Test kit for determining analytes in a sample of a bodily fluid

Abstract

A test kit (110), a test strip (112) and a method for determining analytes in a sample of a bodily fluid (111) are disclosed. The test kit (110) comprises: i) at least one test strip (112) comprising at least one membrane (114), wherein the membrane (114) comprises at least one first test field (116), the first test field (116) comprising at least one first capture component (118), wherein the membrane (114) further comprises at least one second test field (120), the second test field (120) comprising at least one second capture component (122), wherein the second capture component (122) is different from the first capture component (118); and ii) at least one first analyte detection system (124) for contacting with the sample of the bodily fluid (111), wherein the first analyte detection system (124) comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid (111), wherein the first analyte detection system (124) further comprises at least one first capture reagent; wherein the first capture component (118) is configured for capturing the first capture reagent of the first analyte detection system (124), wherein the second capture component (122) is configured for capturing at least one second capture reagent (136) from at least one second analyte detection system (126), the second capture reagent (136) being different from the first capture reagent.

Description

[0001] Test kit for determining analytes in a sample of a bodily fluid

[0002] Technical Field

[0003] The present application refers to a test kit for determining analytes in a sample of a bodily fluid, a test strip for use in the test kit and a method for determining analytes in a sample of a bodily fluid. The devices and the method according to the present invention may be used in medical diagnostics, in order to qualitatively or quantitatively detect one or more analytes in one or more samples of bodily fluids. Other fields of application of the present invention, however, are feasible.

[0004] Background art

[0005] In the field of medical diagnostics, in many cases, one or more analytes have to be detected in samples of a bodily fluid, such as in a sample of blood, blood plasma, interstitial fluid, urine, saliva or other types of bodily fluids. Examples of analytes to be detected comprise, but are not limited to, ions, specifically mineral ions, peptides, antibodies, DNA, electrolytes and / or metabolic compounds, such as creatinine, glucose, triglycerides, lactate, cholesterol or other types of analytes typically present in these bodily fluids. According to the concentration and / or the presence of the analyte, an appropriate treatment may be chosen, if necessary.

[0006] Various types of detectors and / or test elements are known which may specifically be configured for detecting analytes. As an example, optode sensors are used to optically determine the electrolyte concentration in samples of bodily fluids. US 7,651,858 B2 discloses methods of using ion-detecting microspheres containing an ion- phore and a chromoionphore in clinical laboratory instrumentation such as flow cytometry for sample analysis. In one embodiment, the microspheres are contacted with a flowing stream of a sample under conditions that allow the ion-selective ionophores to complex with the ions in the sample, and to cause deprotonation of the chromoionophore. The complexes are then exposed to an excitation wavelength light source suitable for exciting the deprotonated chromoionophore to emit a fluorescence signal pattern. Detection of the fluorescence signal pattern emitted by the deprotonated chromoionophore in microspheres containing the complexes allows for determination of the presence of the target ions in the sample. In one embodiment, lead ion-detecting microspheres are provided that can detect nanomolar levels of lead ions with response times on the order of minutes.

[0007] US 7,208,121 B2 discloses a plasticizer-free ion detective sensor for detecting a target ion in a sample. The sensor comprises a copolymer of methacrylate monomers with pendant alkyl groups of different length, and an ionophore for detecting the target ion. The copolymer matrix may be in a form of membrane or particles. The sensors may be Carrier-based ion- selective electrodes (ISEs) or optodes such as thin film ion-specific optodes or particle-based optodes. The ionophore may be a target ionophore selective for a target ion H+, Li+, Na+, K+, Ca2+, or Mg2+. The ion detective sensor may further include an ion exchanger such as halogenated carboranes. Further described is an ion detective sensor comprising halogenated carboranes as ion exchangers. Particularly, trimethylammonium-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 undecabromocarborane (TMAUBC) is used as ion exchangers.

[0008] US 7,201,876 B2 discloses ion-detecting sensors for detecting a target ion in a sample are provided. The sensor comprises a plasticizer-free copolymer comprised of polymerized units of methacrylate monomers having pendent alkyl groups of different length and a functionalized ionophore of said ion, wherein at least a portion of the functionalized ionophore is grafted into the copolymer through covalent linkages. Sensors may comprise ionophores such as hydrophilic crown ethers or functionalized derivative of 3-oxapentandiaminde-type ionophores. Further described are sensors for detecting target ions in a sample, comprising plasticizer-free molecularly imprinted polymers, wherein the polymers comprise polymerized units of methacrylate monomers having pendent alkyl groups of different length and a functionalized ionophore of said ion. In particular, a magnesium ion sensor comprising a functionalized derivative of a 3-oxapentandiaminde-type calcium ion-selective ionophore is described. The sensors include carrier-based ion-selective electrodes or optodes such as thin film ion-specific optodes, particle-based optodes, or bulk optodes. WO 98 / 03497 Al discloses compounds, constructed in modular manner, of formula

[0009] I — Roi — T — Ro 2 — F wherein I is the monovalent residue of an ionophore, F is the monovalent residue of a fluor- ophore, G is a functional group, Tis a trivalent organic radical and Roi, R02 and R03 are each independently of the others a direct bond or a bridging group. The compounds may be used in immobilised form as active components in polymer membranes of optical sensors for the detection of ions. The sensors are distinguished by a long usable life and a high degree of sensitivity.

[0010] WO 99 / 27371 discloses a pipettable detector comprising an aqueous suspension of particles which are essentially insoluble in a sample of a body fluid containing at least one target ion analyte of interest, the particles having uniformly distributed therein target and indicator ionophores which may be present in the same molecule or separate molecules. The indicator ionophore is capable of giving rise to a detectable signal following complexation of the target ionophore with the target ion from the sample. In a method of detecting an ionic analyte of interest in the sample of a bodily fluid, the sample is contacted with the detector. The indicator ionophore gives rise to a detectable signal following complexation of the target ionophore with the target ion from the sample, and the signal is then detected.

[0011] US 2008 / 0044879 Al discloses systems and methods for optically measuring ion concentrations in biological samples. The systems and methods employ polymer-based optical ion sensors that include ion-selective ionophores and a pH sensitive chromionophore. Electrodes are providing for electrically stimulating the biological samples.

[0012] WO 03 / 042698 Al discloses a reagent mixture, a method and an apparatus, for carrying out simultaneous, automated analysis of multiple analytes in a test sample, particularly a bodily fluid. The reagent mixture, the method and the apparatus are relevant to the field of general clinical chemistry, but may find application in other fields of use.

[0013] Wang L. et al. describe in “Simplified Fabrication for Ion-Selective Optical Emulsion Sensor with Hydrophobic Solvatochromic Dye Transducer: A Cautionary Tale". Anal. Chem., 2019, 91(14), 8973-8978, functionalized polystyrene microbeads, individually isolated by flow cytometry, exhibiting unexpectedly poor fluorescent properties and that the sensor response is instead attributed to the supernatant. A more thorough study reveals that such optical microemulsion sensors can be made operationally functional and chemically selective, seemingly in the absence of any solvent matrix or added surfactant. Instead, it is shown that residual THF used in the fabrication of the emulsified sensors may solubilize the sensing components and give a functional optode response. To evaluate this further, the number of sensing components was stepwise simplified to assess their need. Variation of residual THF levels has no effect on the ion optode response when plasticizer is present, in support of established results. Lipophilic solvatochromic dye transducers are also shown not to require an added surfactant as their nature already endows the emulsified sensors with a stabilizing ionic surface charge. The ionophores are shown to exhibit much larger stability constants in the surfactant-free formulations than surfactant-based ones (valionomycin, log P > 9.2 compared to 6.1; Na+-ionophore X, 6.7 vs 4.7), which is attributed to a less polar solvent environment for the ionophore. Potassium-, sodium-, and calcium-selective sensors were used as model systems in this study.

[0014] Xiaojiang Xie et al. describe in “Potassium-selective optical microsensors based on surface modified polystyrene microspheres'’', Chem. Commun., 2014,50, 4592-4595, ion-selective microspheres based on surface modification of polystyrene particles (0.8 and 2.4 pm, diameter). The lipophilic sensing components of K+selective optodes (chromoionophore, ionexchanger and ionophore) are adsorbed on the surface of the polystyrene particles using a mixed solvent method. The resulting microparticles respond to K+in an exhaustive sensing mode with selectivity and a response time of t95% = 5 s.

[0015] Lookadoo, D. B. et al. describe in “Paper-Based Optode Devices (PODs) for Selective Quantification of Potassium in Biological Fluids", Anal. Chem. 2021, 93, 9383-9389, paperbased optode devices (PODs) for sensing potassium selectively in biological fluids. PODs operate in exhaustive mode and integrate with a handheld, smartphone-connected optical reader. This integrated measuring system provides a linear optical response to potassium concentration via a stackable design and provides a user interface by harnessing a smartphone.

[0016] Mistlberger G. et al. provide in “Ionophore-Based Optical Sensors", Annu. Rev. Anal. Chem. 2014. 7:483-512, an overview of the key aspects of designing ionophore-based optical sensors (IB OS). Despite the advantages achieved by known method and devices, several technical challenges remain. When using optode sensors in a lateral flow assay, the signal is usually developed along the whole membrane, specifically avoiding using the same membrane to develop other assays and / or to include internal controls in the membrane. Thus, there is a need to at least partially avoid these technical challenges.

[0017] Problem to be solved

[0018] It is therefore desirable to provide devices and method which at least partially address aboveidentified technical challenges. Specifically, a test kit for determining analytes in a sample of a bodily fluid, a test strip for use in the test kit and a method for determining analytes in a sample of a bodily fluid shall be proposed which allow combining membrane-based optode sensors with further and / or other sensing mechanisms.

[0019] Summary

[0020] This problem is addressed by a test kit for determining analytes in a sample of a bodily fluid, by a test strip for use in the test kit and by a method for determining analytes in a sample of a bodily fluid, with the features of the independent claims. Advantageous embodiments which might be realized in an isolated fashion or in any arbitrary combinations are listed in the dependent claims as well as throughout the specification.

[0021] As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements.

[0022] Further, it shall be noted that the terms “at least one”, “one or more” or similar expressions indicating that a feature or element may be present once or more than once typically will be used only once when introducing the respective feature or element. In the following, in most cases, when referring to the respective feature or element, the expressions “at least one” or “one or more” will not be repeated, non-withstanding the fact that the respective feature or element may be present once or more than once.

[0023] Further, as used in the following, the terms "preferably", "more preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting alternative possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be optional features, without any restriction regarding alternative embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

[0024] In a first aspect of the present invention, a test kit for determining analytes in a sample of a bodily fluid is disclosed.

[0025] The term “test kit” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an assembly of a plurality of components, wherein the components each may function and may be handled independently from each other, wherein the components of the kit may interact to perform a common function. The components of the test kit may specifically interact with each other to perform at least one measurement, such as for determining a presence and / or an absence of analytes in the sample of the bodily fluid. The test kit may be configured for determining at least two analytes in the sample of the bodily fluid, specifically at least two different analytes, as will be outlined in further detail below. Typically, the components of the test kit are provided in separate containers or within a single container. The container also typically comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code which is capable of carrying out or supports the determination of the analyte referred to in the methods of the present invention when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device or may be provided in a download format such as a link to an accessible server or cloud. Moreover, the test kit may, usually, comprise analyte solutions with stand- ardized amounts for calibration or validation or other reference amounts. The test kit according to the present invention may also comprise further components which are necessary for carrying out the method of the invention such as washing solutions, solvents, and / or reagents required for detection of the detectable label. Further, it may comprise the test strip of the invention either in parts or in its entirety.

[0026] The term “analyte” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an arbitrary chemical or biological substance or species, such as a molecule or a chemical compound. As an example, the at least one analyte may be a chemical compound which takes part in metabolism. The analyte may be or may comprise one or more of ions, specifically mineral ions, peptides, antibodies, DNA, electrolytes and / or metabolic compounds, such as creatinine, glucose, triglycerides, lactate, cholesterol or other types of analytes typically present in these bodily fluids, as will be outlined in further detail below. The analyte e.g. a first and / or further analyte, is suspected to be present in a sample of a bodily fluid. Typically, the analyte, e.g. a first and / or further analyte, may be present in the sample of the bodily fluid.

[0027] The term “determining analytes” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to a quantitative and / or qualitative detection of at least one analyte in an arbitrary sample, more specifically to a quantitative and / or qualitative detection of more than one analyte in an arbitrary sample. The determining of analytes in the sample of the bodily fluid may be or may comprise at least one analytical measurement. The result of the analytical measurement, as an example, may be the presence or absence of the analyte, or of the analytes, to be determined. Alternatively or additionally, the result of the analytical measurement may be a concentration of the analyte, or of the analytes, in the sample of the bodily fluid, specifically a detection of the concentration of the analyte, or of the analytes, in particular to be within the sensitivity of the analyte detection system.

[0028] The term “bodily fluid” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an arbitrary liquid bodily fluid which is present in a body tissue of a human or animal. More specifically, the term may refer to all bodily fluids known to comprise or suspected to comprise an analyte to be determined in line with the present invention, including interstitial fluid, blood, plasma, lacrimal fluid, urine, lymph, cerebrospinal fluid, bile, stool, sweat, and saliva. Typically, the bodily fluid may be or may comprise one or more of blood, interstitial fluid, urine, saliva or the like. More typically, the body fluid is a bodily fluid which is present in a body tissue of a subject, such as in the interstitial tissue.

[0029] The term “sample of a bodily fluid”, also referred to as “sample”, as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an arbitrary aliquot part or aliquant part of a biological fluid which directly is a bodily fluid as defined above or which is derived from a bodily fluid, such as by one or more pre-processing steps, e.g. by centrifugation. The sample of the bodily fluid may comprise one or more analytes of interest. For example, the sample may be a sample of a bodily fluid selected from the group consisting of: a physiological fluid, including blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells or the like. The sample may be used directly as obtained from the respective source or may be subject of a pretreatment and / or sample preparation workflow. For example, the sample may be pretreated by adding an internal standard and / or by being diluted with another solution and / or by having being mixed with reagents or the like.

[0030] The test kit comprises: i) at least one test strip comprising at least one membrane, wherein the membrane comprises at least one first test field, the first test field comprising at least one first capture component, wherein the membrane further comprises at least one second test field, the second test field comprising at least one second capture component, wherein the second capture component is different from the first capture component; and ii) at least one first analyte detection system for contacting with the sample of the bodily fluid, wherein the first analyte detection system comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid, wherein the first analyte detection system further comprises at least one first capture reagent; wherein the first capture component is configured for capturing the first capture reagent of the first analyte detection system, wherein the second capture component is configured for capturing at least one second capture reagent from at least one second analyte detection system, the second capture reagent being different from the first capture reagent. The term “test strip” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an arbitrary device which is capable of measuring or detecting at least one analyte in a sample and / or at least one parameter of the sample or a component of the sample, such as in the sample of the bodily fluid. The test strip may specifically be suited for an ex -vivo measurement, specifically an in-vitro measurement. As an example, the test strip may be an optical test strip. The optical test strip may comprise at least one component or at least one reagent, typically a detectable label, which changes at least one detectable property, typically an optically detectable property, when the analyte is present in the sample, in particular an analyte detection system, as will be outlined in further detail below. The test strip may be or may comprise a strip-shaped test element. As used herein, the term “strip” or "strip-shaped" refers to an element having an elongated shape and a thickness, wherein an extension of the element in a lateral dimension exceeds the thickness of the element, such as by at least a factor of 2, preferably by at least a factor of 5, more preferably by at least a factor of 10 and most preferably by at least a factor of 20 or even at least a factor of 30. The test strip, as an example, may comprise, specifically may be, a lateral flow test strip.

[0031] The term “membrane” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an arbitrary element designed to carry one or more other elements disposed thereon or therein. The membrane may be or may comprise a planar substrate. The membrane may, specifically, have an elongated shape, such as a strip shape, a bar shape or a rectangular shape. However, other kinds of shapes may also be feasible. The membrane, as an example, may be flexible and / or deformable. The membrane may be configured for providing stabilizing means to the test strip, specifically to the first and second test field of the test strip. Typically, said substrate may comprise, more typically may be made of, a polymeric material. Even more typically, the material may be selected from the group consisting of cellulose; nitrocellulose; glass fiber; nylon; a cellulose-based material and the like. In other words, according to the invention, the membrane typically comprises at least one material selected from the group consisting of: cellulose; nitrocellulose; glass fiber; nylon; a cellulose-based material and the like.

[0032] In general, the terms “first” and “second” as used herein are used for the purpose of nomenclature, only, without intention of ranking or numbering and without giving any preferences. The term “test field” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to a region of the membrane of a test strip comprising a capture component which is capable of or configured for capturing at least one analyte in a sample, such as in a sample of a bodily fluid. More specifically, the capture component may be immobilized to the test field, e.g. to said region of the membrane, by use of a chemical linker, typically establishing a covalent chemical bond. In other words, the capture component may be localized to the test field of the membrane, typically by ways of a chemical linker. Even more specifically, said covalent bond may be achieved by use of a chemical linker such as widely used carbodiimide crosslinker chemistry, also referred to as EDC / NHS chemistry involving N-ethyl-N'-(3-(dimethyla- mino) propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), or click chemistry, specific and controllable bioorthogonal reactions as known in the art. Further ways of attaching the capture component by a covalent chemical bond to the membrane may include thiol coupling or conjugations. The skilled artisan is well aware of the procedures necessary for establishing said attachment by said chemical linker molecules. The kind of chemical linker molecules used may in particular be adapted to the capture component to be attached to the membrane and to the membrane material.

[0033] The membrane of the test strip according to the present invention comprises at least a first test field comprising at least one first capture component, and at least a second test field, comprising at least one second capture component, wherein the second capture component is different from the first capture component. Typically, said membrane may comprise exactly two test fields, said first test and said second test field. Said first and second test fields may typically be spaced apart on the membrane.

[0034] The term “capture component” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to a component configured for capturing a capture reagent of an analyte detection system, such as the first capture reagent of the first analyte detection system or the second capture reagent of the second analyte detection system. The term may specifically refer, without limitation, to a component that is capable of capturing a capture reagent. More specifically, the capture component may be immobilized to the test field of the membrane. In a typical embodiment according to the invention, the second capture component immobilizes the at least one second binding agent to the membrane via the second capture reagent prior to the application of the prepared sample of the body fluid to the test strip. According to the invention and as outlined above, the membrane comprises a first and a second test field, comprising at least one first and at least one second capture component respectively, wherein the second capture component is different from the first capture component. In particular, the first capture component and / or the second capture component may be configured for being immobilized, more particularly may be immobilized, at the first test field and the second test field, respectively. Even more particularly first capture component and / or the second capture component may be immobilized by use of a chemical linker as defined elsewhere herein. Typically, the first capture component may specifically bind to the first capture reagent; more typically, the second capture component may specifically bind to the second capture reagent.

[0035] The term “specifically binding” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to the binding affinity and / or binding specificity of the binding partners involved, typically the binding affinity of the capture component to the capture reagent and / or the binding agent, including an optode, to the analyte.

[0036] As commonly known, the affinity of a binding agent, including an optode, as defined elsewhere herein, or a capture component, to its binding partner, such as an antigen, an analyte or a capture reagent, may be inversely related to the dissociation constant (KD). A high affinity of the at least one binding agent, for the analyte, antigen, or capture reagent to be detected or capture according to the invention hence may be characterized by a low KD. Typically, a KD of below 10 pM may represent a suitable affinity for the binding of a binding agent to an analyte to be detected by the immunosensor in line with the present invention. More typically, a KD in the range of in between 10'15M to 10'5M may represent a suitable affinity in line with the present invention. The affinity of the least one first and / or second capture component to the first and / or second capture reagent may be represented by the above-specified ranges for KD.

[0037] The capture component / capture reagent interaction may typically be a reversible coupling system. More typically, the first capture reagent and the first capture component may be elements of reversible coupling systems selected from the group consisting of digoxin / anti- digoxin; streptavidin; avidin; biotin; neutravidin; polystrepatavidin; digoxigenin / anti-di- goxigenin; FITC / anti-FITC; nickel-poly histidine; complementary DNA strands and the like. Still more typically, the second capture reagent and the second capture component may be elements of reversible coupling systems, such as those exemplified for the first capture reagent and the first capture component above. However, the first and second capture reagents differ from each other.

[0038] In line with the invention, the first capture component is configured for capturing the first capture reagent of the first analyte detection system, wherein the second capture component is configured for capturing at least one second capture reagent from at least one second analyte detection system. The phrase “configured for capturing the first or second capture reagent” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to the capability of the first or second capture component, respectively, to capture the first or second capture reagent. Typically, said capturing may relate to a specific binding of the first capture component to the first capture reagent and of the second capture component to the second capture reagent, respectively. As explained elsewhere herein, the term “specific binding” or “specifically binding” is well understood in the art and may typically be related to the affinity of the binding partners to each other.

[0039] According to the invention, the second capture reagent is different from the first capture reagent. Hence, the elements of the first capture reagent and the first capture component and the elements of the second reagent and the second capture component may relate to different reverse coupling systems, i.e. said systems and elements are non-identical. Using different reverse coupling systems may be advantageous as it allows for simultaneous capturing of at least two different capture reagents and hence allows for capturing and in particular detecting at least two different analyte detection systems on a single test strip.

[0040] The test kit according to the invention further comprises at least one first analyte detection system for contacting with the sample of the bodily fluid, wherein the first analyte detection system comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid, wherein the first analyte detection system further comprises at least one first capture reagent.

[0041] The first analyte detection system according to the invention comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid, wherein the first analyte detection system further comprises at least one first capture reagent. The term “optode compound” or “optode” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to an ionophore based ion-sensing compound, more specifically to an optical analyte detection system, in particular an ion-detection system, comprising least one ionophore configured for interacting with the first analyte, such as a first ion, and at least one optical label as defined elsewhere herein. Typically, said at least one optical label comprised in the optode compound may be a chromoionophore. More typically, the first binding agent may be capable of binding to a first analyte, by means of least one ionophore configured for interacting with the first analyte. Even more typically, upon interaction of the first analyte with the ionophore, the optical label, such as the at least one chromoionophore, may emit an optical signal such as light of a specific wavelength, specifically in the visible range, and, optionally, involving the interaction of at least one ion exchanger. Hence, the optode compound may further comprise at least one ion exchanger. Further details on optode compounds are for example given in Mistlberger G. et al., Annu. Rev. Anal. Chem. 2014. 7:483-512.

[0042] The term “ionophore” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to a molecule that selectively binds to an ion; more specifically to an ion-selective molecule. Typically, selectivity by which an ion is bound by a ionophore is depending on the selectivity coefficient defining the displacement of the ionophore from a complex with the ion by an alternative binding agent. Determining selectivity coefficients is well established in the art. For example, selectivity coefficients may be determined experimentally by measuring the two equilibrium constants for the, KAB and KAC, wherein A is the analyte ion and B is one binding agent (such as the ionophore) and C is an alternative binding agent. An ionophore in line with the invention may typically be capable of specifically binding to the first analyte, e.g. an ion of interest. Numerous ionophores are known in the art. These include sodium ionophores, potassium ionophores, calcium ionophores, chloride ionophores, (published for example in Du et al., Biomicrofluidics. 2022 May; 16(3): 031301). In particular, the ionophore may comprise at least one element selected from the group consisting of: nile blue derivatives, hydroxy-azo-compounds, fluorescein derivatives, valinomycin, BME-44, ETH 4120, ETH 129, Crown Ether Ionophores, for example disclosed in Chem. Rev. 1998, 98, 1593-1687. The term “chromoionophore” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. Chromoionophores change color in response to binding or interaction with specific ions. The term may specifically refer, without limitation, to an indicator dye, for example a lipophilic pH indicator. The chromoionophore may typically change color depending on its protonation, and this color change can be detected. Some chromoionophores can also exhibit fluorescence or luminescence upon ion binding, thereby emitting light. Typically chromoionophores suitable for use in an optode compound according to the invention are known in the art and may include for example: Chromoionophore IZETH 5294, Chromoionophore IIZETH 2439, Chromoionophore III / ETH 5350, Chromoionophore VI / ETH 7075, ETH 5350, ETH 5720, ETH 2277 (published for example in Du et al., Biomicrofluidics. 2022 May; 16(3): 031301; and Biihlmann, et al., Chem. Rev. 1998, 98, 1593-1687).

[0043] The optode compound according to the present invention specifically may comprise, more specifically may be, an ion-selective optode.

[0044] The term “ion exchanger” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to a molecule capable of reversibly binding and releasing ions at an interface such as a membrane. More typically, the ion exchanger mediates an exchange of ions with an equivalent amount of protons across an interface such as a membrane. Ion exchangers are known in the art and widely used in the field of analytical science. An ion exchanger suitable for use in the optode compound according to the present invention may comprise at least one element selected from the group consisting of: tridodecylmethylammonium, lipophilic salts, tetraphenylborate.

[0045] The optode compound according to the invention comprises at least one ionophore, and at least one first detectable label. In a particular embodiment according to the invention, the optode compound comprises valinomycin as a potassium selective ionophore, sodium tetrakis(4-fluorophenyl)borate) as ion exchanger, and ETH 5294 as chromoionophore. More particularly, the optode compound may comprise a plasticizer and a polymer membrane such as a PVC membrane and a platicizer, for dioctyl adipate. This type of optode compound set up typically allows for indirect sensing of potassium ions. In another embodiment according to the invention, the optode compound is configured to optically sense pH. This ionophore-based sensor may, as an example, comprise sodium tetrakis(4-fluorophenyl)borate) as ion exchanger, and ETH 5294 as ionophore. In this exampls, the ionophore may be a chromoionophore changing at least one spectral property, such as absorbance and / or fluorescence, upon contact with the analyte. Thus, the optode compound may be configured for a direct sensing scheme.

[0046] The first analyte detection system may, in particular, comprise the optode compound as at least one first binding agent and at least one first detectable label. The first analyte detection system further comprises at least one first capture reagent and, optionally, further components. In particular, the at least one optode may comprise at least one ionophore, at least one first detectable label, typically a chromoionophore, and, optionally, further components. More particularly, the optode compound according to the invention may comprise at least one ionophore configured for interacting with the first analyte and at least one chromoionophore emitting an optically detectable signal upon interaction of the first analyte with the ionophore, and, optionally, at least one ion exchanger. A typical optode compound according to the invention may comprise, typically consist of, an ionophore, a chromoionophore and an ion exchanger. As an example, the optode compound may comprise microparticles, specifically microspheres, comprising the at least one ionophore, the at least one chromoionophore, and, optionally, the at least one ion exchanger. The microparticle may typically encase the optode compound. The first capture reagent may be attached to a surface of the microparticles, specifically of the microspheres, typically be a chemical linker specified elsewhere herein. The microparticles may comprise or may consist of a polymeric material such as polystyrene or modified polystyrene, latex. The use of a microparticle for encasing the optode compound may be of particular advantage as it is thought to contribute to evenly dispersing the optode compound in water or in an aqueous solution. An even dispersion of the optode may in turn contribute to enhanced sensitivity and reliability of analyte detection by the optode compound.

[0047] According to the present invention, the first and / or the second detectable label may, in particular, comprise or consist of an optically detectable label, specifically a fluorophore, an enzymatic label producing a colored product, fluorescent proteins, a chromoionophore, a quantum dot, colloidal silver, a carbon nanoparticle, a gold nanoparticle, a dye-labeled latex particle.

[0048] The term “detectable label” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to a molecule or element of the analyte detection system adapted for making the presence of a molecule or complex comprising said label detectable. Typically, the label may have a detectable property, typically an electrochemical, optical or / and enzymatic property. More typically, said property may be an optically detectable property, such as emission of an optically detectable signal, including radiation, chemiluminescence, fluorescence or FRET, electromagnetic properties, such as interference with electrical or magnetic fields, detectable radioactivity or chemical properties, such as capability of performing or catalyzing a certain chemical reaction. Hence, the label according to the present invention may in particular have an optically detectable property. More particularly, the first and the second detectable label may have the same detectable property, even more particularly the first and the second detectable labels may have an optically detectable property. In other words, both labels may be optical labels. As will be understood, the parameter determined to detect said detectable property will also be referred to as "signal" or "detectable signal". For the avoidance of doubt, it is noted that a signal may also be detected to be zero or below a detection limit.

[0049] A detectable label according to the present invention, such as the first and / or the second detectable label, may specifically refer to a detectable label comprising or consisting of an optically detectable label. Typical detectable labels may include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels or fluorescent labels. Enzymatically active labels may include horseradish peroxidase, alkaline phosphatase, beta-Galactosidase or Luciferase. Suitable substrates for detection of such enzymatically detectable labels may include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzi- dine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phos- phate. A suitable enzyme substrate combination may result in a colored reaction product, fluorescence or chemiluminescence which can be measured according to methods known in the art, e.g., by using a light-sensitive film or a suitable camera system. Typical fluorescent labels include fluorescent proteins, such as GFP, RFP, YFP, BFP or variants thereof, Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes, e.g., Alexa 568. The use of quantum dots as fluorescent labels may also be contemplated. Typical radioactive labels may include 35S, 1251, 32P, 33P and the like. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager. More specifically the optically detectable label may be a fluorophore, an enzymatic label producing a colored product, fluorescent proteins, a chromoionophore, aquantum dot, colloidal silver, a carbon nanoparticle, a gold nanoparticle, or a dye-labeled latex particle. The detectable label may be reversibly or permanently bound to the binding agent. In particular, the second detectable label may be reversibly or permanently, typically permanently, bound to the second binding agent. To this end, the detectable label may be a molecule which is capable of reversibly binding to the second binding agent or it may be a molecule or moiety which is already part of the said second binding agent. In the test kit according to the invention, the second detectable label may comprise or consist of an optically detectable label, specifically a fluorophore, an enzymatic label producing a colored product, fluorescent proteins, a chromoionophore, quantum dots, colloidal silver, carbon nanoparticles, gold nanoparticles, dye-labeled latex particles.

[0050] The test kit according to the invention may further comprise at least one second analyte detection system configured for receiving a second analyte suspected to be present in the sample of the bodily fluid, wherein the second analyte detection system may comprise the at least one second capture reagent.

[0051] Furthermore, in the test kit according to the invention, the first analyte may be different from the second analyte. Hence, advantageously, the test kit may be suitable for determining two different analytes in a single sample of a bodily fluid simultaneously on a single test strip. This may be advantageous as it saves costs and effort that would be required for repeated determining of different analytes.

[0052] Typically, the first analyte may comprise or may be an ion, and the second analyte may be a different analyte such as specified elsewhere herein, More typically, the first analyte may comprise, specifically may be, an ion, specifically a mineral cation, an anion or a polyion.

[0053] Even more typically, the ion may comprise, in particular may be, at least one element selected from the group consisting of: a potassium ion; a sodium ion; a calcium ion; a phosphorous ion; a magnesium ion; an iron ion; a chlorine ion; a copper ion; a zinc ion; a manganese ion; a molybdenum ion; an iodine ion; a selenium ion; an ammonium ion; a phosphate; a carbon dioxide; a nitrate; a nitrite.

[0054] According to a preferred embodiment of the invention, the first analyte is a potassium ion.

[0055] The second analyte may be at least one of, typically is, a peptide, an antibody, a DNA, an electrolyte and a metabolic compound, specifically creatinine, a peptide or protein, or an ion as defined elsewhere herein, more specifically creatinine. The first analyte may be different from the second analyte. However, alternatively, the first analyte may be identical with the second analyte. In said latter case, in particular, the first and second analyte may relate to the same chemical species but may be detected at different concentration ranges.

[0056] Typically, the first analyte may be identical to the second analyte, wherein the first analyte detection system may have a first sensitivity for the analyte suspected to be present in the sample of the bodily fluid, wherein the second analyte detection system may have a second sensitivity for the analyte suspected to be present in the sample of the bodily fluid, wherein the first sensitivity may be different from the second sensitivity.

[0057] Specifically, the second analyte detection system may comprise, more specifically may be, at least one further optode compound. Alternatively, the second analyte detection system may further comprise at least one second binding agent and at least a second detectable label, wherein, specifically, the second binding agent may be different from the optode compound and / or the second detectable label may be different from the first detectable label.

[0058] Typically, the second analyte detection system may be configured for forming a detection complex comprising a second analyte, a second binding agent and the second capture reagent, wherein, specifically, the second capture component may be configured for immobilizing the detection complex onto the second test field of the membrane of the test strip.

[0059] The first and / or second analyte detection system or parts thereof may be provided in a dry form, such as a powder, or in solubilized form in one identical container or in separate containers, typically depending on the affinity of first and second analyte detection system for the analyte. More typically, the second analyte detection system, specifically the second binding agent, may be immobilized, in particular pre-captured, on the second test field of the test strip via the capture reagent, or the capturing may occur after applying the prepared sample of the bodily fluid to the test strip.

[0060] The terms “binding agent” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to any kind of molecule or chemical moiety capable of specifically binding an analyte. The binding agent, in particular the second binding agent, may be selected from antibodies and fragments thereof, nucleic acids, aptamers, peptide nucleic acids (PNAs), receptor or ligand proteins or peptides, enzymes and ionophores, and or mixtures thereof. Typically, the optode compound according to the invention may comprise an ionophore as a first binding agent; and more typically said second binding agent may comprise an antibody or an ionophore. The test strip, as an example, may comprise at least one immunoassay test strip. The membrane may further comprise at least one conjugate pad comprising at least one conjugate for receiving the second analyte suspected to be present in the sample of the bodily fluid. The conjugate may comprise the second capture reagent. Other examples are, however, also feasible.

[0061] The test kit may further comprise at least one incubation unit comprising the first analyte detection system, specifically at least one common incubation unit comprising the first analyte detection system and the second analyte detection system. The term “incubation unit” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may specifically refer, without limitation, to at least one device, such as at least one of a container, vessel, compartment, receptacle or the like, configured for contacting the sample of the bodily fluid with one of the first analyte detection system and the second analyte detection system. The incubation unit may comprise at least one first incubation unit comprising the first analyte detection system. The incubation unit may further comprises at least one second incubation unit comprising the second analyte detection system. Alternatively, the incubation unit may comprise a common incubation unit comprising the first and second analyte detection system. Common incubation units for the first and second analyte detection system may particularly be adequate for similar incubation times.

[0062] As an example, the test kit may comprise at least one test liquid comprising at least one of the first analyte detection and the second analyte detection system, such as at least one common test liquid comprising the first and the second analyte detection system or at least one first test liquid comprising the first analyte detection system and at least one second test liquid comprising the second analyte detection system. The incubation unit may comprise at least one container, vessel, compartment or receptacle comprising the at least one test liquid for contacting the sample of the bodily fluid with the comprising the first and the second analyte detection system. In the case of two different test liquids, the incubation unit may comprise at least one first container, vessel, compartment or receptacle comprising the first analyte detection system and at least one second container, vessel, compartment or receptacle comprising the second analyte detection system. Alternatively or additionally, the first and second analyte detection system may be provided in a dry form, such as in powder form. The incubation unit may comprise at least one container, vessel, compartment or receptacle comprising the dry form of the first and second analyte detection system. For example, the incubation unit may comprise at least one first container, vessel, compartment or receptacle comprising the first analyte detection system and at least one second container, vessel, compartment or receptacle comprising the second analyte detection system. The sample of the bodily fluid may flow through the incubation unit, such as through the container, vessel, compartment or receptacle comprising the dry form of the first and second analyte detection system or stepwise through the first and second container, vessel, compartment or receptacle, for contacting the sample with the respective analyte detection system.

[0063] As an example, the test kit may be part of a microfluidic system. The microfluidic system may further comprise the at least one incubation unit and, optionally, at least one pretreatment unit for sample pretreatment. For example, the sample of the bodily fluid may comprise blood plasma. The sample pretreatment unit may be configured for separating the blood plasma from a blood sample. The microfluidic system may further be configured for transporting the sample of the bodily fluid, e.g. from the pretreatment unit to the incubation unit, and for contacting the sample of the bodily fluid, e.g. the blood plasma, with the first and / or analyte detection system in the incubation unit.

[0064] In a further aspect of the present invention, a test strip is disclosed. The test strip is configured for use in a test kit according to the present invention, such as according to any one of the embodiments disclosed above and / or according to any one of the embodiments disclosed in further detail below. Thus, for definitions of terms and / or possible embodiments of the test strip or any part thereof, reference is made to the description of the test kit above.

[0065] The test strip comprises at least one membrane. The membrane comprises at least one first test field. The first test field comprises at least one first capture component, wherein the first capture component is configured for capturing at least one first capture reagent of at least one first analyte detection system. The first analyte detection system is configured for contacting with the sample of the bodily fluid, specifically outside the test strip. The first analyte detection system comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid. The first analyte detection system further comprises the at least one first capture reagent.

[0066] The test strip may provide a great variety of uses for different analytes to be detected. Specifically, the optode compound for receiving the first analyte may not be part of the test strip, in particular not be present on or attached to the membrane. The optode compound may receive the first analyte outside the test strip, such as in one or more incubation units or the like. Thus, the test strip may be configured for detecting different optode compounds which specifically enhances the variety of test strip with regard to different applications or uses.

[0067] Further, the test strip may also have a higher flexibility with regard to incubation time.

[0068] The membrane may further comprise at least one second test field. The second test field may comprise at least one second capture component. The second capture component may be different from the first capture component. The second capture component may be configured for capturing at least one second capture reagent from at least one second analyte detection system. The second capture reagent may be different from the first capture reagent.

[0069] In a further aspect of the present invention, a method for determining analytes in a sample of a bodily fluid is disclosed. The method comprises using at least one test kit according to the present invention, such as according to any one of the embodiments disclosed above and / or according to any one of the embodiments disclosed in further detail below. Thus, for definitions of terms and / or possible embodiments of the test kit or any part thereof, reference is made to the description of the test kit above.

[0070] The method comprises the following steps, which, as an example, may be performed in the given order. It shall be noted, however, that a different order is also possible. Further, it is possible to perform one or more or even all of the method steps once or repeatedly. Further, it is possible to perform two or more of the method steps simultaneously or in a timely overlapping fashion. The method may comprise additional method steps, which are not listed.

[0071] The method comprises the following steps: a) contacting the sample of the bodily fluid with the first analyte detection system and / or the second analyte detection system to obtain a prepared sample of the bodily fluid; b) applying the prepared sample of the bodily fluid to the test strip; c) determining a capturing of the first capture reagent of the first analyte detection system with the first capture component at the first test field; and d) determining a capturing of the second capture reagent of the second analyte detection system with the second capture component at the second test field.

[0072] Specifically, the contacting of the sample of the bodily fluid with said first analyte detection system may be performed outside of the test strip, for example in an incubation unit or incubation chamber, more specifically thereby generating a prepared sample. Even more specifically, the first analyte detection system may be contacted with the sample of the bodily fluid prior to contacting the resulting mixture with the test strip. Said contacting may be performed using any suitable means and methods known in the art. Said contacting said first analyte detection system with the sample of the bodily fluid, may typically be performed in a suitable container such as an incubation unit, more typically thereby generating the prepared sample. Said contacting may further comprise pipetting; shaking; inverting; stirring; and the like. The contacting may take place for a time sufficient to allow for the formation of at least one analyte detection complex as specified elsewhere herein.

[0073] The method may further comprise at least one sample pretreatment step comprising preparing the sample of the bodily fluid from an aliquot or an aliquant of the bodily fluid, more typically prior to contacting the sample with the first and / or analyte detection system. For example, the sample of the bodily fluid may comprise blood plasma. The sample pretreatment step may comprise separating the blood plasma from a blood sample.

[0074] Alternatively or additionally, the method may further comprise forming at least one analyte detection complex comprising: the first analyte, the optode, the first detectable label and the first capture reagent; or comprising the second analyte, the second analyte binding agent, the second detectable label and the second capture reagent; or forming both of said analyte detection complexes. Typically, said analyte detection complex may form in the incubation unit and, thus, may be present in the prepared sample.

[0075] Applying the prepared sample of the bodily fluid to the test strip may comprise any way of transfer known in the art, such as pipetting, dropping from a container, transferring by microfluidics.

[0076] Typically, the prepared sample being applied to the test strip, is transported in particular through capillary forces in the membrane, to the first and second test field.

[0077] Moreover, the method may comprise a washing step, typically for removing non-bound analyte, binding agent or optode compound from the prepared sample applied to the test strip, more typically with a suitable washing buffer such as those known to the skilled artisan. Said washing may be performed in the incubation unit or incubation chamber.

[0078] Alternatively or additionally, the determining of the capturing of the first capture reagent may comprise a determining a presence and / or an absence of the first analyte in the sample of the bodily fluid. The determining may comprise qualitative determination, semi-quantitative or quantitative determination. In particular, in case the first and second analyte are identical, the determining may refer to a semi -quantitative or quantitative determination. Alternatively or additionally, step c) may comprise detecting the first analyte in the sample of the bodily fluid by using at least one fluorescence signal of the first detectable label at the first test field.

[0079] Alternatively or additionally, the determining of the capturing of the second capture component may comprise a determining a presence and / or an absence of a second analyte in the sample of the bodily fluid.

[0080] Alternatively or additionally, step d) may comprise detecting a second analyte in the sample of the bodily fluid by using at least one optically detectable detection reaction of the second analyte detection system at the second test field.

[0081] The methods and devices according to the present invention may provide a large number of advantages over known methods and devices. Specifically, the test kit and the test strip with the first test field may provide a concentrated and, thus, enhanced signal compared with known optode sensors. Further, the test kit and the test may allow for a combination with other assays, e.g. immunoassays. Further, the test kit and the test strip may allow for performing washing and / or calibration steps.

[0082] For example, optode sensors may be modified with a biotin or other molecule, e.g. digoxin, that can be easily and specifically recognized by other molecule being immobilized on the first test field of the membrane of the test strip, e.g. by streptavidin. Such as test strip may allow having an optode sensor confined in particles that can be specifically trapped in a lateral flow assay. Other examples are also feasible.

[0083] Summarizing and without excluding further possible embodiments, the following embodiments may be envisaged:

[0084] Embodiment 1 : A test kit for determining analytes in a sample of a bodily fluid, wherein the test kit comprises: i) at least one test strip comprising at least one membrane, wherein the membrane comprises at least one first test field, the first test field comprising at least one first capture component, wherein the membrane further comprises at least one second test field, the second test field comprising at least one second capture component, wherein the second capture component is different from the first capture component; and ii) at least one first analyte detection system for contacting with the sample of the bodily fluid, wherein the first analyte detection system comprises at least one ion-selective optode compound comprising at least one ionophore and at least one first detectable label, said ion-selective optode compound being configured for receiving a first analyte suspected to be present in the sample of the bodily fluid, wherein the first analyte detection system further comprises at least one first capture reagent; wherein the first capture component is configured for capturing the first capture reagent of the first analyte detection system, wherein the second capture component is configured for capturing at least one second capture reagent from at least one second analyte detection system, the second capture reagent being different from the first capture reagent.

[0085] Embodiment 2: The test kit according to the preceding embodiment, wherein first capture component and / or the second capture component is configured for being immobilized at the first test field and the second test field, respectively, specifically immobilized by use of a chemical linker.

[0086] Embodiment 3 : The test kit according to any one of the preceding embodiments, wherein the first capture component specifically binds to the first capture reagent, wherein the second capture component specifically binds to the second capture reagent.

[0087] Embodiment 4: The test kit according to any one of the preceding embodiments, wherein the at first analyte is a potassium ion.

[0088] Embodiment s: The test kit according to any one of the two preceding embodiments, wherein the first and / or the second detectable label comprises or consist of an optically detectable label, specifically a fluorophore, an enzymatic label producing a colored product, fluorescent proteins, a chromoionophore, quantum dots, colloidal silver, carbon nanoparticles, gold nanoparticles, dye-labeled latex particles.

[0089] Embodiment 6: The test kit according to any one of the preceding embodiments, further comprising at least one second analyte detection system configured for receiving a second analyte suspected to be present in the sample of the bodily fluid, wherein the second analyte detection system comprises the at least one second capture reagent. Embodiment 7 : The test kit according to the preceding embodiment, wherein the first analyte is different from the second analyte.

[0090] Embodiment s: The test kit according to any one of the two preceding embodiments, wherein the first analyte is identical to the second analyte, wherein the first analyte detection system has a first sensitivity for the analyte suspected to be present in the sample of the bodily fluid, wherein the second analyte detection system has a second sensitivity for the analyte suspected to be present in the sample of the bodily fluid, wherein the first sensitivity is different from the second sensitivity .

[0091] Embodiment 9: The test kit according to any one of the three preceding embodiments, wherein the second analyte detection system comprises, specifically is, at least one further optode compound.

[0092] Embodiment 10: The test kit according to any one of the preceding embodiments, wherein the second analyte detection system further comprises at least one second binding agent and at least a second detectable label, wherein, specifically, the second binding agent is different from the optode compound and / or the second detectable label is different from the first detectable label.

[0093] Embodiment 11 : The test kit according to the preceding embodiment, wherein the second binding agent is selected from the group consisting of antibodies and fragments thereof, nucleic acids, aptamers, peptide nucleic acids (PNAs), receptor or ligand proteins or peptides, and enzymes, and ionophores; typically said first binding agent and / or said second binding agent comprises an antibody, or an ionophore.

[0094] Embodiment 12: The test kit according to any one of the two preceding embodiments, wherein the second detectable label comprises or consist of an optically detectable label; specifically a fluorophore, an enzymatic label producing a colored product, fluorescent proteins, a chromoionophore, quantum dots, colloidal silver, carbon nanoparticles, gold nanoparticles, dye-labeled latex particles.

[0095] Embodiment 13 : The test kit according to any one of the preceding embodiments, wherein the optode compound is an ion-selective optode. Embodiment 14: The test kit according to any one of the preceding embodiments, wherein the first analyte comprises, specifically is, an ion, specifically a mineral cation, an anion or a polyion.

[0096] Embodiment 15: The test kit according to the preceding embodiment, wherein the ion comprises , typically is, at least one element selected from the group consisting of: a potassium ion; a sodium ion; a calcium ion; a phosphorous ion; a magnesium ion; an iron ion; a chlorine ion; a copper ion; a zinc ion; a manganese ion; a molybdenum ion; an iodine ion; a selenium ion; an ammonium ion; a phosphate; a carbon dioxide; a nitrate; a nitrite.

[0097] Embodiment 16: The test kit according to any one of the preceding embodiments, wherein the optode compound comprises at least one ionophore configured for interacting with the first analyte and at least one chromoionophore emitting an optically detectable signal upon interaction of the first analyte with the ionophore, and, optionally, at least one ion exchanger.

[0098] Embodiment 17: The test kit according to the preceding embodiment, wherein the optode compound comprises microparticles, specifically microspheres, comprising the at least one ionophore, the at least one chromoionophore, and, optionally, the at least one ion exchanger.

[0099] Embodiment 18: The test kit according to the preceding embodiment, wherein the first capture reagent is attached to a surface of the microparticles, specifically of the microspheres.

[0100] Embodiment 19: The test kit according to any one of the three preceding embodiments, wherein the ionophore comprises at least one element selected from the group consisting of: nile blue derivatives, hydroxy-azo-compounds, fluorescein derivatives, valino- mycin, BME-44, ETH 4120, ETH 129, Crown Ether Ionophores.

[0101] Embodiment 20: The test kit according to any one of the four preceding embodiments, wherein the chromoionophore comprises at least one element selected from the group consisting of: ETH 5294, ETH 5350, ETH 7075, ETH 5720, ETH 2277. Embodiment 21 : The test kit according to any one of the five preceding embodiments, wherein the ion exchanger comprises at least one element selected from the group consisting of: tridodecylmethylammonium, lipophilic salts, tetraphenylborate.

[0102] Embodiment 22: The test kit according to any one of the preceding embodiments, wherein the first capture reagent and the first capture component are elements of reversible coupling systems selected from the group consisting of: digoxin / antidigoxin; streptavidin; avidin; biotin; neutravidin; polystrepatavidin; digoxigenin / anti-digoxigenin; FITC / anti-FITC; nickel-poly histidine; complementary DNA strands and the like.

[0103] Embodiment 23 : The test kit according to any one of the preceding embodiments, wherein the second analyte detection system is configured for forming a detection complex comprising a second analyte, a second binding agent and the second capture reagent, wherein, specifically, the second capture component is configured for immobilizing the detection complex onto the second test field of the membrane of the test strip.

[0104] Embodiment 24: The test kit according to any one of the preceding embodiments, wherein the test strip comprises at least one immunoassay test strip, wherein the membrane further comprises at least one conjugate pad comprising at least one conjugate for receiving a second analyte suspected to be present in the sample of the bodily fluid, wherein the conjugate comprises the second capture reagent.

[0105] Embodiment 25 : The test kit according to any one of the preceding embodiments, wherein the second analyte is at least one of a peptide, an antibody, a DNA, an electrolyte and a metabolic compound, specifically creatinine.

[0106] Embodiment 26: The test kit according to any one of the preceding embodiments, wherein the membrane comprises at least one material selected from the group consisting of: cellulose; nitrocellulose; glass fiber; nylon; a cellulose-based material and the like.

[0107] Embodiment 27 : The test kit according to any one of the preceding embodiments, wherein the test strip comprises, specifically is, a lateral flow test strip.

[0108] Embodiment 28: The test kit according to any one of the preceding embodiments, wherein the test kit further comprises at least one incubation unit comprising the first analyte detection system, specifically at least one common incubation unit comprising the first analyte detection system and the second analyte detection system. Embodiment 29: The test kit according to the preceding embodiment, wherein the incubation unit comprises at least one first incubation unit comprising the first analyte detection system.

[0109] Embodiment 30: The test kit according to any one of the two preceding embodiments, wherein the incubation unit further comprises at least one second incubation unit comprising the second analyte detection system.

[0110] Embodiment 31 : A test strip for use in a test kit according to any one of the preceding embodiments, wherein the test strip comprises at least one membrane, wherein the membrane comprises at least one first test field, the first test field comprising at least one first capture component, wherein the first capture component is configured for capturing at least one first capture reagent of at least one first analyte detection system, the first analyte detection system being configured for contacting with the sample of the bodily fluid, specifically outside the test strip, wherein the first analyte detection system comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid, wherein the first analyte detection system further comprises the at least one first capture reagent.

[0111] Embodiment 32: The test strip according to the preceding embodiment, wherein the membrane further comprises at least one second test field, the second test field comprising at least one second capture component, wherein the second capture component is different from the first capture component, wherein the second capture component is configured for capturing at least one second capture reagent from at least one second analyte detection system, the second capture reagent being different from the first capture reagent.

[0112] Embodiment 33 : A method for determining analytes in a sample of a bodily fluid, wherein the method comprises using at least one test kit according to any one of the preceding embodiments referring to a test kit, wherein the method comprises the following steps: a) contacting the sample of the bodily fluid with the first analyte detection system and / or the second analyte detection system to obtain a prepared sample of the bodily fluid; b) applying the prepared sample of the bodily fluid to the test strip; c) determining a capturing of the first capture reagent of the first analyte detection system with the first capture component at the first test field; and d) determining a capturing of the second capture reagent of the second analyte detection system with the second capture component at the second test field.

[0113] Embodiment 34: The method according to the preceding embodiment, further comprising at least one sample pretreatment step comprising preparing the sample of the bodily fluid from an aliquot or an aliquant of the bodily fluid, more typically prior to contacting the sample with the first and / or analyte detection system.

[0114] Embodiment 35: The method according to the preceding embodiment, wherein the sample of the bodily fluid comprises blood plasma, wherein the sample pretreatment step comprises separating the blood plasma from a blood sample.

[0115] Embodiment 36: The method according to any one of the preceding method embodiments, further comprising forming at least one analyte detection complex comprising: the first analyte, the first analyte binding agent, the first detectable label and the first capture reagent; or the second analyte, the second analyte binding agent, the second detectable label and the second capture reagent; or both.

[0116] Embodiment 37: The method according to any one of the preceding method embodiments, wherein the determining of the capturing of the first capture reagent comprises a determining a presence and / or an absence of the first analyte in the sample of the bodily fluid.

[0117] Embodiment 38: The method according to any one of the preceding method embodiments, wherein step c) comprises detecting the first analyte in the sample of the bodily fluid by using at least one fluorescence signal of the first detectable label at the first test field.

[0118] Embodiment 39: The method according to any one of the preceding method embodiments, wherein the determining of the capturing of the second capture component comprises a determining a presence and / or an absence of a second analyte in the sample of the bodily fluid.

[0119] Embodiment 40: The method according to any one of the preceding method embodiments, wherein step d) comprises detecting a second analyte in the sample of the bodily fluid by using at least one optically detectable detection reaction of the second analyte detection system at the second test field. Short description of the Figures

[0120] Further optional features and embodiments will be disclosed in more detail in the subsequent description of embodiments, preferably in conjunction with the dependent claims. Therein, the respective optional features may be realized in an isolated fashion as well as in any arbitrary feasible combination, as the skilled person will realize. The scope of the invention is not restricted by the preferred embodiments. The embodiments are schematically depicted in the Figures. Therein, identical reference numbers in these Figures refer to identical or functionally comparable elements.

[0121] In the Figures:

[0122] Figure 1 shows an embodiment of a test kit for determining analytes in a sample of a bodily fluid in a schematic view; and

[0123] Figure 2 shows a flow chart of an embodiment of a method for determining analytes in a sample of a bodily fluid.

[0124] Detailed description of the embodiments

[0125] Figure 1 shows an exemplary embodiment of a test kit 110 for determining analytes in a sample of a bodily fluid 111 in a schematic view. The test kit 110 comprises at least one test strip 112 comprising at least one membrane 114. The membrane 114 comprises a first test field 116. The first test field 116 comprises afirst capture component 118. The membrane 114 further comprises asecond test field 120. The second test field 120 comprises asecond capture component 122. The second capture component 122 is different from the first capture component 118.

[0126] The test kit 110 further comprises at least one first analyte detection system 124 for contacting with the sample of the bodily fluid 111. The first analyte detection system 124 comprises aoptode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid 111. The first analyte detection system 124 further comprises a first capture reagent. The first capture component 118 is configured for capturing the first capture reagent of the first analyte detection system 124. The second capture component 122 is configured for capturing at least one second capture reagent from at least one second analyte detection system 126. The second capture reagent is different from the first capture reagent.

[0127] In the exemplary embodiment shown in Figure 1, the test kit 110 comprises the second analyte detection system 126 configured for receiving the second analyte suspected to be present in the sample of the bodily fluid 111. The second analyte detection system 126 comprises a second capture reagent. The second analyte detection system 126 may further comprise at least one second binding agent and at least a second detectable label, wherein, specifically, the second binding agent may be different from the optode compound.

[0128] Furthermore, the first analyte may be different from the second analyte. Hence, advantageously, the test kit 110 may be suitable for determining two different analytes in a single sample of a bodily fluid 111 simultaneously on a single test strip 112. This may be advantageous as it saves costs and effort that would be required for repeated determining of different analytes. The first analyte may comprise or may be an ion, and the second analyte may be a different analyte, such as specified elsewhere herein. For example, the first analyte may be a potassium ion. The second analyte may be a metabolic compound, specifically creatinine. Other examples are, however, also feasible.

[0129] The first analyte detection system 124 comprises the optode compound as at least one first binding agent and at least one first detectable label. The first analyte detection system 124 further comprises the at least one first capture reagent and, optionally, further components. In particular, the optode comprises at least one ionophore, at least one first detectable label, typically a chromoionophore, and, optionally, further components. More particularly, the optode compound according to the invention may comprise at least one ionophore configured for interacting with the first analyte and at least one chromoionophore emitting an optically detectable signal upon interaction of the first analyte with the ionophore, and, optionally, at least one ion exchanger. A typical optode compound according to the invention may comprise, typically consist of, an ionophore, a chromoionophore and an ion exchanger. As an example, the optode compound may comprise microparticles, specifically microspheres, comprising the at least one ionophore, the at least one chromoionophore, and, optionally, the at least one ion exchanger. The first capture reagent may be attached to a surface of the microparticles, specifically of the microspheres. According to the preferred embodiment shown in the figures, the ionophore comprises potassium ionophores and the first detectable label comprises a dye-labeled latex particle. Thus, in this embodiment, the first analyte detection system 124 comprises latex particles sensitive to potassium, specifically comprising valinomycin, tetrakis borate and a dye pH sensitive, wherein the latex particles may be decorated with digoxin molecules. The potassium-sensitive latex particles having received the first analyte and being captured by the first capture component 118 of the first test field are denoted by reference number 128 in Figure 1.

[0130] Further, the second analyte detection system 126 is configured for forming a detection complex 130 comprising the second analyte 132, the second binding agent 134 and the second capture reagent 136, wherein, the second capture component 122 is configured for immobilizing the detection complex 130 onto the second test field 120 of the membrane 114 of the test strip 112. For example, the second analyte detection system 126 may comprise monoclonal antibodies conjugated with the second optical label, the antibodies being configured for capturing the second analyte 132. The second analyte detection system 126 may further comprise monoclonal antibodies conjugated with biotin against the second analyte 132, in particular recognizing a different epitope.

[0131] In the exemplary embodiment shown in Figure 1, the test strip 112 is a lateral flow test strip 138. The lateral flow test strip 138 comprises a nitrocellulose membrane impregnated with a first recognition line formed with anti digoxin antibodies to form the first test field 116 and a second recognition line with streptavidin antibodies to form the second test field 120.

[0132] As shown in Figure 1, the test kit 110, as an example, may be part of a microfluidic system 140. As schematically shown in Figure 1, the microfluidic system 140 further comprises at least one incubation unit 142 and at least one pretreatment unit 144 for sample pretreatment. For example, the bodily fluid may comprise a blood sample 146 and the sample of the bodily fluid 111 may comprise blood plasma. The sample pretreatment unit 144 is configured for separating the blood plasma from the blood sample 146. The microfluidic system 140 is further configured for transporting the sample of the bodily fluid 111, e.g. from the pretreatment unit 144 to the incubation unit 142, and for contacting the sample of the bodily fluid 111, e.g. the blood plasma, with the first analyte detection system 124 and the second analyte detection system 126 in the incubation unit 142. The exemplary embodiment of Figure 1 shows a single incubation unit 142 comprising the first and second analyte detection system 124, 126. However, separated incubation units, such as a first incubation unit for the first analyte detection system 124 and a second incubation unit for the second analyte detection system 126, are also feasible. The exemplary test kit 110 may be used in a method for determining analytes in a sample of a bodily fluid 111. An exemplary embodiment of the method is shown in Figure 2. Thus, for a detailed description of a method of using the test kit 110, reference is made to the description of Figure 2.

[0133] Figure 2 shows a flow chart of an embodiment of a method for determining analytes in a sample of a bodily fluid 111. The method comprises using at least one test kit 110 according to the present invention, such as according to the exemplary embodiment of Figure 1 and / or according to any other embodiment disclosed herein. Thus, for a detailed description of the test kit 110, reference is made to the description of Figure 1.

[0134] The method comprises the following steps, which, as an example, may be performed in the given order. It shall be noted, however, that a different order is also possible. Further, it is possible to perform one or more or even all of the method steps once or repeatedly. Further, it is possible to perform two or more of the method steps simultaneously or in a timely overlapping fashion. The method may comprise additional method steps, which are not listed.

[0135] The method comprises the following steps: a) (denoted by reference number 148) contacting the sample of the bodily fluid 111 with the first analyte detection system 124 and / or the second analyte detection system 126 to obtain a prepared sample of the bodily fluid; b) (denoted by reference number 150) applying the prepared sample of the bodily fluid to the test strip 112; c) (denoted by reference number 152) determining a capturing of the first capture reagent of the first analyte detection system 124 with the first capture component 118 at the first test field 116; and d) (denoted by reference number 154) determining a capturing of the second capture reagent 136 of the second analyte detection system 126 with the second capture component 122 at the second test field 120.

[0136] As shown in Figure 2, the method may comprise at least one sample pretreatment step (denoted by reference number 156). The sample pretreatment step may be an optional method step and may be performed prior to the contacting of the sample of the bodily fluid 111 with the first analyte detection system 124 and / or the second analyte detection system 126. The sample pretreatment step may comprise preparing the sample of the bodily fluid 111 from an aliquot or an aliquant of the bodily fluid. For example, the sample of the bodily fluid 111 may comprise blood plasma. The sample pretreatment step may comprise separating the blood plasma from the blood sample 146.

[0137] Further, the contacting of the sample of the bodily fluid 111 with said first analyte detection system 124 may be performed outside of the test strip 112, for example in the incubation unit 142, more specifically thereby generating the prepared sample. Even more specifically, the first analyte detection system 124 may be contacted with the sample of the bodily fluid 111 prior to contacting the resulting mixture with the test strip 112. Said contacting may be performed using any suitable means and methods known in the art. Said contacting said first analyte detection system 124 with the sample of the bodily fluid 111 may typically be performed in a suitable container, such as the incubation unit 14, more typically thereby generating the prepared sample. Said contacting may further comprise pipetting; shaking; inverting; stirring; and the like. The contacting may take place for a time sufficient to allow for the formation of at least one analyte detection complex as specified elsewhere herein.

[0138] The method may further comprise forming at least one analyte detection complex comprising: the first analyte, the optode, the first detectable label and the first capture reagent; or comprising the second analyte 132, the second analyte binding agent 134, the second detectable label and the second capture reagent 136; or forming both of said analyte detection complexes. Typically, said analyte detection complex may form in the incubation unit 142 and, thus, may be present in the prepared sample.

[0139] Applying the prepared sample of the bodily fluid to the test strip 112 may comprise any way of transfer known in the art, such as pipetting, dropping from a container, transferring by microfluidics.

[0140] Further, the method may comprise a washing step (denoted by reference number 158). The washing step may be an optional method step and may typically be performed prior to the determining of the determining of the capturing of the first and second capture reagents. The washing step may typically comprise removing non-bound analyte, binding agent or optode compound from the prepared sample applied to the test strip 112, more typically with a suitable washing buffer such as those known to the skilled artisan.

[0141] Further, the determining of the capturing of the first capture reagent may comprise a determining a presence and / or an absence of the first analyte in the sample of the bodily fluid 111. The determining of the capturing of the second capture component may comprise a determining a presence and / or an absence of the second analyte 132 in the sample of the bodily fluid 111.

[0142] For example, the method may start with the sample pretreatment step in the pretreatment unit 144, e.g. for separation of blood plasma for the blood sample 146. The microfluidic system 140 may transfer the blood plasma to the incubation unit 142 where the sample of the bodily fluid 111 contacts the first and second analyte detection system 124, 126. During the incubation, the immunoassay may take place and the potassium ions may dissolve in the sample 111. In a next step, the sample 111 may be transferred to the nitrocellulose membrane. The nitrocellulose membrane may specifically be modified by line impregnation prior to the method and the sample 111 may diffuse through the membrane 114. After the chromatography is finished, the potassium-sensitive latex particles may be bound to the antidigoxin antibodies at the first test field 116 and the biotin-conjugated antibodies to the streptavidin molecules at the second test filed 120 arranged on the membrane 114. Subsequently, the washing step may remove non-bound reagents. A readout of the test strip 112 may involve an optical detector recognizing the intensity of the different recognition lines at the first test field 116 and the second test field 120.

[0143] EXAMPLES

[0144] In an example scenario, potassium and NTproBNP are simultaneously determined using a microfluidic-based sensor. The process begins with sample preparation, resulting in plasma of a defined volume. In the first step, the sample is incubated with a first antibody targeting NT-proBNP. In the second incubation step, the sample is incubated with a secondary antibody and ion selective microparticles. Following the second incubation, the mixture is transferred onto a chromatographic membrane.

[0145] During the chromatography process, the ion selective microparticles selectively bind to a recognition line via digoxin-antidigoxin interaction (the anti digoxin antibodies are immobilized in the membrane). Simultaneously, the antibodies bind to another recognition line using a streptavidin-based approach (streptavidin is immobilized in the membrane). This ensures that only biotin-conjugated antibodies are immobilized in the recognition line. The signal is then read out, and a washing step may be performed if needed. List of reference numbers

[0146] 110 test kit

[0147] 111 sample of a bodily fluid

[0148] 112 test strip

[0149] 114 membrane

[0150] 116 first test field

[0151] 118 first capture component

[0152] 120 second test field

[0153] 122 second capture component

[0154] 124 first analyte detection system

[0155] 126 second analyte detection system

[0156] 128 potassium-sensitive latex particles

[0157] 130 detection complex

[0158] 132 second analyte

[0159] 134 second binding agent

[0160] 136 second capture reagent

[0161] 138 lateral flow test strip

[0162] 140 microfluidic system

[0163] 142 incubation unit

[0164] 144 pretreatment unit

[0165] 146 blood sample

[0166] 148 contacting the sample of the bodily fluid with the first analyte detection system

[0167] 150 applying the prepared sample of the bodily fluid to the test strip

[0168] 152 determining a capturing of the first capture reagent

[0169] 154 determining a capturing of the second capture reagent

[0170] 156 sample pretreatment step

[0171] 158 washing step List of cited literature

[0172] Biihlmann, et al., Chem. Rev. 1998, 98, 1593-1687.

[0173] Du et al., Biomicrofluidics. 2022 May; 16(3): 031301.

[0174] Lookadoo, et al., Anal. Chem. 2021, 93, 9383-9389.

[0175] Mistlberger G. et al. “ Ionophore-Based Optical Sensors’", Annu. Rev. Anal. Chem. 2014. 7:483-512. Wang et al., Anal. Chem., 2019, 91(14), 8973-8978.

[0176] Xiaojiang Xie et al., Chem. Commun., 2014,50, 4592-4595

Claims

Claims1. A test kit (110) for determining analytes in a sample of a bodily fluid (111), wherein the test kit (110) comprises: i) at least one test strip (112) comprising at least one membrane (114), wherein the membrane (114) comprises at least one first test field (116), the first test field (116) comprising at least one first capture component (118), wherein the membrane (114) further comprises at least one second test field (120), the second test field (120) comprising at least one second capture component (122), wherein the second capture component (122) is different from the first capture component (118); and ii) at least one first analyte detection system (124) for contacting with the sample of the bodily fluid (111), wherein the first analyte detection system (124) comprises at least one ion-selective optode compound comprising at least one ionophore and at least one first detectable label, said ion-selective optode compound being configured for receiving a first analyte suspected to be present in the sample of the bodily fluid (111), wherein the first analyte detection system (124) further comprises at least one first capture reagent; wherein the first capture component (118) is configured for capturing the first capture reagent of the first analyte detection system (124), wherein the second capture component (122) is configured for capturing at least one second capture reagent (136) from at least one second analyte detection system (126), the second capture reagent (136) being different from the first capture reagent.

2. The test kit (110) according to the preceding claim, wherein first capture component (118) and / or the second capture component (122) is configured for being immobilized at the first test field (116) and the second test field (120), respectively.

3. The test kit (110) according to any one of the preceding claims, wherein the first capture component (118) specifically binds to the first capture reagent, wherein the second capture (122) component specifically binds to the second capture reagent (136).

4. The test kit (110) according to any one of the preceding claims, further comprising at least one second analyte detection system (126) configured for receiving a second analyte (132) suspected to be present in the sample of the bodily fluid (111), wherein the second analyte detection system (126) comprises the at least one second capture reagent (136); typically wherein the first analyte is different from the second analyte (132) or wherein the first analyte is identical to the second analyte (132), wherein the first analyte detection system (124) has a first sensitivity for the analyte suspected to be present in the sample of the bodily fluid (111), wherein the second analyte detection system (126) has a second sensitivity for the analyte suspected to be present in the sample of the bodily fluid (111), wherein the first sensitivity is different from the second sensitivity; more typically, wherein the second analyte detection system (126) comprises, specifically is, at least one further optode compound.

5. The test kit (110) according to any one of the preceding claims, wherein the second analyte detection system (126) further comprises at least one second binding agent (134) and at least a second detectable label, wherein, specifically, the second binding agent is different from the optode compound and / or the second detectable label is different from the first detectable label.

6. The test kit (110) according to any one of the preceding claims, wherein the first capture reagent and the first capture component (118) are elements of reversible coupling systems selected from the group consisting of: digoxin / antidigoxin; streptavidin; avidin; biotin; neutravidin; polystrepatavidin; digoxigenin / anti-digoxigenin; FITC / anti- FITC; nickel-poly histidine; complementary DNA strands and the like.

7. The test kit (110) according to any one of the preceding claims, wherein the second analyte detection system (126) is configured for forming a detection complex (130) comprising a second analyte (132), a second binding agent (134) and the second capture reagent (136), wherein, specifically, the second capture component (122) is configured for immobilizing the detection complex (130) onto the second test field (120) of the membrane (114) of the test strip (112).

8. The test kit (110) according to any one of the preceding claims, wherein the second analyte (132) is at least one of a peptide, an antibody, a DNA, an electrolyte and a metabolic compound, specifically creatinine.

9. The test kit (110) according to any one of the preceding claims, wherein the test strip (112) comprises, specifically is, a lateral flow test strip (138).

10. The test kit (110) according to any one of the preceding claims, wherein the test kit (110) further comprises at least one incubation unit (142) comprising the first analyte detection system (124).

11. A test strip (112) for use in a test kit (110) according to any one of the preceding claims, wherein the test strip (112) comprises at least one membrane (114), wherein the membrane (114) comprises at least one first test field (116), the first test field (116) comprising at least one first capture component (118), wherein the first capture component (118) is configured for capturing at least one first capture reagent of at least one first analyte detection system (124), the first analyte detection system (124) being configured for contacting with the sample of the bodily fluid (111), wherein the first analyte detection system (124) comprises at least one optode compound configured for receiving a first analyte suspected to be present in the sample of the bodily fluid (111), wherein the first analyte detection system (124) further comprises the at least one first capture reagent.

12. A method for determining analytes in a sample of a bodily fluid (111), wherein the method comprises using at least one test kit (110) according to any one of the preceding claims referring to a test kit (110), wherein the method comprises the following steps: a) contacting the sample of the bodily fluid (111) with the first analyte detection system (124) and / or the second analyte detection system (126) to obtain a prepared sample of the bodily fluid; b) applying the prepared sample of the bodily fluid to the test strip (112); c) determining a capturing of the first capture reagent of the first analyte detection system (124) with the first capture component (118) at the first test field (116); and d) determining a capturing of the second capture reagent (136) of the second analyte detection system (126) with the second capture component (122) at the second test field (120).

13. The method according to the preceding claim, further comprising at least one sample pretreatment step (156) comprising preparing the sample of the bodily fluid (111) from an aliquot or an aliquant of the bodily fluid.

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