[0077] In some embodiments, two or more dry reaction components (e.g. assays e.g. comprising transducing and signalling components) may be provided for detecting two or more analytes in the same preloaded SAP (e.g. SAP particles) and / or two or more analytes in different preloaded SAP (e.g. in preloaded SAP particles) may be provided.

[0078] In one or more embodiments, the at least one reaction component (e.g. the transducing and / or the signalling component) comprises (e.g. comprises the components of) at least one of an electrochemical assay; a colorimetric assay; a nitrate assay; a ketone assay; a glucose assay; a bilirubin assay; a component capable of photometric detection; a component capable of spectrometric detection; a component of a mechanical resonator (e.g. a piezoelectric resonator).

[0079] Preferably, the at least one reaction component (e.g. the transducing and / or the signalling component) comprises a colorimetric assay comprising a chromogen (and / or dye).

[0080] Preferably, the at least one dry reaction component (e.g. the at least one transducing component) is selected from: one or more enzymes;

[0081] Glucose Oxidase (GOD);

[0082] Glucose Oxidase (GOD) and Horseradish Peroxidase (HRP) and the at least one signalling component is selected from a chromogen a colourless chromogen potassium iodide (e.g. to provide brown colours)

[0083] 3, 3’, 5, 5’ tetramethylbenzidine (TMB) (e.g. to provide blue colours). In one or more example embodiments, for example, a Benedict’s solution will change from a transparent blue colour to orange or brick red precipitate depending on the level of glucose present, whereas green represents normal glucose.

[0084] Preferably, the superabsorbent polymer is a synthetic superabsorbent polymer, or a naturally-derived superabsorbent polymer, or a biodegradable superabsorbent polymer, or any combination thereof.

[0085] In one or more embodiments the superabsorbent polymer comprises, or consists of, one or more SAPs recognised or determined by international standards such as: ISO 17190:2019 which refers to ‘Polymeric material capable of absorbing and retaining large quantities of aqueous solutions, saline solutions, and body fluids’ or ISO 12687:2019 which refers to ‘Polymeric materials, based on acrylic acid, acrylamide or their derivatives, that can absorb and retain water and aqueous solutions to a degree that the resulting hydrogel products can absorb at least 10 times their own weight of water under pressure’ or ASTM F2221-15 which refers to ‘polymeric material that, when hydrated, swells and retains large amounts of liquid, such as water or aqueous solutions, as a result of the action of capillary forces or hydrogen bonding or both.’

[0086] Preferably, the superabsorbent polymer comprises at least one of: polymerised acrylic acid or salts thereof or the like, sodium polyacrylate, polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, polyvinyl alcohol copolymers, cross-linked polyethylene oxide, and starch grafted copolymer of polyacrylonitrile, e.g. at least 50%, or at least 75%, or at least 90%, or at least 95% by weight, of one or more of these.

[0087] In one or more further embodiments, the apparatus for sample (e.g. bodily) fluid interrogation may be configured for sampling urine, or interstitial fluid, or tears, or saliva, or plasma, or serum, or blood.

[0088] Preferably, the limited volume (e.g. per particle) is less than an expected volume of sample liquid for a particular application. Preferably, the limited volume (e.g. per particle) is at least an order of magnitude less than an expected volume of sample liquid. Thus, the size of the subsidiary sample chamber provided by the sample size may be configured accordingly, e.g. by controlling the size of the dry SAP used.

[0089] In one or more embodiments, at least a portion of the (modified e.g. pre-loaded) dry superabsorbent polymer (SAP) of the absorbent material comprises particles of (modified e.g. pre-loaded) superabsorbent polymer (SAP). In some embodiments, at least a portion of the dry superabsorbent polymer (SAP) of the absorbent material comprises superabsorbent polymer (SAP) fibres.

[0090] Preferably, the one or more main sample chambers has / have a predetermined limited volume. Preferably, the one or more main sample chamber has at least one impervious wall (e.g. a rear wall, or rear and one or both side walls) e.g. to resist further ingress of sample liquid once full. Preferably, the one or more main sample chamber is rigid and / or comprises one or more rigid walls (e.g. roof, floor, side walls etc.).

[0091] Thus, in some embodiments the superabsorbent polymer may be used as a subsidiary sample chamber to absorb a predetermined amount of at least one reaction component (e.g. during manufacture), and / or in which the superabsorbent polymer may be used as a subsidiary sample chamber to absorb a predetermined amount of sample liquid. In a further aspect, there is provided a method of manufacturing an apparatus as described herein comprising: preparing SAP (e.g. particles of a predetermined dimension and / or range of dimensions e.g. volume or length or diameter to provide, when swollen, a predetermined swollen volume or range of swollen volumes), optionally, of known or predictable size or range of sizes as discussed in various example herein; drying SAP particles; in the absence of an analyte, introducing at least one wet reaction component for detecting and signalling the presence of the analyte, to the dry SAP; drying the SAP to form dry SAP pre-loaded with the at least one dry reaction component for detecting and signalling the presence of an analyte.

[0092] In some embodiments, the method may comprise adding a predetermined amount (e.g. number and / or volume and / or mass) of dry, pre-loaded SAP (e.g. SAP particles) to at least one main sample chamber of fixed and / or predetermined volume.

[0093] In a further aspect, there is provided a process for detecting analyte in a fluid (e.g. in vitro) comprising: a) providing apparatus as described herein; b) introducing a sample liquid to the at least one sample chambers, preferably at the same time, optionally at different times; c) exposing a or the predetermined amount of (e.g. modified) SAP in a main sample chamber to sample liquid, preferably a predetermined amount of sample liquid, preferably the same predetermined amount in each main sample chamber, optionally a different amount; d) rehydrating (e.g. at least partially) the at least one reaction component (e.g. within the SAP) to detect and signal (e.g. from within the SAP) the presence (and / or concentration, and / or level, and / or amount) of (e.g. at least one) analyte in the sample liquid; e) detecting and signalling the presence (and / or concentration, and / or level, and / or amount) of (e.g. at least one) analyte in the sample liquid (e.g. from within the SAP e.g. by visual indication within the SAP such as a change of colour of the SAP).

[0094] In some embodiments, carrying out (e.g. commencing or completing) a step of detecting and signalling the presence of analyte is carried out within <30 mins, <20 mins, or <15 mins, or <10 mins, or <5 mins, or <3 mins from initial exposure to sample liquid. In some embodiments, the step of detecting and signalling (e.g. determining and / or measuring) takes, e.g. a period of time, of <1 min, or <30 sec, or <20 sec, or <10 sec (e.g. to complete).

[0095] In a further aspect, there is provided a kit comprising a precursor apparatus for interrogation of sample liquid to detect analyte, the precursor apparatus comprising: at least one main chamber of predetermined volume; and the kit further comprising, for placing within the main reaction chamber, a predetermined amount of dry, absorbent, sensing material comprising (e.g. comprising substantially) dry superabsorbent polymer (SAP) (preferably a predetermined amount thereof); and, within the dry superabsorbent polymer, at least one dry reaction component (preferably a predetermined amount thereof) for detecting and for signalling the presence of analyte (e.g. within the SAP).

[0096] In one or more embodiments of any aspect of the invention, the analyte to be detected may comprise (or contain, or consist of) one or more bacteria, such as one or more types of bacteria, and / or one or more viruses, such as one or more types of virus, and / or a component of bacteria, and / or a component of a virus, and / or an emission of a bacteria, and / or an emission of a virus, and / or a property of a bacteria, and / or a property of a virus.

[0097] In one or more embodiments, bacteria and / or viruses may be captured and / or retained within the initially dry absorbent material e.g. within the modified SAP e.g. for analysis of the one or more (e.g. types) bacteria and / or one or more (e.g. types of) virus(es), a component and / or emission and / or property of the one or more (e.g. types) bacteria and / or one or more (e.g. types) virus(es). By emission is meant a substance, e.g. a fluid such as liquid and / or a detectable phenomenon e.g. light. By property is meant a detectable state of the bacteria and / or virus e.g. colour and / or volume for temperature.

[0098] In one or more embodiments, the apparatus and / or method(s) is / are configured for the detection of one or more bacteria and / or one or more viruses and / or the growth of one or more bacteria and / or one or more viruses and / or capture of one or more bacteria and / or one or more viruses and / or retention of one or more bacteria and / or one or more virus(es).

[0099] In one or more embodiments, the (e.g. initially dry) dry absorbent material comprises a growth medium for bacteria. In one or more embodiments, the sample liquid may be any one or more bodily fluid(s).

[0100] Several embodiments of the invention are described and any one or more features of any one or more embodiments may be used in any one or more aspects of the invention as described herein as would be understood by someone skilled in the art. Thus, the features of the claims and / or those described herein may be used in any aspect of the invention, the different aspects of the invention making use of the features of embodiments described with reference to a different aspect. For example, whilst SAP particles and in particular SAP beads are described in several examples below, it will be understood that technical features of the SAP particles and in particular the SAP may be adapted to SAP in non-particulate form or in other shapes (e.g. SAP bulk medium, and / or SAP fibres etc.) with suitable changes, mutatis mutandis, as would be understood by someone skilled in the art. Further, capturing and / or retention of bacteria and / or virus for analysis, e.g. as an analyte to be detected, or as a precursor to an analyte to be detected (such as component and / or emission and / or property of the bacteria and / or virus, is applicable to any aspect of the invention, mutatis mutandis.

[0101] In one or more embodiments of any aspect of the invention, the apparatus, and / or method and / or kit may be configured for the detection of one or more bacteria and / or virus(es).

[0102] In one or more embodiments of any aspect of the invention, the apparatus, and / or method and / or kit may be configured for promoting and / or resisting growth of bacteria and / or virus(es).

[0103] In one or more embodiments of any aspect of the invention, (e.g. the apparatus, and / or method and / or kit), the dry absorbent material may comprise a growth medium and / or antimicrobial component and / or anti-viral component for bacteria and / or virus(es).

[0104] In one or more embodiments of any aspect of the invention, (e.g. the apparatus, and / or method and / or kit), the at least one reaction component may comprise one or more antibiotic(s) and / or antiviral component(s).

[0105] Brief Description of the Invention

[0106] The present invention will now be described, by way of example only, with reference to the following figures.

[0107] Figure 1A shows a schematic, side, cross-sectional view of a reaction chamber with a known multiple, here three, of dry pre-loaded SAP particles generally of spherical shape, here beads.

[0108] Figure 1 B shows the reaction chamber of Figure 1A with an added biological fluid of, preferably, known volume. Figure 1C shows the reaction chamber of Figures 1A and 1 B after a, preferably predetermined, period of time illustrating a known multiple of beads now swollen having absorbed an amount (e.g. a volume), typically a predictable amount (e.g. over a predetermined period of time), of fluid e.g. biological fluid.

[0109] Figure 2A shows a schematic, side, cross-sectional view of an alternative apparatus with three sample chambers, each with differing numbers of SAP particles (e.g. beads).

[0110] Figure 2B shows the apparatus of Figure 2A with differing known quantities of sample liquid Vf11, Vf21, Vf31.

[0111] Figure 2C shows the apparatus of Figure 2B after, preferably, a predetermined amount of time, during which absorption has taken place.

[0112] Figure 2D shows the apparatus of Figures 2A to 2C in which different methods of interrogation are applied to different sample chambers.

[0113] Figure 3A shows a schematic, plan view of an alternative apparatus of rectangular shape in which six sample chambers of the same substantially square shape, and of substantially equal size with equal numbers of pre-loaded SAP particles of the same size are provided.

[0114] Figure 3B shows a schematic plan view of an alternative apparatus in which four equisized sample chambers are provided, here each forming a segment, and in particular a quarter, of a circle when viewed from above, each sample chamber having a differing number of preloaded SAP particles.

[0115] Figure 3C shows yet a further alternative embodiment of the apparatus in which four differently sized and shaped sample chambers are provided within a single housing, each having the same number of preloaded SAP particles.

[0116] Figures 4A, 4B, and 4C show, respectively, the apparatus of Figures 3A, 3B, and 3C after a sample fluid has been applied, and a sufficient period of time allowed for e.g. a predetermined period of time, for the preloaded SAP particles to swell e.g. to a predetermined size, and for the detection and signalling to take place.

[0117] Figure 5A shows a method of manufacturing an apparatus as described herein.

[0118] Figure 5B shows a method of using an apparatus as described herein. Figure 6 shows an example chemical detection and signalling regime for detection of glucose, in one or more example embodiments of any aspect of the invention.

[0119] Figures 7A, 7B and 7C show, respectively, schematic views of first, second and third chambers for detection of bacteria and / or assessment of antimicrobial resistance of bacteria.

[0120] Figure 8 shows experimental results: reactive SAP with Bacterial Reagent (e-coli) - (L-R Beads - Control (32A-0) (No Bacteria), reacted beads (32A-1 , 32A-2) showing presence of bacteria (e-coli), and control liquid 52 sample in test vial 50).

[0121] Figure (9A, 9B and 9C show respectively a bottle, an upside-down bottle, and a cap of a bottle showing a series of windows and / or beads indicating results for one or more tests, for use with one or more embodiments of the present invention.

[0122] Detailed Description of the Invention

[0123] Any quantities, dimensions, or shapes used herein are to be understood to lie within the tolerances, ranges, and variations expected or understood within the particular field. For example, beads are usually spherical but need not be so, and spherical beads may vary in their sphericity. In this disclosure, the term ‘beads’ is used as one example of the more general term ‘particles’, which are discrete, independently movable from one another, each having a limited mass, which may vary from one to the next. Further within the following description SAP may refer to SAP alone, or, where the context so indicates, to modified SAP material comprising both SAP and at least one reaction component and the present text is to be read with this in mind. SAPs are defined in several standards including ISO standards referred to elsewhere in this text.

[0124] Hydrogel Biosensors have generated considerable interest over the past few years, with respect to the excellent alternative systems to detect a wide range of biomolecules, including small biochemicals, pathogenic proteins, and disease-specific genes.

[0125] Due to the excellent physical properties of hydrogels, such as the high water-content and stimuli-responsive behavior of cross-linked network structures, this system can offer substantial improvement for the design of novel detection systems for various diagnostic applications. The other main advantage of hydrogels is the role of biomimetic three- dimensional (3D) matrix immobilizing enzymes and aptamers within the detection systems, which enhances their stability. This provides ideal reaction conditions for enzymes and aptamers to interact with substrates within the aqueous environment of the hydrogel.

[0126] In comparison, a super absorbent polymer (SAP) is a hydrogel without the water content. In other words, a SAP is dry. SAP comprises water-absorbing polymers which when crosslinked, absorb aqueous solutions through hydrogen bonding with water molecules. A SAP’s ability to absorb water, liquid or a biological fluid depends on the ionic concentration of the aqueous solution.

[0127] In this disclosure, a SAP (superabsorbent polymer) is intended to refer to such a polymer in dry form. This may be entirely dry (H2O <0.5% by weight), essentially dry (<5% H2O by weight), substantially dry (<15% H2O by weight), or sufficiently dry (e.g. <20% H2O by weight) such that the stability of the SAP and its contents (such as its reaction components) do not degrade to any significant extent over the period of preferably > 6 months, more preferably > 1 year, even more preferably > 18 months, even more preferably > 2 years. It is preferred that the SAP does not comprise enough or sufficient water to enable the transducer component e.g. enzyme to activate or to activate the signalling component.

[0128] Further, in this disclosure, a hydrogel is intended to refer to a SAP (superabsorbent polymer) in wet form. This may be partially swollen with liquid e.g. (>25% H2O by weight), or substantially swollen with liquid e.g. (>50% H2O by weight or >60% H2O by weight or >70% H2O by weight) or sufficiently swollen with liquid e.g. H2O that further absorption of liquid is substantially reduced and / or prevented e.g. within the timescale of exposure to liquid.

[0129] In deionized and distilled water, a SAP may absorb 300 times its weight (from 30 to 60 times its own volume), but when put into a 0.9% saline solution, the absorbency may drop to 50 times its weight. Of note is the swift initial swelling in the first 200 seconds and the much slower absorption thereafter (see Zohuriaan-Mehr et al 2008). The present inventors have appreciated that this variation in speed of swelling can be used in short-lived disposable hygiene absorption products or interrogation mechanisms in sensors such as test sensors e.g. test strips to deliver an indicator of the presence of an analyte or of a disease or state or, indeed in some embodiments, a test result. Examples of analytes include but are not limited to glucose, lactate, urea and cholesterol.

[0130] Biosensor systems typically comprise: i. a biologically active receptor element e.g. enzymes, optionally with one or more cofactors), antibodies, antigens, cells, nucleic acids, DNA etc.; ii. a base material which may be a base substrate in the form of a layer of polymer or paper or other suitable material; iii. a transducer element based on, for example, optical signalling (e.g. colour change, absorbance, luminescence, electrochemical (e.g. amperometric, conductometric, potentiometric, impedimetric, piezoelectric resonator etc.; iv. a detection or observation element.

[0131] In the remainder of this document, we shall talk about glucose and glucose oxidase in a colorimetric system as examples of an analyte I receptor system with a transducer and signalling capability, but it will be understood that in other embodiments, other biosensor chemistries and / or components such as piezoelectric resonators may be used in the invention.

[0132] Unlike the prior art, in this invention it is the absorptive material itself, the superabsorbent polymer, that provides a subsidiary ‘vessel’ or subsidiary ‘sample chamber’ within which the reaction takes place between the biologically-active receptor and an analyte and, preferably also, within which the preferably visual (or other) signalling occurs. It has been appreciated by the present inventors that this combination of the dry biologically-active receptor and optionally dry transducer element within a single dry component (the superabsorbent polymer) greatly simplifies the manufacture of biosensors which provide a disease state indication or, indeed, measurement and is particularly useful when used (e.g. in known amounts, in combination with a main sample chamber.

[0133] The approach of the present inventors is to use the properties of a SAP and combine this with a number of known bio reagents so that, when presented with a biological fluid sample, the super absorbent reagent will, firstly, capture and contain the biological sample and, secondly, will react to provide an indication of the presence or concentration of the analyte e.g. of elevated glucose and / or ketones. The indication may be directly observable e.g. visible by eye such as a change in colour, or may be provided after interrogation e.g. direct or indirect interrogation e.g. electrochemical interrogation. Thus, by using preferably a predefined e.g. known or predefined quantity of SAP, then volume (e.g. a predefined volume) of subsidiary sample chamber(s) with (preferably) a predefined quantity of at least one reaction component, within same, are provided within a main sample chamber of known size, thus various advantages are realized and more complex biosensing structures (test sensors, or lab on a chip structures) and measurement regimes can be developed more simply. Industry-recognised assay techniques and reagent chemicals may be combined with SAP to provide this new approach. Superabsorbent polymers are commonly made from the polymerization of acrylic acid blended with sodium hydroxide in the presence of an initiator to form a poly-acrylic acid sodium salt (sometimes referred to as sodium polyacrylate). This polymer is the most common type of SAP made in the world today.

[0134] Other materials are also used to make a superabsorbent polymer, such as polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, polyvinyl alcohol copolymers, cross-linked polyethylene oxide, and starch grafted copolymer of polyacrylonitrile to name a few. The latter is one of the oldest SAP forms created (see CALO et al 2015).

[0135] Today, superabsorbent polymers are made using one of three primary methods: gel polymerization, suspension polymerization or solution polymerization. Each of the processes has their respective advantages but all yield a consistent quality of the product.

[0136] Gel polymerization - A mixture of acrylic acid, water, cross-linking agents and UV initiator chemicals are blended and placed either on a moving belt or in large tubs. The liquid mixture then goes into a ‘reactor’ which is a long chamber with a series of strong UV lights. The UV radiation drives the polymerization and cross-linking reactions. The resulting ‘logs’ are sticky gels containing 60-70% water. The logs are shredded or ground and placed in various sorts of driers. Additional cross-linking agent may be sprayed on the particles' surface; this ‘surface cross-linking’ increases the product’s ability to swell under pressure — a property measured as Absorbency Under Load (AUL) or Absorbency Against Pressure (AAP). The dried polymer particles are then screened for proper particle size distribution and packaging.

[0137] Solution polymerization - Solution polymers offer the absorbency of a granular polymer supplied in solution form. Solutions can be diluted with water prior to application, and can coat most substrates or used to saturate them. After drying at a specific temperature for a specific time, the result is a coated substrate with super absorbency. For example, this chemistry can be applied directly onto wires and cables, though it is especially optimized for use on components such as rolled goods or sheeted substrates.

[0138] Solution-based polymerization is commonly used today for SAP manufacture of copolymers, particularly those with the toxic acrylamide monomer. This process is efficient and generally has a lower capital cost base. The solution process uses a water-based monomer solution to produce a mass of reactant polymerized gel. The polymerization’s own exothermic reaction energy is used to drive much of the process, helping reduce manufacturing cost. The reactant polymer gel is then chopped, dried and ground to its final granule size. Any treatments to enhance performance characteristics of the SAP are usually accomplished after the final granule size is created.

[0139] Suspension polymerization - The suspension process is practiced by only a few companies because it requires a higher degree of production control and product engineering during the polymerization step. This process suspends the water-based reactant in a hydrocarbon-based solvent. The net result is that the suspension polymerization creates the primary polymer particle in the reactor rather than mechanically in post-reaction stages. Performance enhancements can also be made during, or just after, the reaction stage.

[0140] In one or more embodiments, the invention provides a system in which a biological fluid is diffused into a modified (also known as preloaded) SAP, e.g. into initially dry SAP-based absorbent material, and more preferably into (preferably) initially dry SAP particles, which has / have been bonded with a wide variety of chemical reagents during or after manufacture e.g. as outlined in any of the three types of SAP manufacture above. Thus, an initially dry SAP pre-loaded with one or more reaction components can be provided within one or more main sample chambers.

[0141] Thus, in one or more embodiments, the invention provides SAP + Reagent which when insulted by a sample liquid e.g., a biological fluid provides a hydrogel sample for interrogation. The biological sample acts as the wetting material within the superabsorbent polymer.

[0142] The invention aims to solve and improve upon methods of capturing a biological sample within a specific mechanical carrier (e.g. microfluidic or nanofluidic cell, paper-based assay I dipstick) to measure target analytes including small biochemicals, pathogenic proteins, and disease-specific genes.

[0143] One or more embodiments of the invention allow for diffusion of the specific sample that is under interrogation and allows for the act of absorption to mix the biological sample with the predefined reagent. By combining this technology with known amounts of sample fluid and / or reagent and / or sample volume and / or SAP within a main sample chamber of well- defined dimensions and / or characteristics (e.g. volumetric capacity, and / or shape, and / or number of inlets and outlets etc.), optionally within two or more such main sample chambers, various advantages are provided. SAPs, once swollen with liquid, particularly once swollen to near or final capacity, are resistant to further absorption. The present inventors have recognised the usefulness of this capability in developing embodiments of the present invention, particularly within such a mechanical carrier having one or more such main sample chambers. See, for example, Figure 4B in which in one segment-shaped main sample chamber (20-2), liquid absorption by a cell volume-limiting condition arising in other into SAP particles may be halted because there is no room for SAP particles to swell further, the cell 20-2 being full. This technique may be used to limit further absorption and half the further development and / or dilution of a signal from within the SAP e.g. dilution of a colour / fluorescence is substantially prevented.

[0144] Whereas typical absorbent materials such as wood or paper fibres can be ‘wrung out’ by applying pressure when wet. This does not occur to any significant extent in swollen SAP. Liquid, once absorbed by SAP particles, remains absorbed within the main sample chamber(s). Furthermore, SAPs, once swollen with liquid, even partially, demonstrate a far lower speed of absorption e.g. swelling rate. The present inventors have recognised the usefulness of this capability in combination with one or more rigid main sample chambers to develop test sensor and methods

[0145] Furthermore, since SAP particles have a limited mass, they therefore also have a limited, although great, ability to absorb liquid. So, in this sense, they also have a limited, predefined volume, in other words a limited volume into which sample can be absorbed. This may be thought of as a subsidiary sample chamber. Thus, once the SAP particles are ‘full’ they cannot ‘easily’ absorb more liquid. Further, they do not give up their liquid contents easily either, particularly against a diffusion gradient provided by a surrounding liquid sample. As explained above, once even partially swollen, their swelling rate decreases (see Zohuriaan-Mehr et al 2008). Once the sample chamber is full, further insults of liquid sample bypass the main sample chamber and the already swollen SAP particles. These, therefore, represent an initial insult of sample liquid, unaffected by later insults. In other words, the swollen SAP particles (preloaded with reagent) present a measure of analyte that is frozen in time at least for a short period of time, say 10 to 30 minutes. This property can be exploited, particularly when any further absorption is substantially prevented (see Figure 4B for example).

[0146] When in vivo SAP particles are constantly surrounded by wet conditions with constant flow about them, diffusion may occur into and out of the particles. However, in in vitro conditions, in one or more embodiments of the present invention which provides one or more main sample chambers as well as subsidiary chambers in the form of SAP particles, the particles are, in essence, isolated from further insults of fluid of differing analyte concentrations, particularly when spherical or swollen within a main sample chamber. Indeed, no matter the initial shape, sphericity is increased in swollen SAP particles. If there is no flow around the SAP particles, diffusion into and out of each SAP particle is also very low and may be negligible over the time frames of the test. What has been absorbed, stays absorbed, at least for a time. In effect, each SAP particle provides a limited volume which the present inventors have appreciated can be thought of as a subsidiary sample chamber resistant to further ingress of liquid, particularly when a main chamber holding the SAP is full and therefore resistant to further ingress of fluid.

[0147] Even in bulk SAP, for example, one notional subdivision of SAP (e.g. with a mass) lies next to another subdivision of SAP (with the same predefined mass) and these are both swollen with sample liquid, then diffusion from one subdivision to the next is minimal, as there is no diffusion gradient. In effect, each subdivision of SAP provides a limited volume for absorption related to its mass within that subdivision. In effect, each subdivision forms a sample chamber, each sample chamber being adjacent to a neighbouring one.

[0148] This is easier to contemplate and, indeed, is more definitive where the SAP is actually physically subdivided into individual particles, preferably beads, more preferably spherical beads. Particles of other such shapes may be used such as, grains, rods, flakes and the like etc.

[0149] So, volume limitation to the assay is provided by notional, or actual, subdivision of SAP into particles. These subdivisions may be thought of as first sample chambers.

[0150] Thus, in the present invention there is provided one or more main sample chambers within the physical configuration of a carrier e.g. a test sensor such as a test strip. Such main sample chamber(s) may have one or more impermeable walls and / or a closable entrance and so on. Such main sample chamber(s) preferably have one or more rigid walls. These may be of micrometre dimensions. These may be configured to operate by capillary action to deliver fluid inside the main sample chamber.

[0151] These preferably have a pre-defined sample chamber volume and where one or more than one is provided, these may have the same sample chamber volume. By providing a main, rigid-walled, well-defined volume main sample chamber with at least one rigid wall, and preferably a rigid floor and rigid wall(s) and preferably also a rigid roof, the main sample chamber volume is carefully defined. Alternative methods of defining volumetric capacity may be used (e.g. hydrophilic layers and / or capillary stops etc.). Nevertheless, the use of absorbent sensing material of the invention in combination with such one or more main sample chamber(s) provides opportunities for various analyte investigations within a single apparatus (e.g. a test sensor). This may be testing for different analytes using SAP, e.g. the same or different preloaded SAP in different main sample chambers of the same size, and / or testing for the same analyte using different quantities of reagent within the same and / or different quantities of SAP, and / or within the same and / or different main sample chambers using the same or one or more different detection methods with respect to main sample chambers and so on.

[0152] After an initial insult, the liquid that has entered the SAP particles (or subdivisions of bulk SAP), notionally subsidiary sample chambers, remains there. It is not easily washed away or diluted even by further insults of liquid sample. This is particularly the case in point-of- care or home test sensors where results are expected within seconds up to around 30 minutes to an hour.

[0153] Thus, by providing a limited volume of liquid sample in contact with a predetermined amount of reaction chemistry, a more accurate indication of the amount (e.g. the concentration) of analyte in the sample can be obtained. By providing multiple main sample chambers, one or more measurements (e.g. the same or different measurements) may be repeated, or adjusted, as described herein to provide further insight. This insight offers some practical solutions to qualitative indications of disease and may offer opportunities for interrogation in a quantitative manner using the absorbent material and techniques of the invention.

[0154] In one or more embodiments, a main lateral dimension of dry SAP particles e.g. a length or diameter, may be <0.1 mm (<100 micrometres, which would give a volume of a cube or sphere of or of the order of, 1 nL), or < 0.05 mm (<50 micrometres, which would give a volume of a cube or sphere of, or of the order of, 65 picolitres), or <0.01 mm (<10 micrometres, which would give a volume of a cube or sphere of, or of the order of, 1 picolitre), or <0.005 mm (<5 micrometres, which would give a volume of a cube or sphere of, or of the order of, 0.065 picolitres). At least e.g. 90% of the particles (or other proportion of the particles as disclosed elsewhere) may be of the stated dry volume e.g. within expected tolerances.

[0155] In one or more embodiments, a suitable dimension of swollen SAP particles into which a liquid sample has been absorbed, e.g. a length or diameter, may be <5 mm (<5000 micrometres which would give a volume of a cube or sphere of, or of the order of, 6.5 microlitres), <1 mm (<1000 micrometres which would give a volume of a cube or sphere of, or of the order of, 1 microlitres), or < 0.5 mm (<500 micrometres which would give a volume of a cube or sphere of, or of the order of, 65 nanolitres). The volume and / or other suitable dimension of the corresponding dry SAP particles which would absorb these amounts of liquid as a liquid sample of generally or substantially predetermined size is related to this swollen volume by the swelling ratio. The swelling ration depends on, amongst other things, the material from which the SAP is made. Examples of materials are shown in Table 1. Table 1

[0156] In one or more embodiments, the volume of the subsidiary sample chamber provided by a single SAP particle is defined to be a particular size or in a particular range for example by defining a particular dimension e.g. diameter or other suitable dimension.

[0157] Examples are shown in table 2 in which example cube and sphere sizes are shown, and table 3 in which ranges of sphere sizes, here swollen sphere sizes Vmax, are shown: Table 2

[0158] Table 3

[0159] In one or more embodiments the purpose of selecting such dimension, in some embodiments at one end of the range e.g. a small dimension, for the majority (e.g. >50%, or >75%, or >90%, or >95% >99%, in other words, preferably most) SAP particles is so that the volume of the subsidiary sample chamber provided by each particle is more defined to be a particular size for a selected application.

[0160] To explain, the actual volume of such a sample chamber is in effect limited by the size of the initial dry particle. Thus, the volume of a fully swollen SAP bead is closely based on its initial dimensions when dry and so, for spherical particles, its dry diameter as well as, to a lesser extent, the SAP type, the sample liquid, and its surroundings. This means that the maximum volume of the fully swollen particle (Vmax) depends on the SAP material, the swelling factor of the SAP material, the initial dry particle size, and surroundings.

[0161] In the present invention, it is intended to immerse preloaded SAP / SAP particles in sample liquid and retain these immersed in that same sample liquid.

[0162] The benefits of this may be particularly useful where a very limited amount of subsidiary sample chamber volume is available within one SAP particle, or a majority, or most, or all, where more than one is provided, and a less limited amount of main sample chamber volume remains.

[0163] The calculation for determining the diameter of a spherical SAP bead to achieve a certain volume can be done based on

[0164] V = 1 / 6 TT D3F where V is the volume, D is the diameter, F is the Swelling Factor, or swelling ratio, is the ratio of the volume of the fully swollen SAP bead to its dry volume, TT (pi) is a mathematical constant with a value of approximately 3.14.

[0165] Swelling factor F is a dimensionless quantity that represents the amount of liquid (e.g. water) absorbed per gram of dry SAP. The swelling factor F can vary depending on the specific type of SAP used and the conditions of the swelling. Therefore, in certain embodiments or scenarios, or for certain applications, it can be important to use the appropriate swelling ratio for the specific SAP material being used and / or specific conditions.

[0166] Table 4 shows various example of particle size (assuming as an example spherical particles, and various swelling factors F).

[0167] Table 4

[0168] Diameter D of dry SAP bead (ftm) _

[0169] Therefore, assuming a consistent density and composition of material, a dry SAP bead with a diameter D of 12.4 microns has a volume of approximately 1 picolitres. If this has a swelling ratio of 500, then it absorbs (1 x 500) picolitres of liquid, so 500 picolitres. This is in effect the sample volume per particle.

[0170] Therefore, a dry SAP bead with a diameter D of 0.5 microns has a volume of approximately 0.065 picolitres. If this has a swelling ratio of 500, then it absorbs (0.065 x 500) picolitres of liquid, so 3270 picolitres or 3.27 nanolitres. This is in effect the sample volume per particle. Thus, SAP particles of various anticipated sample chamber volumes can be provided for different purposes, and / or different diagnostic techniques and / or different sample types.

[0171] Indeed, a dry SAP bead of 1.24 cm could provide absorption of 100cmA3 if the swelling factor is 100. The volume of the main sample chamber in any test sensor apparatus may be determined by any known method. For example, a test sensor may be formed as a test strip having a substrate on which one or more electrodes may be formed, an insulation layer providing side walls to one or more main sample chambers, and a hydrophilic layer as a roof to fill the chamber via capillary action. Such main chambers may, for example, be <1 pl, or <5 pl, or <10 pl, or <20 pl in size, or other sizes as known in the art, in which case the SAP is sized and / or shaped accordingly.

[0172] Typically, chronic conditions are diagnosed using measurements of the concentration of analytes in biofluids. In the case of diabetes, the most relevant analyte is glucose in blood or urine. Such measurements may be made using either disposable test strips or laboratory analysers. Disposable test strips can measure glucose in both blood and urine.

[0173] To measure glucose in blood it is necessary to extract a blood sample and introduce it to the test strip or laboratory analyser. This involves puncturing the skin, which causes pain, rendering such an approach unsuitable for routine measurements in populations of infants, the vast majority of whom will not display any symptoms. Using a laboratory analyser has the additional disadvantage that the infant or the sample must be transported to a central analysis facility.

[0174] The measurement of glucose in urine does not result in any pain, but in infants who are not potty trained it is extremely challenging to obtain a suitable sample of urine for measurement in the home using a disposable test strip. If a laboratory analyser is used the same practical limitations as noted above apply.

[0175] Diabetes screening shows 60% of under 2s are already in diabetic ketoacidosis at the time of diagnosis, which is undesirable. Faster, more convenient, more reliable testing is desirable.

[0176] In some embodiments of the invention, two or more other analytes may be detected in urine based on one or more sensing element(s) (e.g. comprising preloaded SAP in one or more respective main sample chambers) which indicate, e.g. change colour or otherwise provide an indication, when in contact with the relevant analyte.

[0177] A key challenge to overcome is discriminating between a small amount of more concentrated sample fluid e.g. urine and a larger amount of less concentrated sample fluid e.g. urine, both of which may contain the same absolute quantity of analyte. This challenge is addressed using a fluid capture system such as the absorbent sensing material within a main sample chamber which, as explained elsewhere, over the time scale of use is capable of receiving a finite amount of urine. Each SAP particle is in one sense capable of receiving only a defined volume of sample liquid e.g. urine. Similarly, each main chamber is capable of receiving only a defined volume of sample liquid. Therefore, the amount of analyte that is delivered to each SAP particle is more closely proportional to the concentration in the bulk fluid and the bulk liquid surrounding the SAP particles (as these are immersed) remains. Once the SAP particle is full or partially full, it resists further ingress of fluid.

[0178] In one or more embodiments which effect a colour change, the reaction components consist of a sensing chemistry immobilised internally. For example, in the case of glucose, a two-stage dual enzyme system may be used in which Glucose Oxidase (GOD) and Horseradish Peroxidase (HRP) are provided. In the first stage Glucose Oxidase (GOD) catalyses the reaction of glucose to gluconic acid and hydrogen peroxide. In the second stage, Horseradish Peroxidase (HRP) catalyses the reaction of peroxide with the initially colourless chromogen (dye precursor) and a colour develops. The colour will depend on the chromogen selected. For example, with potassium iodide, green to brown colours are accessible. With 3,3’,5,5’-tetramethylbenzidine (TMB) blue colours are accessible. Other colours are accessible using different chromogens (see Figure 8 and Examples below).

[0179] This sensing regime is within the SAP and may also be present within the main sample chamber. In this case, the surrounding sample liquid may change colour and / or fluoresce to match the SAP particles.

[0180] An alternative implementation is possible, in which the sample liquid in the main sample chamber does not change colour, in effect, highlighting the now coloured and / or fluorescing SAP particles.

[0181] In another variant, the same level of the same type of chromogen may be present in each group of SAP particles within different main sample chambers, with the sensing performance of each differentiated on the basis of the enzyme system and loadings, such that the sensing SAP particles in each compartment reach a different colour end point. Other variations can be envisaged from this disclosure.

[0182] In one or more embodiments, at least two groups of SAP particles are provided, at least one group in a first main sample chamber configured to be indicative of a first level of a first analyte, and a second group in a second main sample chamber configured to be indicative of a second level of a first analyte or of a different second analyte.

[0183] Preparation of sensing elements Co-pending applications GB2204051.3 and PCT / GB2023 / 05063 HOWARTH describe the preparation of sensing elements (SAP beads) and this description is briefly incorporated here.

[0184] Example 1

[0185] SAP beads were prepared using two-stage dip process in which SAP beads were first impregnated with enzymes, a dye fixer and a colour enhancer, then impregnated with the chromogen and a film-forming agent.

[0186] Specifically, SAP beads were dipped into Solution 1 , a 25 % ethanol solution buffered to pH 4.8 with citrate buffer, containing the enzymes GOD and HRP, the dye fixer Gantrez AN-139, and the colour enhancer polyvinylpyrrolidone. The SAP beads were then allowed to dry.

[0187] The SAP beads were then dipped into Solution 2, consisting of the chromogen TMB and a film forming agent ethyl cellulose in chloroform. The SAP beads were then dried.

[0188] The steps above were performed three times with increasing chromogen concentration to create the differing sensitivities. Each of the three portions of sensing elements contains increasing loadings of the same chromogen, TMB.

[0189] Example 2

[0190] Absorbent polymer (SAP) beads were stably loaded with reagents that would react with a glucose solution to produce a visually detectable colour change. A pre-existing commercial assay was loaded onto the beads using a reaction mix containing an enzyme and a reagent which produces a visible colour change in the presence of the product of the reaction between the enzyme and glucose.

[0191] The stability of the loaded beads was tested for freezing at -80°C and subsequently for loaded beads that underwent freeze-drying or thermal drying.

[0192] As will be understood by those skilled in the art, the sensitivity of this approach can be demonstrated using a number of glucose concentrations (relatively low to relatively high) to transect the physiological glucose concentration range. Furthermore, the beads remain active after drying. Furthermore, long-term stability and potential for suitability for long term storage is indicated after early stage accelerated stability testing. Absorbent SAP beads in the form of a super absorbent polymer consisting of sodium polyacrylate was used. This has no known toxicity or hazard.

[0193] A glucose assay kit (colorimetric / fluorometric) was used e.g. from Abeam (product ab65333). This is a well-characterized, simple and rapid assay used to quantify glucose levels in biological samples. The supplied enzyme mix oxidises glucose, generating a product that reacts with a dye which generates a red colour which is visible to the naked eye. The detection regime is shown in Figure 6. The sensitivity of the assay, relative to a range of concentrations of glucose provide a different depth of colour, or using Benedict’s solution, a range of colours. Coated beads were freeze dried or heat dried.

[0194] The retention of activity of treated, freeze-dried, dry SAP beads over time (“shelf-life”) is much longer than for undried loaded, swollen hydrogel beads.

[0195] It has been demonstrated that:

[0196] • The SAP beads can absorb the glucose assay reaction mix.

[0197] • The reaction mix retains activity after absorption.

[0198] • Heat-dried test beads retain their reactivity with glucose.

[0199] • Freeze-dried test beads retain their reactivity with glucose.

[0200] • Freeze-drying was a much more effective method than heat-drying.

[0201] • Test beads retain their structure and function after undergoing both freezing at -80°C and the freeze-drying process.

[0202] • Freeze-drying changes the appearance of test beads when buffer is used in the reaction mix and this may be due to buffer salts.

[0203] • Freeze-drying appearance changes are not observed where water is substituted for buffer in the reaction mix.

[0204] • Water should be used as a substitute for buffer in reaction mix in future experiments to avoid background colour that could represent a false positive.

[0205] Preferably, buffer is not used in the reaction mix. Preferably water is used, particularly if freeze-drying is to be used.

[0206] It has been shown that partially swollen beads containing reaction mix can swell further upon insult with analyte. The limited mass of each particle provides a limit to the total amount that can be absorbed. It has been shown that swollen beads containing reaction mix can be dried and retain activity when rewetted. Use of dry superabsorbent polymer particles containing dry reaction component(s) in the detection of analytes in vitro liquids has been demonstrated. Referring now to the Figures, Figure 1A shows a single sample chamber (reaction) chamber 20 with rigid side walls and no roof having a predetermined volume Vc. A multiple, here a known multiple, of pre-loaded SAP particles, here beads 30, are provided. SAP beads 30 have been prepared using the methods described above and in our co-pending patent applications GB2204051.3 and PCT / GB2023 / 05063, or may be prepared by other methods envisaged by those skilled in the art. Three SAP beads 30A, 30B, 30C are provided. In this case, these preloaded SAP beads are identical and have the same volume, and so the same expected volumetric capacity for absorption of liquid, and have been preloaded with the same quantities of at least one reaction component (e.g. typically comprising at least one dry transducing component for detecting analyte, and at least one dry signalling component responsive to the detection of analyte for signalling the presence of an analyte).

[0207] The internal walls of main sample chamber 20 may be provided with a hydrophilic coating up to a certain level e.g. height (not shown) and also with an inlet (not shown) and, optionally, an outlet (not shown) so that fluid may be received within sample chamber 20, up to a predetermined volume. For example, the overall volume of chamber 20 Vc may be larger than the anticipated usable volume Vc’ (sVc) within which fluid may be received.

[0208] Figure 1 B shows sample chamber 20 with sample fluid 22 of volume VF1within it. Liquid volume VF1is less than or equal to the total volume Vc of the sample chamber 20 and typically equals Vc’. Figure 1 B shows beads 30A, 30B, and 30C immediately after fluid application and before these have had an opportunity to swell.

[0209] Figure 1C shows apparatus 10 approximately 20 minutes later by which time preloaded SAP beads 30A, 30B, and 30C have swollen in size to volume VB3, VB4, and VB5. The volume of free fluid remaining within sample chamber 20 has now reduced to VF2. The sample volume before VF1equals the total volume after, that is VF2plus the increase in bead volume VB3+ VB4+ VB5in other words the overall level of liquid should not change.

[0210] Thus, in this example embodiment, the SAP beads are entirely immersed within sample liquid 22 and continue to be surrounded by sample liquid 22 for a period of time. The quantity of reaction components that the sample liquid encounters within the preloaded SAP beads is, in this example embodiment, substantially constant from one bead to the next, and furthermore the beads are essentially of the same initial size and density. Thus, it is expected that beads 30A, 30B, and 30C will increase in size by absorbing essentially the same amount of liquid as the other beads. Further, these are not prevented from swelling further by lack of sample liquid, or volumetric constraint of the main sample chamber to further swelling), the volume Vc of the main sample chamber 20 being larger, typically significantly larger, than the volume of fluid absorbed by beads 30. Where a colorimetric reaction regime is used, each of the beads 30A, 30B, and 30C should therefore change colour by corresponding amounts, in other words these change to the same degree or to the same depth of colour.

[0211] Figures 2A, 2B, 2C, and 2D show an alternative apparatus 10A, typically having a rigid housing within which are provided a plurality of sample chambers 20, here three main sample chambers 20-1 , 20-2, and 20-3. These are identical, having identical working volumes, provided by rigid walls and floors. In this case, each sample chamber is open but it need not be, a roof may be provided, preferably a rigid roof. Alternatively, the side walls of sample chambers 20-1 , 20-2, and 20-3 may each be provided with a hydrophilic coating to a certain level to define a sample volume within which sample liquid can be received from an inlet (not shown). Typically, the use of a hydrophilic coating on the side walls to a certain level facilitates, for relatively small sample chambers of microlitre or tens of microlitre size, a capillary stop that prevents further ingress of fluid once the height of the hydrophilic coating on the side walls has been reached. Thus, despite the absence of a roof, the anticipated volume of liquid receivable within main sample chambers 20 is predefined.

[0212] Whilst three main sample chambers 20 are provided and these are identical, a different number of preloaded SAP beads are provided in each one. In first sample chamber 20-1 , four beads 30-1 A, 30-1 B, 30-1 C, and 30-1 D are provided. In the second sample chamber 20-2, two SAP beads 30-2A and 30-2B are provided. In a third main sample chamber 20- 3, a single SAP bead 30-3A is provided. It will be understood that these are mere representative numbers and the exact number of SAP particles e.g. beads or quantity of SAP provided may vary significantly from this, these figures being for illustrative purposes only. For example, for a main sample chamber 20 (20-1 , 20-2 etc) being on a 10-100pl scale, 40, 20 or 10 SAP beads, respectively in each chamber, may be provided e.g. of the same (e.g. approximately) 1 l initial size when dry. In this way, a variety of components as in claim 1 and / or described herein can be combined in a variety of ways, e.g. in a kit, to develop differing sizes and types of biosensors.

[0213] Figure 2B shows biological sample of volume V1 being applied. Volume V1 is typically sufficient to exceed the combined volumetric capacity of all the sample chambers 20. Each sample chamber receives a quantity of sample, typically predefined either by the volumetric size of the sample chamber or by a hydrophilic coating or by other means, as understood by those skilled in the art. Thus, sample chamber 20-1 receives a volume F1of fluid, second sample chamber 20-2 receives VF2, and third sample chamber receives volume VF3. These volumes may be the same, but they need not be.

[0214] Thus, the ability to provide sensing particles in the form of preloaded SAP particles e.g. beads is particularly helpful in developing apparatus for sensing the same analyte in different manner and / or different analytes in the same or different manners. It is the combination of providing known quantities of reagent in the form of preloaded absorbent sensing material, here in the form of preloaded SAP particles, together with one or more, preferably two or more sample chambers of constant e.g. fixed and / or predetermined size, or rather constant volumetric capacity, and / or of fixed and / or predetermined size that enables the design of many different kinds of apparatus for biosensing, particularly microfluidic biosensing apparatus comprising microfluidic channels for directing fluid into and / or out of main sample chambers.

[0215] Figure 2C shows the SAP particles 30-1 A to 30-3A in sample chambers 20-1 , 20-2, and 20-3 after a period of time, typically a predetermined period of time, say 10 or 20 minutes.

[0216] Figure 2D shows an example embodiment in which a first method of detection is used with respect to a first sample chamber 20-1 , whereas first and second methods of detection are used with respect to second sample chamber 20-2, and a third method of detection is used with respect to a third sample chamber 20-3. Again, this indicates how flexible the arrangements can be made for designing biosensor apparatus and methods described here using the absorbent sensing material particularly in the form of individual preloaded SAP particles of known and, preferably, substantially the same size, and optionally of known size, in designing biosensing apparatus.

[0217] This is further illustrated in Figures 3A, 3B and 3C and Figures 4A, 4B and 4C in which schematic apparatus 10B, 10C and 10D are shown.

[0218] In Figure 3A, apparatus 10B comprises a rigid housing forming a sample chamber 20 having six wells each functioning as individual main sample chambers 20-1 , 20-2, 20-3, 20-4, 20-5, and 20-6. Each main sample chamber 20-1 , 20-2 etc. has respective sample volume Vc1, Vc2etc. These may the same or substantially the same as illustrated here in Figure 3A.

[0219] Figure 4A shows apparatus 10B after fluid has been applied to sample chamber 20 and, in particular, to individual main sample chambers 20-1 , 20-2 etc. Preferably and typically, apparatus 10 is designed so that sample fluid arrives at each main sample chamber at substantially the same time so that beads 30 within each are exposed to sample fluid 22 at substantially the same time. This need not be the case and embodiments can be envisaged in which the reactions in each cell start at different times.

[0220] Beads 30 typically have an initial volume of Vb when dry but, as shown in Figure 4A, these swell substantially, absorbing sample liquid, dissolving dry reaction components within these and signalling the presence and / or level and / or concentration of analyte. In the case shown in Figures 3A and 4A, the number of beads in each sample chamber is the same and the beads within each sample chamber are also of the same construction and therefore swell to the same amount, e.g. to final volume VB (e.g. within a given time period).

[0221] Figures 3B and 4B show apparatus 10C comprising a housing forming a sample chamber 20 with four equally spaced equally sized wells, here forming four quadrants of a circular sample chamber 20. A first main sample chamber 20-3 has a volume of VC1and three preloaded SAP particles. A second main sample chamber 20-2 has two linear side walls and one curved side wall and a volume of Vc2and is provided with 24 preloaded SAP particles, here identical preloaded SAP particles. A third sample chamber 20-3 has a volumetric capacity of Vc3and comprises six preloaded SAP particles identical to those in the other sample chambers. A fourth main sample chamber 20-4 has a volumetric capacity of Vc4and comprises one preloaded SAP particle.

[0222] As seen in Figure 4B, once sample fluid 22, in this case an equal amount of sample fluid 22, to each of the four main sample chambers 20-1 , 20-2, 20-3, and 20-4 is provided, beads 30 swell over a period of time, typically over 10, 20 or 30 minutes. Thus, it is preferable to wait up to 10, 20, or even 30 minutes before commencing a measurement step to allow time for the reaction components within the preloaded SAP particles to dissolve in the sample fluid and to initiate a signal, e.g. a change in colour and / or fluorescence, or other signal e.g. electrochemical, mechanical (e.g. due to change in mass or weight of the particles) etc.

[0223] Whilst SAP particles 30 were initially identical, in this case sufficient SAP particles were provided in sample chamber 20-2 that some were prevented from swelling due to the limitation of the volumetric capacity of main sample chamber 20-2. This might result in a mechanical pressure on an outer wall of main sample chamber 20-2 which could be used to initiate a signal or carry out an activity such as exert pressure on a contact etc.

[0224] In alternative embodiments other forms of signalling over different e.g. shorter timescales may be used. Turning to Figures 3C and 4C, here an apparatus 10D, having, when viewed from above, a sample chamber 20 having an outer wall of hexagonal shape. In this case, four differently sized sample chambers 20-1 , 20-2, 20-3, and 20-4 are provided, 20-1 and 20-3 having a triangular shape and 20-2 and 20-4 having a quadrilateral shape. The volumetric capacity of each sample chamber 20-1 , 20-2, 20-3, and 20-4 is Vc1, Vc2, Vc3, Vc4. It can be seen that the volumes of each are different, as are their shapes. In this case, upon application of sample fluid 22 to fill, e.g. to its respective volumetric capacity, each sample chamber, beads 30 swell. In the case of sample chamber 20-1 , beads 30 rearrange themselves to precisely occupy in an efficient way e.g. by hexagonal close packing the internal shape of main sample chamber 20-1. Occurrence of this rearrangement may be used to indicate a start time for a measurement step or indeed an end time.

[0225] It will be appreciated from the enclosed that there are many different types of sensors, biosensors, particularly microfluidic sensors such as microfluidic biosensors, that might make use of the technology disclosed herein and, in particular, the combination of absorbent material preloaded SAP particles and sample chambers of limited, typically predefined, preferably known, volumetric capacity. Thus, the invention provides methods and apparatus for (e.g. multiple) sample interrogation using a superabsorbent reagent carrier of fixed volume for fluid sample e.g. bodily fluid sample interrogation.

[0226] The invention is a method for (e.g. multiple) samples to be collected with bodily fluid from a source. The bodily fluid delivered to one or more main sample chambers and is absorbed into one or more than one superabsorbent polymer (SAP) (e.g. more than one type of preloaded SAP) which has been doped or bonded with a variety of reagents (e.g. the same or a different type or set of reagents within preloaded SAP provided in each of two or more main sample chambers). The result is a range of fixed volume reacting systems contained within at least one primary holding chamber (also referred to as a main sample chamber) which preferably has a fixed volume, which is ready for one or more methods of sample interrogation.

[0227] The bodily fluid is presented into a primary holding chamber where more than one reacting (preloaded) SAP bead is present. The biological fluid is absorbed into more than one reacting bead over a time duration where a reaction occurs. At least one type of method of interrogation can then occur within the primary holding chamber. The sample interrogation can be used within a variety of chemical analysis, including but not limited to electrochemical, photometric, spectrophotometry, resonator-based biosensing. One method may be used on each cell, or more than one. This means that multiple results can be obtained from the primary sample using multiple reacting SAPs, preferably each within its own main sample chamber (also known as a cell).

[0228] An embodiment where more than one type of reacting SAP bead, each of which preferably have the same fixed volume, can be provided within at least one primary collection chamber, or within multiple (e.g. respective) collection chambers or cells, for conducting at one or more methods of interrogation within a supra collection chamber.

[0229] The invention aims to solve and improve upon the methods of capturing a biological sample within a specific mechanical carrier (e.g. microfluidic or nanofluidic cell, paper-based assay / dipstick) while enabling more than one type of small biochemicals, pathogenic proteins, and disease-specific genes to be measured.

[0230] The invention improves previous techniques by allowing for diffusion of the specific sample that is under interrogation and allows for the act of absorption to mix the biological sample with the predefined reagent due to the limitations of sample volume within the SAP bead itself. Resulting in increased sensitivity in the reaction to occur.

[0231] In some embodiments, the present invention allows multiple measurements to be carried out within one main sample chamber or a series of main sample chambers within a main housing, forming a supra sample collection chamber.

[0232] The invention provides a method for manufacturing absorbent articles or test sensors and a method for carrying out detection of analyte using the absorbent articles or test sensors as shown in Figure 5. In step 10, initially dry SAP I SAP particles are pre-loaded with at least one wet reaction component, e.g. in solution, to cause the SAP particles to form swollen hydrogel particles.

[0233] In step 20, primarily water is driven off from the SAP I SAP particles by drying these e.g. by heat and / or freeze-drying e.g. until a desired residual water content is achieved.

[0234] In step 30, a predetermined quantity of preloaded dry SAP I SAP particles with at least one reaction component are added to a main sample chamber e.g. of fixed volume in a test sensor, during manufacture of same.

[0235] In step 40, the predetermined amount of dry pre-loaded SAP I SAP particles is exposed to sample liquid within one or more main sample chambers (also known as reaction chambers). By exposing this / these to a limited quantity of sample liquid, detecting and indicating the presence or concentration of the analyte is simplified, because the SAP I SAP particles are trapped within the main sample chamber(s). In step 50, the measurement that results in determination of the level of analyte commences and preferably also completes within a predetermined amount of time e.g. <30 mins, or <20 mins, or <10 mins, or <5 mins, or <3 mins from first application of sample liquid to the main sample chamber.

[0236] In step 60, the period of time over which the measurement takes place, e.g. after commencement, is preferably <60 secs, or <45 secs, or <30 secs, or <20 secs.

[0237] Thus, the use of dry superabsorbent polymer (SAP) I SAP particles containing dry reaction component(s), in other words dry modified (preloaded) SAP can be extended to other detection scenarios, and interrogation techniques for example those that require a limited sample chamber volume.

[0238] EXAMPLES

[0239] Figure 7A shows a reaction chamber (chamber 1) comprising a growth medium, four sample beads 32 to 38. E-coli bead 32 absorbs sample and bacteria and swells here changing colour typically once bacteria above a threshold amount and / or concentration is present (here from no colour to a green colour).

[0240] Figure 7B and 7C shows second and third chambers, typically identical to chamber 1 , except each also contains one or more specific antibiotic. The bacterial growth is exposed to an antibiotic, as and / or before it interacts with the reactant compenent(s) within the detection beads, giving once reacted, a different colour (here pink).

[0241] In one or more embodiments, the invention relates to an apparatus designed for the interrogation of sample liquids intended to detect one or more specific analytes within those liquids. The apparatus incorporates materials and / or modular components, in one or more embodiments, to provide precise, and / or rapid, and / or reliable detection of one or more analytes. Thus, one or more embodiments can be highly useful in medical diagnostics, and / or environmental monitoring, and / or other areas requiring analysis of fluids such as liquids.

[0242] Key Components:

[0243] 1 . Main Reaction Chamber (20): o Purpose: Acts as the primary containment area where the sample liquid interacts with the absorbent sensing material. Design: The chamber is of a predetermined volume to standardise the amount of sample processed, providing more consistent results. ent Sensing Material: Composition: comprising (e.g. consists of) a modified dry superabsorbent polymer (SAP) comprising (e.g. unmodified) SAP, and one or more reaction components, for absorbing the sample liquid and facilitating the interaction with reaction components. The dry absorbent sensing material may be referred to as modified SAP, the modified SAP comprising (or consisting (e.g. essentially) of) dry SAP and one or more reaction components. Integrated Reaction Component(s): Pre-loaded within the modified SAP, these component(s) are responsible for detecting and signalling the presence of specific analytes. The modified SAP material can include various percentages and / or types of one or more reaction components to meet different testing requirements. e Main Sample Chambers (Optional): Design: The apparatus may be configured with multiple sample chambers, each designed to be of the same or different volumes, and / or sizes, and / or shapes, and / or configurations depending on the testing needs. This feature allows for parallel testing and / or multiplexing, which can enhance throughput and efficiency. rement Modules: Function: Individual measurement modules, optionally separated, more, preferably in fluid flow connection with one another, may be provided to detect and / or determine one or more results, within testing apparatus e.g. within a single testing apparatus having a common (e.g. single) housing. These modules may be integrated into the apparatus (e.g. within the housing) to measure the signals generated within the reaction chamber(s) e.g. within respective reaction chamber(s). The device can include single or multiple measurement modules (which could be beads 32A-D) or separate sample chambers e.g. with respective beads e.g. 32A-D) to capture various signals, which could be indicative of different analytes or testing conditions.bsorbent Polymer (SAP): Variants: The SAP can be synthetic, and / or naturally-derived, and / or biodegradable, offering flexibility in environmental impact and application. The particles of SAP can be of specific sizes, and / or shapes to improve (e.g. optimise) the absorption and / or reaction dynamics. o Subsidiary Sample Chambers: Separate Modified SAP elements (e.g. particles) may each also serve as a secondary containment area within the main reaction chamber, holding a controlled amount of sample liquid for specific reaction pathways.

[0244] 6. Dry Reaction Components: o Types: The reaction components may include a variety of assays (e.g., electrochemical, colourimetric, photometric, spectrometric) and / or components capable of resonance detection. This versatility allows the apparatus to be tailored for detecting different analytes such as nitrates, ketones, glucose, or bilirubin.

[0245] Operation:

[0246] • Sample Introduction: The sample liquid is introduced into the main reaction chamber, where it is absorbed by the modified SAP (e.g. modified SAP particles).

[0247] • Reaction Process: The SAP rehydrates the pre-loaded dry reaction components, initiating the detection process.

[0248] • Signal Detection: The apparatus, e.g. the modified SAP particles and in particular the reaction component(s), detect(s) and signal(s) the presence of the analyte through the integrated measurement modules. The process is designed to be quick, with potential detection times as short as a few seconds.

[0249] Applications:

[0250] • Medical Diagnostics: In one or more embodiments, the present invention may be used for detecting biomarkers in bodily fluids, providing rapid and accurate diagnostics.

[0251] • Environmental Monitoring: In one or more embodiments, the present invention may be capable of detecting contaminants or analytes in water, soil, or air samples.

[0252] • Industrial Testing: In one or more embodiments, the present invention may be suitable for quality control in various industries where fluid analysis is crucial.

[0253] Kit Option:

[0254] The apparatus can also be offered as part of a kit, where the main reaction chamber is provided along with a separate packet containing the dry absorbent sensing material of one or more embodiments of the invention, allowing for user assembly or customisation before use. This high-level product description summarises the primary functions and components of the apparatus, highlighting its versatility and potential applications in various fields of fluid analysis.

[0255] The method of interrogation may be electrochemical and / or colorimetric etc.

[0256] In one or more embodiments, modified SAP (e.g. with one or more reaction components within it) and in particular modified SAP elements, e.g. particles, may be provided in bead form.

[0257] Example Product embodiments

[0258] Embodiment: Urinary Tract Infection (UTI) Diagnostic Apparatus with Antibiotic Susceptibility Testing

[0259] Overview:

[0260] Referring to Figures 7A to 7C, this embodiment describes a diagnostic apparatus designed to identify the presence of bacterial pathogens in a urine sample and simultaneously assess the bacteria's susceptibility to various antibiotics. The apparatus comprises multiple chambers, each containing reactive beads according to one or more embodiments of the invention tailored for specific functions — accelerating bacterial growth, detecting specific bacteria, and testing antibiotic effectiveness.

[0261] Key Components:

[0262] 1. Chamber 1 : Bacterial Growth Acceleration and Initial Detection o Function: A first chamber 20, here Chamber 1 is designed to accelerate bacterial growth and initiate detection. It contains multiple beads, each preloaded with a reactive component that promotes the rapid growth of bacteria within the urine sample. Each bead is also functionalized to detect a specific type of bacterium, allowing for a broad-spectrum analysis of the urine sample. o Reactive Component: The reactive component within Chamber 1 is (preferably) optimized for rapid bacterial growth, ensuring that even low concentrations of bacteria can be quickly identified. o Detection Mechanism: As the bacteria grow, they interact with the beads, leading to a detectable signal that indicates the presence of specific bacterial species. Chambers 2, 3 or 4, or 5 or more (here only 3 are shown): Antibiotic Susceptibility Testing o Function: One or more further chambers 20 ((e.g. sub-chambers) preferably fluidly connected together) may be provided e.g. here chambers 2 to 3 may be identical in structure but differ in the antibiotics integrated within each chamber. These chambers are configured to assess the susceptibility of the detected bacteria to different antibiotics. o Beads: Similar to Chamber 1 , each of these chambers contains beads (e.g. a predetermined number and / or amount (e.g. mass)) capable of detecting one or more specific bacteria. However, the reactive environment within these chambers is altered by the presence of one or more antibiotic(s). o Antibiotic Integration: One or more or each chamber may be preloaded with one or more specific antibiotic(s). As the urine sample moves through these chambers, the bacterial growth is exposed to the antibiotic environment before interacting with the detection beads. o Analysis: The response in each chamber allows for the determination of whether the bacteria are susceptible or resistant to the antibiotic present. A diminished or absent signal would indicate effective antibiotic action, while a strong signal would suggest resistance. Sample Flow and Reaction Process:

[0263] Option 1 o Sequential Flow: The urine sample is introduced into the apparatus and sequentially flows into Chambers 1 through 3. o Chamber 1 Reaction: The sample interacts with the reactive component(s), promoting bacterial growth and initiating detection via the beads. o Chamber 2-3 Antibiotic Interaction: In one or more each subsequent chamber, the sample is exposed to a specific antibiotic before being absorbed into the detection beads. Groups of one or more detection beads may be of different construction and / or components to one another, and / or to any beads comprising components promoting bacterial growth. o Final Detection: Each chamber may provide data on both the presence of bacteria and the effectiveness of the antibiotic present, allowing for a comprehensive analysis of the bacterial infection and potential treatment options.

[0264] Option 2 o Sample Introduction: The urine sample is introduced into the apparatus and first enters Chamber 1. o Chamber 1 Reaction: The sample interacts with the reactive component, promoting bacterial growth and initiating detection via the beads. o Sequential Flow: After processing in Chamber 1 , the sample sequentially flows into Chambers 2 through to 3 (or 4 or 5 if provided). o Antibiotic Interaction: In in one or more or each subsequent chamber, the sample is exposed to a specific antibiotic before being absorbed into the detection beads (which may be separate e.g. of different construction to each other and / or to any beads promoting bacteria growth). o Final Detection: One or more or each chamber provides data on preferably both the presence of bacteria and the effectiveness of the antibiotic present, allowing for a comprehensive analysis of the bacterial infection and potential treatment options.

[0265] Applications:

[0266] • Urinary Tract Infection (UTI) Diagnostics: The apparatus is particularly useful for diagnosing UTIs, providing not only the identification of bacterial pathogens but also critical information on which antibiotics would be most effective for treatment.

[0267] • Antibiotic Resistance Monitoring: This device is valuable in clinical settings where monitoring antibiotic resistance is crucial, helping to guide appropriate treatment decisions.

[0268] Advantages:

[0269] • Rapid Diagnosis: The integrated bacterial growth acceleration in Chamber 1 allows for faster detection, leading to quicker diagnosis and treatment.

[0270] • Comprehensive Testing: The inclusion of multiple antibiotics in separate chambers enables a broad assessment of antibiotic susceptibility, improving patient outcomes by identifying the most effective treatment.

[0271] • Compact Design: The multi-chambered design is efficient, allowing for streamlined sample processing in a single device.

[0272] This embodiment leverages the multi-chamber design to provide a robust solution for both the identification of bacterial infections and the assessment of antibiotic effectiveness, offering a valuable tool in the management of UTIs and similar conditions.

[0273] Embodiment: Diagnostic Apparatus for Detection of Sexually Transmitted Infections (STIs) in Menstrual Blood Overview:

[0274] This embodiment describes a diagnostic apparatus designed for the detection of sexually transmitted infections (STIs) in menstrual blood. The apparatus features multiple chambers, each containing one or more specialized beads and associated reactive components that allow for the identification of specific pathogens associated with STIs. The design leverages the unique properties of menstrual blood as a sample matrix to detect STIs with high sensitivity and specificity.

[0275] Key Components:

[0276] 1. Chamber 1 : Initial Sample Processing and Pathogen Detection o Function: A Chamber 1 , similar to Figure 7A, is designed to process the menstrual blood sample and initiate the detection of STIs. It contains one or more multiple beads, preferable each tailored to detect a specific pathogen associated with STIs, such as HIV, HPV, chlamydia, gonorrhoea, syphilis, and others. o Reactive Component: The chamber includes a reactive component that facilitates the breakdown of blood cells, releasing the target pathogens and their genetic material (e.g., DNA or RNA) into the chamber for subsequent detection. o Detection Mechanism: The beads within this chamber are functionalized with antibodies, nucleic acid probes, or other specific binding molecules that target and capture the pathogens or their biomarkers. The interaction leads to a detectable signal, indicating the presence of specific STIs.

[0277] 2. Chambers 2-3: Multiplexed Pathogen Identification o Function: One or more further chambers e.g. Chambers 2 to 3 (similar to Figures 7B and 7C) are designed to provide further validation and specificity for the detected pathogens. One or more or each further chamber focuses on confirming the presence of a particular STI by using a secondary detection method or targeting different biomarkers associated with the pathogen. o Beads: One or more or each further chamber contains beads similar to those in Chamber 1 but specialized for different aspects of pathogen detection, such as genetic material, surface antigens, or specific enzymes. o Enhanced Specificity: By using multiple chambers with different detection strategies, the apparatus ensures high specificity in STI detection, reducing the likelihood of false positives. o Sequential Detection: The menstrual blood sample moves sequentially through these chambers, allowing for comprehensive screening of multiple STIs in a single run.

[0278] 3. Sample Flow and Reaction Process: o Sample Introduction: The menstrual blood sample is introduced into the apparatus and first enters Chamber 1. o Chamber 1 Reaction: The sample interacts with the reactive component that breaks down blood cells, enabling the release and capture of STI pathogens or their markers by the detection beads. o Sequential Flow: After processing in Chamber 1 , the sample sequentially flows into Chambers 2 through 3 (or more). o Multiplexed Detection: Each subsequent chamber provides additional confirmation and specificity for different STIs, leveraging various detection mechanisms to ensure accurate results.

[0279] 4. Chamber 4, 5 or 6 (Optional): Confirmatory Testing and Cross-Validation o Function: A further chamber is optional and can be used for confirmatory testing or cross-validation of results obtained from the previous chambers. This chamber could use a different detection methodology, such as PCR (polymerase chain reaction) amplification of pathogen DNA or RNA, to confirm the presence of specific STIs. o Advanced Detection: By incorporating advanced molecular techniques, this chamber ensures the highest level of accuracy in detecting and confirming the presence of STIs.

[0280] Applications:

[0281] • Detection of STIs in Menstrual Blood: This apparatus is specifically designed for use with menstrual blood, providing a non-invasive method for the early detection of STIs in women.

[0282] • Multiplexed STI Screening: The multi-chamber design allows for the simultaneous screening of multiple STIs, offering a comprehensive diagnostic tool in one device.

[0283] Advantages:

[0284] • Non-lnvasive Sample Collection: Using menstrual blood as the sample matrix allows for a non-invasive, patient-friendly approach to STI detection, which can be self-collected, increasing accessibility and compliance. • High Sensitivity and Specificity: The use of multiple detection chambers ensures that even low concentrations of pathogens are detected, while the sequential testing reduces the likelihood of false positives.

[0285] • Broad Coverage: The device is capable of detecting a wide range of STIs, providing a thorough assessment of the patient's health status.

[0286] • Compact and Efficient: The multi-chambered apparatus is designed for streamlined processing of the menstrual blood sample, ensuring quick and reliable results.

[0287] This embodiment represents an innovative approach to STI detection, leveraging the unique properties of menstrual blood and a multi-chamber design to provide a comprehensive, non-invasive diagnostic tool.

[0288] Embodiment: Diagnostic Apparatus for Detection and Quantification of a Specific Virus

[0289] Overview:

[0290] This embodiment describes a diagnostic apparatus specifically designed to detect and quantify a particular virus in a biological sample, such as blood, saliva, or nasal swab fluid. The apparatus consists of multiple chambers, each tailored to perform specific functions — initial viral capture, amplification of viral markers, and precise quantification of viral load.

[0291] Key Components:

[0292] 1 . Chamber 1 : Viral Capture and Initial Detection o Function: Chamber 1 is designed to capture and initiate the detection of the target virus from the biological sample. It contains beads coated with antibodies or nucleic acid probes that specifically bind to the virus or its genetic material. o Reactive Component: The chamber includes a reactive medium that facilitates the lysis of cells (if present), releasing viral particles or viral RNA / DNA into the chamber for capture by the beads. o Detection Mechanism: Once the virus binds to the beads, a detectable signal is generated, indicating the presence of the virus in the sample. This signal may be fluorescence, chemiluminescence, or another easily measurable marker.

[0293] 2. Chamber 2: Viral Amplification Function: Chamber 2 is designed to amplify the viral markers to enhance detection sensitivity. This chamber uses nucleic acid amplification techniques, such as PCR (polymerase chain reaction) or isothermal amplification, to increase the quantity of viral RNA / DNA. Amplification Process: The viral genetic material captured in Chamber 1 is transferred to Chamber 2, where it undergoes exponential amplification. The amplification process is carefully controlled to ensure that it reflects the initial viral load, maintaining accuracy in quantification. Intermediate Detection: An intermediate detection system monitors the amplification process, providing real-time data on the progress and ensuring that the amplification is proceeding as expected. er 3: Quantification and Measurement Function: Chamber 3 is designed for the precise quantification of the viral load in the sample. This chamber contains beads or sensors containing initially dry absorbent sensing material according to the invention specifically calibrated to measure the amount of amplified viral genetic material or viral proteins. Quantitative Analysis: The chamber uses a quantitative detection method, such as real-time PCR (qPCR) or ELISA (enzyme-linked immunosorbent assay), to determine the exact concentration of the virus in the sample. The results are processed to provide an indication, optionally, numerical value for the viral load. Calibration and Standards: The chamber may include internal standards or calibration curves to ensure that the quantification is accurate and reliable. er 4 (Optional): Validation and Cross-Checking Function: Chamber 4 serves as an optional validation step, where the quantification results from Chamber 3 are cross-checked using a different detection method. This redundancy ensures the accuracy and reliability of the viral load measurement. Alternative Detection: This chamber might use a different assay type (e.g., digital PCR or a different type of immunoassay) to validate the results, further increasing confidence in the measurements. Flow and Reaction Process: o Sample Introduction: The biological sample is introduced into the apparatus and first enters Chamber 1 for viral capture. o Sequential Processing: After the initial detection in Chamber 1 , the sample moves to Chamber 2 for amplification, followed by Chamber 3 for quantification. o Data Output: The apparatus provides a final quantitative measurement of the viral load, displayed on a digital interface or transmitted to a connected device for further analysis.

[0294] Applications:

[0295] • Viral Load Monitoring: The apparatus is ideal for monitoring the viral load in patients, particularly in the context of chronic viral infections like HIV, hepatitis B or C, or COVID-19, where quantification of the virus is critical for disease management.

[0296] • Diagnostic Testing: The device can be used in clinical settings to diagnose acute viral infections and determine the severity of the infection based on the viral load.

[0297] • Epidemiological Studies: Useful in research settings to study viral dynamics in populations, providing data on viral prevalence and transmission rates.

[0298] Advantages:

[0299] • High Sensitivity: The use of amplification techniques in Chamber 2 can enable even low levels of viral RNA / DNA to be detected and quantified, making the apparatus highly sensitive.

[0300] • Specificity: The beads in Chamber 1 are designed to specifically capture the target virus, reducing the likelihood of cross-reactivity and false positives.

[0301] • Accurate Quantification: The quantitative methods used in Chamber 3 provide precise measurements of viral load, crucial for monitoring disease progression and treatment efficacy.

[0302] • Integrated Process: The multi-chamber design streamlines the entire diagnostic process from detection to quantification, reducing the need for multiple devices and handling steps.

[0303] This embodiment offers a comprehensive solution for detecting and measuring the viral load of a specific virus, providing both diagnostic and monitoring capabilities in a single, compact apparatus.

[0304] The present inventors have appreciated a simple solution for indicating or monitoring a disease state using a body fluid or, more generally, for indicating the presence of an analyte in a sample liquid. In one or more embodiments, the present invention does not use a separate sensor. In one or more embodiments, the present invention does not (initially at least) use hydrogel (in the sense of wet SAP). Where SAP particles such as beads are used, their shape may be spherical, cylindrical, irregular, regular, cubic, flakes, prismatic, fibre-like, hollow, or any 2 or 3-dimensional structure. In one or more embodiments, SAP particles are positioned within a rigid sample chamber which has a window e.g. a clear, preferably colourless, panel for observing the SAP particles (e.g. before and / or during and / or after insult with a sample liquid).

[0305] The residual moisture content in preloaded dry SAP for use in one or more embodiments of the invention is typically 0.5% to 15% by weight, or 1 % to 10% by weight, or less than 15% by weight or less than 10% by weight or less than 5% by weight. The residual moisture content may be determined by EDANA (European Disposables and Non-Woven Association) recommended test method No. NWSP 230.0. R2 ‘Moisture Content’.

[0306] The SAP particle size may be determined by means of EDANA (European Disposables and Non-Woven Association) recommended test method No. NWSP 220.0.R2 ‘Particle Size Distribution’. The swelling capacity of the SAP e.g. of particles may be determined by EDANA recommended test method No. NSWP 240.02. R2 ‘Free Swell Capacity’. The fluid retention capacity may be determined by the EDANA recommended test method No. NWSP 241.0.R2. Other parameters may be determined from the EDANA test methods or adapted test methods if required e.g. swelling rate. Other recognised standard test methods may be used.

[0307] The art discusses how to make SAP beads but not how to use SAP beads in practical, commercial methods for analyte detection in body liquids or, indeed, in other liquids. The use of e.g. a fixed quantity of SAP beads as subsidiary sample chambers in combination with one or more main sample chambers has not previously been discussed in the art.

[0308] In some embodiments, a separate fluid compartment may be provided for absorbent material, separate from the one or main sample chambers containing preloaded SAP I SAP particles. Different main sample chambers may be provided with different sensing capabilities e.g. different reagent components within SAP or SAP particles for different assays (e.g. urine and ketone) or of different sensitivities, so a higher concentration of analyte is required before a signalling component (e.g. a colour change is seen). It will be understood by those skilled in the art that the apparatus of the invention may be used as a test strip or other sensor technologies for interrogation by visual and / or other methods. It will be further clear to those skilled in the art that the invention is not limited to sensing elements which are based on colorimetric detection. The SAP particles are of limited volume and solve the problem of requiring a limited volume in a different way to that of the main sample chamber - the internal volume or rather swelling capacity of each SAP bead limits the volume of sample that can be interrogated in each particle. Whilst use of dry SAP particles is known, its use as a medium for interrogating sample liquids, particularly for interrogating sample liquids within sample chambers in sensor technologies, is not known. This ability to combine the use of SAP as an absorbent and as a carrier for one or more dry detection and signalling components, facilitates very simple use and many different embodiments when combined with one or multiple main sample chamber(s) of predetermined volume. Typically, the signalling component is observable by eye, although other measurable or observable regimes can be used.

[0309] The invention may be applied to other analytes besides glucose and other disease states besides diabetes. Staying with diabetes as an example, a similar dual enzyme detection method may be used to detect ketones in urine, indicative of diabetic ketoacidosis. Broadening to other disease states, protein in urine is indicative of kidney inflammation, nitrite and white blood cells are indicative of bacterial infection, bilirubin and urobilinogen are indicative of liver damage. Each analyte may be detected in a manner analogous to that described for glucose above. Multiple analytes may be detected simultaneously by combining a series of main sample chambers two or more or each with different analyte detection capabilities in its SAP or SAP particles within it.

[0310] It will also be clear to those skilled in the art that the invention is not limited to the detection of analytes in urine. It is appropriate to any biofluid, for example it could also be applied in a transdermal patch in which interstitial fluid is drawn into the fluid capture system, or indeed a dry tissue for blowing a nose or wiping away tears, or a nasal or throat swab for sampling nasal or throat fluids.

[0311] In one or more embodiments, the present invention at least partially addresses one or more problems of the art: variability of concentration of analyte in sample liquid, and / or a variable flow, e.g. of urine, and / or a variable contact time do not render the test invalid; additional insults of bodily fluid do not render the test invalid; has less effect on the element;

[0312] SAP e.g. SAP particles do not break, they also relatively cheap; dry SAP sensing systems are stable over the time periods for long term storage required for commercial products; a controlled sample volume of one or more main sample chambers be more easily manufactured. In one or more embodiments, the invention provides apparatus with one or more main sample chambers containing dry preloaded superabsorbent polymer, at least one reaction component within the SAP for detecting and signalling the presence of analyte, and / or, in some embodiments, for detecting and signalling the concentration, e.g. the relative level of concentration of analyte, and / or, in some embodiments, for detecting and signalling the relative quantity of analyte; wherein upon insult of sample liquid, the dry super absorbent polymer absorbs sample liquid, the dry reaction component, dissolves, preferably within the SAP, and if analyte is present in the sample liquid, its presence is detected and signalled within the SAP itself, within its respective main sample chamber.

[0313] 10, 10A, 10B, 10C, 10D apparatus

[0314] 20, 20-1 , 20-2, 20-3 main sample (reaction) chamber

[0315] 30, 30A, 30B, 30C SAP particles (e.g. beads) typically individual SAP particles

[0316] 30-1 A, 30-1 B, 30-2A etc.

[0317] N no. of SAP particles, N1 (in chamber 1), N2 (in chamber 2 etc)

[0318] Vc, Vc1, Vc2volume of sample chamber Vcor subchamber (Vc1, Vc2etc.)

[0319] Vf volume of fluid, Vf1initially, Vf2later e.g. post-absorption v6volume of particle (e.g. bead) when dry

[0320] VB volume of particle (e.g. bead) when swollen e.g. fully swollen

[0321] (VB-Vb) x N = Vf2- Vf1

[0322] 32 - unreacted (initially dry) Escherichia coli particle e.g. bead 32A - reacted (now swollen) Escherichia coli particle e.g. bead 32A-0 - swollen unreacted e-coli bead - control clear colour 32A-1 - reacted swollen e-coli bead - dark yellow - e-coli detected 32A-2 - reacted swollen e-coli bead - dark yellow - e-coli detected 34 - klebsiella pneumoniae (initially dry) particle e.g. bead

[0323] 36 - proteus mirabilis particle e.g. bead

[0324] 38 - staphylococcus saprophyticus particle e.g. bead

[0325] 50 - test vial

[0326] 52 - sample liquid control

[0327] 60 - sample bottle

[0328] 61 - cap

[0329] 62 - beads (or windows through which beads can be seen) in cap

Claims

Claims1 . Apparatus (10) for interrogation of sample liquid to detect analyte, comprising: at least one main chamber (20) of predetermined volume; and, within the main reaction chamber (20), a predetermined amount of dry, absorbent, sensing material comprising (e.g. modified) dry superabsorbent polymer (SAP); and, within the (e.g. modified) dry superabsorbent polymer, dry SAP and at least one dry reaction component for detecting and for signalling the presence of analyte.

2. Apparatus according to claim 1 in which the predetermined amount of absorbent sensing material comprises a predetermined amount of (e.g. modified) super absorbent polymer (SAP).

3. Apparatus according to claim 2 in which the predetermined amount of (e.g. modified) superabsorbent polymer with at least one dry reaction component within it comprises a predetermined amount of superabsorbent polymer.

4. Apparatus according to any of claims 1 to 3 in which the predetermined amount of absorbent sensing material comprises a predetermined amount of at least one reaction component.

5. Apparatus according to any preceding claim in which a plurality of main sample chambers (20) are provided.

6. Apparatus according to claim 4 in which the main sample chambers (20) have the same volume; and / or in which the main sample chambers (20) have the same size and / or shape and / or configuration as each other.

7. Apparatus according to any of claims 5 to 6 in which the same predetermined amount of the dry, absorbent, sensing material is provided in each main sample chamber.

8. Apparatus according to any preceding claim in which one or more or all of the at least one main sample chamber is or are provided with one or more rigid walls.

9. Apparatus according to any preceding claim comprising: at least one first measurement module for measuring a signal from the at least one main reaction chamber.

10. Apparatus according to claim 9 comprising: a first and a second measurement modules for measuring a first and / or second signal from at least one main reaction chamber.

11. Apparatus according to any preceding claim in which the absorbent sensing material comprises at least 50%, or at least 75%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% by weight of dry superabsorbent polymer comprising the at least one dry reaction component.

12. Apparatus according to any preceding claim in which the dry superabsorbent polymer material comprises at least 95%, or at least 97%, or at least 98%, or at least 99% by weight of dry superabsorbent polymer, and up to 5%, or up to 3%, or up to 2%, or up to 1 % by weight of the at least one dry reaction component(s).

13. An absorbent sensing material according to any preceding claim in which the dry superabsorbent polymer provides at least one subsidiary sample chamber.

14. Apparatus according to any preceding claim in which the dry superabsorbent polymer comprises particles.

15. Apparatus according to claim 14 in which the dry particles are of similar or same mass and / or size and / or shape.

16. Apparatus according to claim 14 or 15 in which at least 90%, or at least 95%, or at least 98%, or at least 99% by weight of the particles are within <±10% or <± 5% or <± 2% of the average dry weight of the particles.

17. Apparatus according to claim 16 in which, when dry, the particles have a main dimension of: <100 nanometres, or 100 to 1000 nanometres, or 100 to 5000nanometres, or 5 to 1000 microns, or 5 to 500 microns, or 5 to 250 microns, or < 500 microns, or <0.1 mm, or < 0.05 mm, or <0.01 mm or <0.005 mm or 0.5 to 5 mm, or from 1 mm to 4 mm, or from 0.5 mm to 3 mm, or from 0.5 mm to 2 mm, or from 1 mm to 2 mm.

18. Apparatus according to any preceding claim in which the at least one dry reaction component comprises at least one dry transducing component for detecting an analyte; and, at least one dry signalling component responsive to the detection of analyte for signalling the presence of analyte.

19. Apparatus according to claim 18 in which the at least one reaction component comprises at least one of an electrochemical assay; a colorimetric assay; a nitrate assay; a ketone assay; a glucose assay; a bilirubin assay; a component capable of photometric detection; a component capable of spectrometric detection; a component capable of resonance.

20. Apparatus according to any preceding claim in which the superabsorbent polymer is synthetic superabsorbent polymer, a naturally-derived superabsorbent polymer, a biodegradable superabsorbent polymer.

21. An absorbent sensing material according to claim 19 in which the superabsorbent polymer comprises at least 50%, or at least 75%, or at least 90%, or at least 95% by weight of at least one of: polymerised acrylic acid or salts thereof or the like, sodium polyacrylate, polyacrylamide copolymer, ethylene maleic anhydride copolymer, crosslinked carboxymethylcellulose, polyvinyl alcohol copolymers, cross-linked polyethylene oxide, and starch grafted copolymer of polyacrylonitrile, e.g. at least 50%, or at least 75%, or at least 90% by weight.

22. Apparatus according to any of claims 1 to 21 in which at least two groups of SAP particles are provided, at least one group configured to be indicative of a level of a first analyte, and a second group configured to be indicative of a level of a second analyte.

23. Apparatus according to claim 22 in which the first group are provided in a first main sample chamber and the second are provided in a second main sample chamber.

24. Apparatus according to any preceding claim in which the superabsorbent polymer is used as a subsidiary sample chamber provided with a predetermined amount of the at least one reaction component.

25. Apparatus according to any preceding claim in which the superabsorbent polymer is used as a subsidiary sample chamber to absorb a predetermined amount of sample liquid in use.

26. Apparatus according to any preceding claim, in which the apparatus is configured for the detection of one or more bacteria and / or virus(es).

27. Apparatus according to any preceding claim in which the apparatus is configured for promoting and / or resisting growth of bacteria and / or virus(es).

28. Apparatus according to claim 27 in which the dry absorbent material comprises a growth medium and / or antimicrobial component and / or anti-viral component for bacteria and / or virus(es).

29. Apparatus according to any preceding claim in which the at least one reaction component comprises one or more antibiotic(s) and / or antiviral component(s).

30. A method of manufacturing an apparatus according to any of claims 1 to 25, comprising: a. preparing SAP; b. drying SAP particles; c. in the absence of an analyte, introducing at least one wet reaction component for detecting and signalling the presence of the analyte, to the dry SAP; d. drying the SAP to form dry SAP pre-loaded with the at least one dry reaction component for detecting and signalling the presence of an analyte.

31. A method according to claim 30, comprising:adding a predetermined amount of dry, preloaded SAP to at least one main sample chamber of predetermined and / or fixed volume.

32. A process for detecting analyte in a fluid (e.g. in vitro) comprising: a) providing apparatus according to any of claims 1 to 24; b) introducing sample to the one or more main sample chambers, optionally at the same time; c) exposing a predetermined amount of (e.g. modified) SAP to sample liquid, optionally a predetermined amount of sample liquid; d) rehydrating the at least one reaction component to detect and signal the presence and / or concentration of analyte in the sample liquid; e) detecting and signalling the presence of analyte in the sample liquid (e.g. from within the SAP).

33. A process according to claim 32 comprising carrying out a step of detecting and signalling the presence of analyte within <20 mins, or <15 mins, or <10 mins, or <5 mins, or <3 mins from initial exposure to sample liquid.

34. A process according to claim 32 or 33 in which the step of detecting and signalling takes <1 min, or <30 sec, or <20 sec, or <10 sec.

35. A kit comprising a precursor apparatus for interrogation of sample liquid to detect analyte, the precursor apparatus comprising: at least one main chamber (20) of predetermined volume; and the kit further comprising, for placing within the main reaction chamber (20), a predetermined amount of dry, absorbent, sensing material comprising (e.g. modified) dry superabsorbent polymer (SAP); and, within the dry superabsorbent polymer, at least one dry reaction component for detecting and for signalling the presence of analyte.

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