Compositions, kits and methods for multiplex assays to correct for biotin interference in target analyte measurements

Abstract

Disclosed are kits containing a multiplexed chemiluminescent detection system and microfluidic devices and methods for detecting the presence and / or concentration of biotin and at least one target analyte in a sample. The kits, microfluidic devices, and methods utilize a combination of singlet oxygen-activatable chemiluminescent compounds and two or more fluorescent molecules that emit light at different wavelengths.

Description

[0001] Cross-reference to related applications / incorporation of claims by reference

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 735,905, filed September 25, 2018, pursuant to 35 USC § 119(e). The entire contents of the above-cited patent application are hereby expressly incorporated herein by reference.

[0003] Statement regarding federally funded research or development

[0004] not applicable.

[0005] background

[0006] Diagnostic assays often consist of conjugates of antibodies or other small drug molecules with haptens (such as biotin and fluorescein). The tight binding of these haptens to large protein molecules coated on a solid support or surface (e.g., anti-Biotin / streptoacidin for biotin and anti-FITC for fluorescein) provides a convenient method for immobilizing hapten-antibody or hapten-drug conjugates on a solid support / surface.

[0007] Biotin is well known in the art for its use as a food supplement; for example, it is used to promote healthy hair and nail growth and to treat various medical conditions. Given this use, significant levels of biotin can be found in biological samples, such as (but not limited to) blood. Since biotin is used in many diagnostic assays (e.g., with a solid-coated substrate), high levels of biotin in the test sample can interfere with the assay signal in any assay employing a biotinylated assay component. This is especially true for assays such as those found in Siemens' ADVIA CENTAUR® Immunoassay System, DIMENSION EXL™ Integrated Chemistry System, and DIMENSION VISTA® LOCI® System (Siemens HealthcareDiagnostics Inc., Tarrytown, NY), where the biotinylated assay component is expected to bind to a streptavidin-coated solid-coated substrate.

[0008] Because interference has been observed in various assays for different biotin concentrations, the biotin level present in patient samples should be quantified to determine whether the sample is suitable for a particular assay. However, most currently available biotin assays have a narrow range (i.e., 0–50 ng / ml), which is unsuitable for detecting the wide dynamic range of biotin concentrations (i.e., 0–1500 ng / ml) typically found in real patient samples.

[0009] Using biotin as an example, there are three ways to mitigate the problem of hapten interference. One is to use pre-prepared reagents, where the biotinylated assay component is “pre-bound” to a streptavidin-coated solid support during reagent production. Due to the tight binding and slow dissociation rate between streptavidin and biotin, replacing the already bound biotin with biotin from the patient sample is not a primary process. The second approach is to increase the streptavidin binding sites on the solid support, providing additional binding sites for biotin molecules in the sample besides the biotinylated assay component. The third approach is a combination of the above two. However, unless the use of biotin-streptavidin as the active assay component is completely avoided, none of these three strategies truly solve the problem of biotin interference. Another major problem with all the above solutions is that the assay component's involvement in preventing interference can easily affect the amplitude of the assay signal itself.

[0010] U.S. Patent 5,212,063 teaches the use of polymer particles having a biotin-binding core and a coating of protein, carbohydrate, or copolymer for the purpose of filtering free biotin, but not biotin conjugated to large molecules. This method may be effective for some assay formats, but it also introduces particles that may generate additional absorbance that interferes with the assay signal.

[0011] The medical diagnostics field utilizes many different forms of assay techniques. One example of a commercially available assay is the luminescent oxygen channel assay (LOCI®). The LOCI® advanced chemiluminescence assay is described, for example, in U.S. Patent No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference. Currently available LOCI® techniques are highly sensitive and utilize several reagents. Specifically, the LOCI® assay requires two of these reagents (referred to as “sensibead” and “chemibead”) to be held together by other specifically binding pair assay reagents in such a manner that the sensibead and chemibead are brought close to each other to achieve a signal. Upon exposure to light of a specific wavelength, the sensibead releases singlet oxygen, and if the two beads are brought close together, the singlet oxygen is transferred to the chemibead; this triggers a chemical reaction that causes the chemibead to emit light, which can be measured at different wavelengths.

[0012] However, there are no currently available methods for biotin assays in the form of LOCI® or for eliminating interference caused by biotin in the form of LOCI®.

[0013] Therefore, there is a need in the art for new and improved assays for detecting and correcting biotin interference that overcome the shortcomings and defects of existing technologies. This disclosure relates to such assays, as well as kits and microfluidic devices containing them, and methods of using them. Brief description of the attached diagram

[0015] Figure 1 The multiple determination components used in one non-limiting embodiment of this disclosure are schematically depicted.

[0016] Figure 2 schematically depicting by Figure 1 The complex formed by multiple assay components depends on the presence of biotin and target analytes in the test sample.

[0017] Figure 3 Two non-limiting embodiments are schematically depicted, including receptor beads (top small figure) that can be utilized according to this disclosure and multiple determinations performed according to this disclosure (bottom small figure).

[0018] Figure 4 The following describes the assay sequence of a non-limiting embodiment of a multiplex assay constructed according to this disclosure, wherein two analytes are measured: protein X (PrX) and biotin. S, sample. S-biotin, sample biotin. Ab1, primary antibody against PrX. Ab2, secondary antibody against PrX. CB, chemibead. SB, sensibead. “::” represents binding.

[0019] Figure 5 The graph depicts how the biotin signal is converted into concentration ([biotin] ng / ml) through appropriate calibration.

[0020] Figure 6 The graphic depicts various situations related to the interference of biotin on the target analyte signal and its correction in a non-limiting embodiment of multiple determination according to this disclosure.

[0021] Figure 7 It includes a table that provides hypothetical scenarios for data obtained through multiple assays according to this disclosure and shows the percentage of interference of sample biotin with the target analyte signal.

[0022] Figure 8 The invention includes a non-limiting method by which a target analyte signal can be corrected based on the sample biotin signal or concentration.

[0023] Figure 9 It contains another non-limiting method by which the target analyte signal can be corrected based on the sample biotin signal or concentration.

[0024] Detailed description

[0025] Before explaining in detail at least one embodiment of this disclosure by way of exemplary language and results, it should be understood that this disclosure is not limited to its application of the details of the construction and arrangement of components set forth in the following description. This disclosure can have other embodiments, or can be practiced or implemented in various ways. Therefore, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Moreover, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0026] Unless otherwise defined herein, the scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. The foregoing techniques and procedures are generally practiced according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature and laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0027] All patents, published patent applications, and non-patent publications mentioned in this specification indicate the level of skill of a person skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the extent that each individual patent or publication is specifically and individually indicated to be incorporated by reference.

[0028] In view of this disclosure, all articles, compositions, kits, and / or methods disclosed herein can be prepared and performed without excessive experimentation. Although the articles, compositions, kits, and / or methods have been described in particular embodiments, it will be apparent to those skilled in the art that variations may be applied to the articles, compositions, kits, and / or methods, as well as the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that are considered obvious to those skilled in the art are within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0029] As used in accordance with this disclosure, unless otherwise instructed, the following terms shall be understood to have the following meanings:

[0030] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the terms "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Therefore, the terms "a," "an," and "the" include plural indicators unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "multiple" means "two or more."

[0031] The term "at least one / type" will be understood to include one / type and any quantity of more than one / type, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100 / type, etc. The term "at least one / type" may be extended to 100 or 1000 or more / types, depending on the term it is attached to; furthermore, a quantity of 100 / 1000 is not considered limiting, as higher limits can also produce satisfactory results. Furthermore, the term "at least one / type of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing two or more items and does not imply any order or sequence of importance of one item relative to another, or any order of addition.

[0032] The term "or" is used in claims to mean inclusive "and / or" unless explicitly stated to refer only to alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfy the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0033] As used herein, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “an example” means that a specific element, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, the phrases “some embodiments” or “one example” appearing in various places in the specification do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as non-limiting to the claims.

[0034] Throughout this application, the term "about" is used to indicate that the value includes inherent variations in the error of the composition / instrument / device used to determine the value, or variations present among study subjects. For example, but not limited to, when using the term "about," the specified value may differ from the listed values ​​by plus or minus 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, because such variations are applicable to performing the disclosed methods and are understood by one of ordinary skill in the art.

[0035] As used in this specification and one or more claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or method steps.

[0036] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is significant in the particular context. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless it is apparent from the context.

[0037] As used herein, the term "substantially" means that the subsequently described event or situation occurs completely or extensively. For example, when relating to a specific event or situation, the term "substantially" means that the subsequently described event or situation occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are closely adjacent to each other but not 100% adjacent, or that a part of one of the two items is not 100% adjacent to the other item but is closely adjacent to it.

[0038] As used herein, the phrases “associated with” and “coupled to” include both direct association / binding of two parts to each other and indirect association / binding of two parts to each other. Non-limiting examples of association / coupling include, for example, covalently binding one part to another through a direct bond or through a spacer group; non-covalently binding one part to another directly or by means of a specific binding pair member that binds to said part; incorporating one part into another by dissolving one part in another or by synthesis; and coating one part onto another.

[0039] The terms “analog” and “derivative” are used interchangeably herein and refer to substances that contain the same basic carbon skeleton and carbon functionality as a given compound in their structure, but may also contain one or more substitutions thereof. As used herein, the term “substitution” will be understood to mean replacing at least one substituent on a compound with residue R. In some non-limiting embodiments, R may include H, hydroxyl, thiol, a halide selected from fluorides, chlorides, bromides, or iodides, and a C1-C4 compound selected from: optionally substituted straight-chain, branched, or cyclic alkyl groups, and straight-chain branched or cyclic alkenyl groups, wherein the optional substituents are selected from one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylhexacycloalkyl groups. The group, wherein each is optionally substituted, wherein the optional substituent is selected from one or more of the following: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl and arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxyl and -C(O))-alkyl.

[0040] As used herein, the term "sample" will be understood to include any type of biological sample that may be utilized under this disclosure. Examples of available fluid biological samples include, but are not limited to: whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0041] As used herein in the terms “biotin-specific binding coupler” or “target analyte-specific binding coupler” (but not by way of limitation), the term “specific binding coupler” should be understood to mean any molecule capable of specifically binding to either biotin or a target analyte, respectively. For example, but not limited to, the binding coupler can be an antibody, receptor, ligand, aptamer, molecularly imprinted polymer (i.e., an inorganic matrix), combinations thereof, or derivatives thereof, and any other molecule capable of specifically binding to either biotin or a target analyte, respectively.

[0042] The term "antibody" is used in the broadest sense herein and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates (such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, biantibodies, single-chain antibodies, and other antibody fragments and conjugates that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. The antibody may be any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0043] As used herein, the term "hapten" refers to a small protein or non-protein antigenic determinant (or "epitope") that can be recognized by a target analyte-specific binding partner, such as (but not limited to) an antibody. The term "multiple hapten" as used herein should be understood to refer to a synthetic molecule containing multiple epitopes / antigenic determinants to which it is attached.

[0044] An analyte is a large molecule that can be recognized by an analyte-specific binding partner, such as (but not limited to) an antibody. Both analytes and haptens contain at least one antigenic determinant or "epitope," which is a region of the antigen or hapten that binds to an analyte-specific binding partner (i.e., an antibody). Typically, the epitope on a hapten is the entire molecule.

[0045] Certain non-limiting embodiments of this disclosure relate to multiplex assays for detecting both biotin and one or more target analytes in a sample, as well as kits containing the same and methods of use thereof. In some assay embodiments, the signal generation system (SPS) member includes a sensitizer, such as, for example, a photosensitizer, and two or more chemiluminescent-fluorescent molecular compositions (wherein a first chemiluminescent composition generates a signal associated with the presence of biotin, and at least a second chemiluminescent composition generates a signal associated with the presence of the target analyte); in these assay embodiments, activation of the sensitizer produces a product that activates one or more chemiluminescent compositions, thereby generating a detectable signal relating to the amount of bound target analyte and bound biotin detected. An exemplary (but non-limiting) embodiment of an assay platform upon which this disclosure may be based is the luminescent oxygen channel assay (LOCI®; Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The LOCI® assay is described, for example, in U.S. Patent No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0046] Any target analyte that can be detected by the assays described herein or otherwise considered can be detected by the multiplex assays of this disclosure. Non-limiting examples of target analytes include: procalcitonin (PCT), BNP, NT-proBNP, D-dimer, CKMB, myoglobin, myeloperoxidase, ST2, hCG, LH, FSH, iPTH, TSH, fT4, T4, PSA, fPSA, and cPSA, and combinations thereof.

[0047] Some non-limiting embodiments of this disclosure relate to kits for performing multiplex assays utilizing a chemiluminescence detection system for determining the concentrations of biotin and at least one additional target analyte in a sample. The kit comprises: (a) a composition comprising a chemiluminescent compound having a singlet oxygen-activated chemiluminescent compound having a biotin or an analogue thereto directly or indirectly bound thereto, and a fluorescent molecule excited by the activated chemiluminescent compound; (b) a composition comprising a singlet oxygen-activated chemiluminescent compound having a first analyte-specific binding pair of a target analyte having a first analyte-specific binding pair of the target analyte having a first analyte-specific binding pair of the target analyte having a first analyte-specific binding pair of the target analyte having a second ...

[0048] In some non-limiting embodiments, the kit further comprises: (e) a composition comprising a singlet oxygen-activated chemiluminescent compound having a first analyte-specific binding partner of a second target analyte that binds directly or indirectly thereto, and a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) a biotinylated second analyte-specific binding partner of a second target analyte, wherein the second analyte-specific binding partner binds an epitope of the second target analyte that is different from the first analyte-specific binding partner.

[0049] Chemiluminescent compounds (chemiluminescent agents) are chemically activatable compounds that emit light of a specific wavelength as a result of such activation. Examples of chemiluminescent agents, by way of example and not limitation, include, for instance: alkenes capable of reacting with singlet oxygen or peroxides to form hydroperoxides or dioxetanes that can decompose into ketones or carboxylic acid derivatives; stable dioxetanes that can decompose under light; alkynes capable of reacting with singlet oxygen to form diketones; hydrazones or hydrazides capable of forming azo compounds or azocarbonyl compounds, such as (but not limited to) luminol; and aromatic compounds capable of forming internal peroxides. As a result of the activation reaction, chemiluminescent agents directly or indirectly induce luminescence.

[0050] In some embodiments, the singlet oxygen-activated chemiluminescent compound can be a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate, which can decompose and emit light simultaneously or subsequently. A composition containing a chemiluminescent compound can be directly excited by the activated chemiluminescent compound; or, the composition may further contain at least one fluorescent molecule excited by the activated chemiluminescent compound.

[0051] A sensitizer is a molecule, typically a compound, that generates a reactive intermediate (such as, for example, singlet oxygen) for activating a chemiluminescent compound. In some non-limiting embodiments, the sensitizer is a photosensitizer. Other sensitizers that can be chemically activated (by, for example, enzymes and metal salts) by example, and not by limitation, include other substances and compositions that can generate singlet oxygen with or without activation by an external light source. For example, certain compounds have been shown to catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Non-limiting examples of other sensitizer substances and compositions include oxides of alkaline earth metals Ca, Sr, and Ba; d 0 Derivatives of elements in groups 3A, 4A, SA, and 6A in the configuration; oxides of actinides and lanthanides; and the oxidizing agent CIO. - BrO - Au 3+ IO3 - and IO4 - Specifically, molybdate, peroxymolybdate, tungstate, and peroxytungstate ions, and acetonitrile. The following references (which are expressly incorporated herein by reference in their entirety) provide further disclosure regarding sensitizing substances and compositions that also fall within the scope of this disclosure: Aubry, J. Am. Chem. Soc. , 107:5844-5849 (1985); Aubry, J. Org. Chem ., 54:726-728 (1989); Böhme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foote, Inorg. Chem., 31:3472-3476 (1992); Nardello et al., Inorg. Chem., 34:4950-4957 (1995); Aubry and Bouttemy, J. Am. Chem. Soc., 119:5286-5294 (1997); and Almeida et al., Anal. Chim. Acta , 482:99-104 (2003); the full contents of each are hereby expressly incorporated by reference.

[0052] The scope of photosensitizers also includes compounds that are not true sensitizers, but which release singlet oxygen molecules upon excitation by heat, light, ionizing radiation, or chemical activation. Members of this class include, for example (but not limited to), internal peroxides, such as 1,4-dicarboxyethyl-1,4-naphthalene internal peroxide, 9,10-diphenylanthracene-9,10-internal peroxide, and 5,6,11,12-tetraphenylnaphthalene-5,12-internal peroxide. Heating these compounds or direct absorption of light by these compounds releases singlet oxygen.

[0053] A photosensitizer is a sensitizer used to activate a photoactive compound by, for example, photoexcitation to generate singlet oxygen. Photosensitizers are photoactivated and include, for example, dyes and aromatic compounds, and are typically compounds composed of covalently bonded atoms, usually having multiple conjugated double or triple bonds. The compound should absorb light in the wavelength range of about 200 nm to about 1,100 nm, such as (but not limited to) about 300 nm to about 1,000 nm, or about 450 nm to 950 nm, and at the excitation wavelength, have an extinction coefficient greater than 500 M at its maximum absorbance. -1 cm -1 or greater than 5,000 M -1 cm -1 or greater than 50,000 M -1 cm -1 Photosensitizers should be relatively photostable and should not react effectively with singlet oxygen. Examples of photosensitizers, by way of example and not limitation, include: acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metalloporphyrins such as (but not limited to) hematoporphyrin; phthalocyanine; chlorophyll; rose red; and barkminsterfullerene and derivatives of these compounds.

[0054] Specific, non-limiting examples of chemiluminescent compounds and photosensitizers utilized in this disclosure are described in U.S. Patent No. 5,340,716 (Ullman, et al.), the entire contents of which are expressly incorporated herein by reference.

[0055] Any target analyte-specific binding conjugate known in the art or otherwise considered herein may be utilized in accordance with this disclosure. Non-limiting examples of target analyte-specific binding conjugates include antibodies, receptors, ligands, aptamers, molecularly imprinted polymers (i.e., inorganic matrices), any combination or derivative thereof, and any other molecule capable of specifically binding to a target analyte. In one specific (but non-limiting) example, the first and second analyte-specific binding conjugates of (a) and (b) are each antibodies against the target analyte. Similarly, in one specific (but non-limiting) embodiment, if present, the first and second analyte-specific binding conjugates of (e) and (f) may each be antibodies against a second target analyte.

[0056] Any biotin-specific binding partner known in the art or otherwise considered herein may be used in accordance with this disclosure. In some non-limiting embodiments, the biotin-specific binding partner is an antibody against biotin. In other non-limiting embodiments, the biotin-specific binding partner is an anti-biotin protein or an analogue thereof.

[0057] According to this disclosure, any avidin analogue known in the art or otherwise considered herein can be used, provided that the avidin or avidin analogue: (1) is capable of associating with a sensitizer; (2) is capable of binding a biotinylated analyte-specific binding partner; and (3) is capable of binding biotin that may be present in the sample. Non-limiting examples of avidin analogues that can be used according to this disclosure include Kang et al. ( J Drug Target Those disclosed in (1995) 3:159-65 (the entire contents of which are expressly incorporated herein by reference). Specific, non-limiting examples of avidin analogues include avidin, streptoavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, esters, salts, and / or derivatives of any of the foregoing.

[0058] In some non-limiting embodiments, the singlet oxygen of (a) and / or (b) can activate chemiluminescent compounds having haptens that bind directly or indirectly thereto.

[0059] Any fluorescent molecule known in the art that can be excited by an activated chemiluminescent compound and emit light at a specific, detectable wavelength may be used, according to this disclosure, as the fluorescent molecule in (a) and (b) (and (e), if present), provided that the signal generated by each fluorescent molecule is detectable differently from the signal generated by the other fluorescent molecules utilized. That is, the fluorescent molecule in (a) must emit light at a wavelength sufficiently different from the wavelength emitted by the fluorescent molecule in (b), such that the two signals can be distinguished from each other when detected simultaneously. In a specific (but not limiting) example, each fluorescent molecule utilized according to this disclosure is independently selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not in a limiting way), regarding the generation of two or three signals that can be distinguished from each other when detected simultaneously, terbium emits light at a wavelength of about 545 nm, uranium emits light at a wavelength of about 612 nm, and samarium emits light at a wavelength of about 645 nm.

[0060] The assay components / reagents of the composition / kit / microfluidic device / method can be provided in any form that allows them to function according to the concepts of this disclosure. For example, but not limitingly, each reagent may be provided in liquid form and presented in the kit in bulk and / or as a single aliquot. Alternatively, in a particular (but not limiting) embodiment, one or more reagents may be presented in the kit as a single aliquot lyophilized reagent. The use of dried reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0061] In addition to the assay components / reagents described in detail above, the kit may further contain other reagents for performing any specific assays described herein or otherwise considered. The properties of these additional reagents will depend on the specific assay format, and their identification is entirely within the skill of a person skilled in the art; therefore, it is not considered necessary to describe them further. Furthermore, the components / reagents present in the kit may each be in separate containers / compartments, or various components / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0062] The relative amounts of the various components / reagents in the kit can be widely varied to provide component / reagent concentrations that substantially optimize the reactions that need to occur during the assay and further substantially optimize the sensitivity of the assay. Where appropriate, one or more components / reagents in the kit may be provided as dry powders, such as lyophilized powders, and the kit may further include excipients for dissolving the dried reagents; in this way, a reagent solution having an appropriate concentration for performing the method or assay according to this disclosure can be obtained from these components. The kit may also include positive and / or negative controls. Furthermore, the kit may further include a set of written instructions explaining how to use the kit. Kits of this nature can be used in any method described herein or otherwise considered.

[0063] Certain additional non-limiting embodiments of this disclosure relate to microfluidic devices that include components of any of the kits described above. Specifically, certain non-limiting embodiments include microfluidic devices for determining the concentrations of biotin and at least one additional target analyte in a sample. The microfluidic device includes: (i) an inlet channel through which a sample is applied; and (ii) at least one first compartment capable of fluid communication with said inlet channel. (ii) contains: (a) at least a first composition comprising a singlet oxygen-activated chemiluminescent compound having a biotin or an analogue thereto directly or indirectly bound thereto, the first composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound; (b) at least a second composition comprising a singlet oxygen-activated chemiluminescent compound having a first analyte-specific binding pair of a target analyte that has a direct or indirect binding pair thereto, the second composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a); (c) a biotinylated second analyte-specific binding pair of a target analyte, wherein the second analyte-specific binding pair binds an epitope of the target analyte that is different from the first analyte-specific binding pair of (b); and (d) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding pair thereto directly or indirectly bound thereto. In some specific (but not limiting) embodiments, the compartment of (ii) further contains at least a third composition comprising a singlet oxygen-activated chemiluminescent compound and having a second target analyte or analogue thereto directly or indirectly bound thereto, the second composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b).

[0064] In the microfluidic devices of this disclosure, any singlet oxygen can be used to activate chemiluminescent compounds, sensitizers, fluorescent molecules, biotin or analogues thereof, target analytes or analogues thereof, and target analyte-specific binding couplers, as described in detail above or otherwise considered herein.

[0065] For example, in some specific (but not limiting) embodiments, the singlet oxygen-activated chemiluminescent compounds of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate that can decompose and emit light simultaneously or subsequently.

[0066] In a specific (but not limiting) implementation, the sensitizer is a photosensitizer.

[0067] In a specific (but not limiting) implementation, the first and second analyte-specific binding couples in (b) and (c) are each antibodies against the target analyte.

[0068] In a specific (but not limiting) implementation, the biotin-specific binding partner of (iii) is an anti-biotin protein or an analogue thereof, or an antibody against biotin.

[0069] In specific (but not limiting) implementations, the singlet oxygen of (a) and / or (b) can activate chemiluminescent compounds having haptens that bind directly or indirectly to them.

[0070] In specific (but not limiting) embodiments, the fluorescent molecules in (a) and (b) are each independently selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not in a limiting manner), terbium emits light at a wavelength of about 545 nm, uranium emits light at a wavelength of about 612 nm, and samarium emits light at a wavelength of about 645 nm.

[0071] In a specific (but not limiting) embodiment, the compartment of (ii) further comprises: (e) at least a third composition comprising a singlet oxygen-activated chemiluminescent compound and having a first analyte-specific binding partner for the second target analyte that binds directly or indirectly thereto, the second composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) a biotinylated second analyte-specific binding partner for the second target analyte, wherein the second analyte-specific binding partner binds to an epitope of the second target analyte that is different from the first analyte-specific binding partner of (e). In one specific (but not limiting) embodiment, the fluorescent molecule of the third composition is selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium.

[0072] In some non-limiting embodiments, all elements (a)-(d) (and (e) and (f), if present) of (ii) are present in the same compartment. In alternative non-limiting embodiments, elements (a)-(d) (and (e) and (f), if present) are separated between two or more compartments.

[0073] The device can provide any arrangement of compartments and the distribution of various components therebetween, which allows the device to function according to this disclosure.

[0074] Any compartment of a microfluidic device can be sealed to maintain the reagents contained therein in a substantially hermetically sealed environment until they are used; for example, a compartment containing lyophilized reagents can be sealed to prevent any unintentional remodeling of the reagents. An inlet channel and a compartment, as well as two compartments, can be described as “capable of fluid communication with each other”; this phrase indicates that each of the compartments can still be sealed, but that the two compartments are able to have fluid flow between them after a seal formed therein or between them is punctured.

[0075] The microfluidic device of this disclosure may provide any other desired features known in the art or otherwise considered herein. For example, but not limitingly, the microfluidic device of this disclosure may further include a reading chamber; the reading chamber may be any compartment containing the reagents described above, or the reading chamber may be in fluid communication with the compartment. The microfluidic device may further include one or more additional compartments containing other solutions, such as (but not limited to) washing solutions, diluents, excipients, interfering solutions, positive controls, negative controls, mass controls, etc. These additional compartments may be in fluid communication with one or more other compartments. For example, the microfluidic device may further include one or more compartments containing washing solutions, and these compartments may be in fluid communication with any other compartment of the device. In another example, the microfluidic device may further include one or more compartments containing excipients for dissolving one or more drying reagents, and these compartments may be in fluid communication with any other compartment of the device. In yet another further example, the microfluidic device may include one or more compartments containing diluents, and these compartments may be in fluid communication with any other compartment of the device.

[0076] Furthermore, any kit / microfluidic device described herein or otherwise considered may include more than one target analyte assay reused with the biotin assay in a single kit / device. When multiple target analyte assays are present, each assay may be constructed and function as described herein. Alternatively, the biotin and target analyte assays described herein may be reused with any other target analyte assays known in the art that can be included in the kits / microfluidic devices disclosed herein. Non-limiting examples of other assays that may be reused with the assays disclosed and claimed herein include BNP, NT-proBNP, D-dimer, CKMB, myoglobin, myeloperoxidase, ST2, PCT, hCG, LH, FSH, iPTH, TSH, fT4, T4, PSA, fPSA, and cPSA, and combinations thereof.

[0077] When multiple target analyte determinations are present in a single microfluidic device, multiple inlet channels can be connected to the sample application chamber. In some embodiments, portions of the sample can pass through the sample application chamber to multiple inlet channels, regardless of their contents. Alternatively, structures may exist in the connection between the sample application chamber, the inlet channels, and / or the inlet channels, allowing the separation of certain components from the whole sample and delivery of said components to different determinations. Non-limiting examples of sample dispensing devices utilized in this disclosure can be described in detail in U.S. Patent No. 9,416,776 (Ledden, et al.), the entire contents of which are expressly incorporated herein by reference.

[0078] Some non-limiting embodiments also relate to methods for detecting the presence and / or concentration of biotin and at least one additional target analyte in a sample. The methods include the following steps.

[0079] In the first step, a sample suspected of containing biotin and at least one additional target analyte is sequentially combined with the following, simultaneously or in whole or in part: (a) at least a first composition comprising a singlet oxygen-activated chemiluminescent compound having biotin or an analogue thereof directly or indirectly bound thereto, the first composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound; (b) at least a second composition comprising a singlet oxygen-activated chemiluminescent compound having a first analyte-specific binding pair of the target analyte directly or indirectly bound thereto, the second composition... The composition further comprises (c) a fluorescent molecule excited by an activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a); (d) a biotinylated second analyte-specific binding partner of the target analyte, wherein the second analyte-specific binding partner binds an epitope of the target analyte that is different from the first analyte-specific binding partner of (b); and (d) an excess of the composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner that binds directly or indirectly thereto.

[0080] In the second step, the components are incubated together to allow (b) and (c) to bind to the target analyte present in the sample, and (c) to bind to (d), wherein the indirect binding of (b) and (d) (via (c) and the target analyte) results in the formation of a target analyte complex, wherein the sensitizer is brought into close proximity to the chemiluminescent compound. Additionally, (a) binds to (d) in the absence of biotin in the sample and results in the formation of a biotin complex, wherein the sensitizer is brought into close proximity to the chemiluminescent compound.

[0081] In the third step, the sensitizer is activated to generate singlet oxygen, wherein the activation of the sensitizer present in the biotin complex and the target analyte complex causes the activation of the chemiluminescent compound present in each complex.

[0082] In the fourth step, the amount of chemiluminescence generated by the chemiluminescent compound activated in the biotin complex is determined by measuring the amount of light emitted by the fluorescent molecules in (a), wherein the amount of biotin in the sample is inversely proportional to the amount of emitted light.

[0083] In the fifth step, the amount of target analyte in the sample is determined by measuring the amount of light emitted by the fluorescent molecules in (b) to determine the amount of chemiluminescence generated by the chemiluminescent compound activated in the target analyte complex.

[0084] In step six, steps two through five may be repeated if desired.

[0085] In the seventh step, the result of step (5) is corrected based on any biotin interference detected in step (4). Alternatively, if the biotin concentration detected in step (4) is higher than the maximum threshold level, the result of step (5) is marked as unreliable.

[0086] In some non-limiting embodiments of the method, step (1) further comprises, in combination with elements (a)-(d): (e) at least a third composition comprising a singlet oxygen-activated chemiluminescent compound having a first analyte-specific binding partner for the second target analyte that is directly or indirectly bound thereto, the third composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) a biotinylated second analyte-specific binding partner for the second target analyte, wherein the second analyte-specific binding partner binds an epitope of the second target analyte that is different from the first analyte-specific binding partner of (e). In this case, the method may further include the following steps: (8) determining the amount of chemiluminescence generated by the chemiluminescent compound activated in the second target analyte complex by measuring the amount of light emitted by the fluorescent molecules of (e) to determine the amount of target analyte in the sample; and (9) correcting the result of step (8) based on any biotin interference detected in step (4), and / or marking the result of step (8) as unreliable if the biotin concentration detected in step (4) is higher than the maximum threshold level.

[0087] In the methods of this disclosure, any singlet oxygen can be used to activate chemiluminescent compounds, sensitizers, fluorescent molecules, biotin or analogues thereof, target analytes or analogues thereof, and target analyte-specific binding couplers, as described in detail above or otherwise considered herein.

[0088] For example, in certain specific (but not limiting) embodiments, the singlet oxygen-activated chemiluminescent compounds (a) and (b) (and (e), when present) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate that can decompose and emit light simultaneously or subsequently.

[0089] In a specific (but not limiting) implementation, the sensitizer is a photosensitizer, and the activation of the sensitizer in step (3) includes irradiation with light (such as, but not limited to, irradiation at about 680 nm).

[0090] In a specific (but not limiting) implementation, the first and second analyte-specific binding couples of (b) and (c) (and (e) and (f), if present) are each antibodies against the target analyte.

[0091] In a specific (but not limiting) implementation, (d) the biotin-specific binding partner is an anti-biotin protein or an analogue thereof, or an antibody against biotin.

[0092] In specific (but not limiting) implementations, the singlet oxygen of (a) and / or (b) can activate chemiluminescent compounds having haptens that bind directly or indirectly to them.

[0093] In specific (but not limiting) embodiments, the fluorescent molecules in (a) and (b) (and (e), if present) are each independently selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not in a limiting manner), terbium emits light at a wavelength of about 545 nm, uranium emits light at a wavelength of about 612 nm, and samarium emits light at a wavelength of about 645 nm.

[0094] Any sample for which the presence of biotin can be determined using a target needle can be used as a sample according to the method of this disclosure. Non-limiting examples of samples include biological samples such as, but not limited to, whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder lavage fluid, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0095] As mentioned above, the various components of the method are provided in combination (simultaneously or sequentially). The order in which the components are added can be varied when they are added sequentially; those skilled in the art can determine the specific desired order in which different components are added to the assay. The simplest order of addition is, of course, to add all substances simultaneously and measure the signal generated therefrom. Alternatively, each component, or a group of components, can be combined sequentially. In some embodiments, an incubation step may be included after one or more additions.

[0096] In an alternative (but non-limiting) embodiment, step (1) of the method includes first combining the sample with a biotinylated target analyte-specific binding coupler and a composition containing a sensitizer, incubating it, and then adding a composition containing a singlet oxygen-activated chemiluminescent compound. Alternatively, step (1) of the method may include first combining the sample with a composition containing a singlet oxygen-activated chemiluminescent compound, incubating it, and then adding a composition containing a sensitizer. In this latter embodiment, the biotinylated target analyte-specific binding coupler may be added before or after the incubation step.

[0097] Although specific embodiments of this disclosure are described as having a LOCI® assay, it should be understood that this disclosure also relates to other assays (and kits, microfluidic devices, and methods of execution thereof) for which it is desirable to eliminate biotin interference in the sample. For example (but not in a limiting way), this disclosure also includes assays in which different signaling molecules (such as (but not limited to) different antibodies linked to different enzymes that generate signals at different wavelengths) can be used instead of the composition containing the chemiluminescent compound described above. Example

[0098] Examples are provided below. However, it should be understood that the application of this disclosure is not limited to the specific experiments, results, and laboratory procedures disclosed herein. Rather, these examples are provided merely as one of various implementations and are intended to be exemplary, not exhaustive.

[0099] Example 1

[0100] Figure 1 The assay components present in kits and microfluidic devices according to certain non-limiting embodiments of this disclosure and used in methods are depicted. These components include (from left to right):

[0101] (i) A first chemibead having biotin or an analogue thereto directly or indirectly and containing a first fluorescent molecule excited by an activated chemiluminescent compound present in the chemibead;

[0102] (ii) sensibead, which is capable of generating singlet oxygen in its excited state and has a biotin-specific binding partner that binds directly or indirectly to it;

[0103] (iii) Biotinylated target analytes – specifically binding couplers; and

[0104] (iv) A second chemibead having a target analyte or its analogue that is directly or indirectly bound thereto and containing a second fluorescent molecule excited by an activated chemiluminescent compound present in the chemibead.

[0105] The first and second fluorescent molecules are different from each other and emit light at different wavelengths; in this way, both biotin and the target analyte can be measured simultaneously in the same reaction, since the complex containing (i) is detected at a different wavelength than the complex containing (iv).

[0106] When biotin is absent in the sample, a complex forms between (i) and (ii), as in Figure 2 As shown on the left side of the upper inset, a signal is generated after the sensitizer is activated. However, when biotin is present in the sample, it competes with (i) for binding to (ii), as... Figure 2 The lower small diagram is shown on the left. Therefore, the presence of biotin in the sample is detected as a reduction in the signal detected at the first wavelength used to detect light emitted from the first fluorescent molecule.

[0107] Additionally, in the same reaction, (iii) binds to the target analyte present in the sample, and then (iv) binds to (iii), as... Figure 2 The lower right of the small diagram is shown. Therefore, the presence of the target analyte in the sample is detected as an increase in the signal detected at the second wavelength used to detect light emitted from the second fluorescent molecule. Conversely, when the target analyte is absent, only (iii) can bind to (ii), but (iv) cannot, and therefore no signal is generated in the absence of the target analyte. Figure 2 (The left side of the upper small image).

[0108] If biotin is detected in the sample, the results obtained from the target analyte determination can be corrected for any biotin interference detected using the first chemibead, and / or if the detected biotin concentration is above the maximum threshold level, it is marked as unreliable.

[0109] Furthermore, in addition to detecting biotin interference, the multiplex assay of this disclosure is also suitable for the simultaneous detection of more than one target analyte. For each additional target analyte to be detected, an additional component (such as (iii) and (iv)) is added, wherein the fluorescent molecule present in the additional component (such as (iv)) differs from the first and second fluorescent molecules in that it emits light at a wavelength that is different from and can be detected separately. In this way, two, three, four, five, six, seven, eight, nine, ten or more target analytes can be detected in a single reaction, provided that each chemibead used for detection contains its own different fluorescent molecule that emits light at different and can be detected separately; therefore, the limiting factor for how many target analytes can be detected in a single reaction is the number of available fluorescent molecules that function as described herein.

[0110] Example 2

[0111] This embodiment provides multiplex assays of biotin and procalcitonin (PCT). Biotin is covalently conjugated to terbium-stained chemibead (Chemibead). Figure 3 (Left side of the upper small image), and the capture antibody for PCT is covalently conjugated to chemibead stained with europium ( Figure 3 (See the right side of the upper inset). Multiplex methods are calibrated using analytes containing at least both biotin and PCT. The same streptavidin protein sensibead is used to detect both biotin and PCT.

[0112] like Figure 3 As shown in the lower inset, sensibeads are activated by excitation light at 680 nm to generate singlet oxygen, which diffuses into both types of chemibeads. When complexed with biotin, the biotin chemibead emits light at 545 nm, which is measured as the biotin signal. When complexed with PCT, the PCT chemibead emits light at 612 nm, which is measured as the PCT signal. Two calibration curves are then generated from the multiple measurements: one for biotin and one for PCT.

[0113] Biotin concentration measured in the sample using terbium as the fluorescent molecule and biotin LOCI measured using europium as the fluorescent molecule. ® Signal correlation. The LOCI was obtained experimentally at various biotin concentrations, measured by europium. ® The signal can then be used to predict (extrapolate) the biotin LOCI at a specific biotin concentration via europium. ®The signal is then subtracted (if a positive effect on the signal is observed) or added (if an adverse effect on the signal is observed) to correct for the results of the target analyte, if necessary.

[0114] In this way, the biotin signal, derived from biotin measurement in multiple ways, can be used to correct the target analyte determination results. Alternatively, if the detected biotin concentration is above the maximum threshold level, the result can be marked as unreliable.

[0115] Example 3

[0116] Figure 4 The following describes the assay sequence of a non-limiting embodiment of a multiplex assay constructed according to the present disclosure, wherein two analytes are to be measured: a target analyte (referred to in this embodiment as protein X (PrX)) and biotin. The assay for the target analyte PrX is a sandwich assay requiring the use of two antibodies; a first antibody (Ab1) is coated onto a chemibead (Ab1-CB), and a second antibody (Ab2) is biotinylated. The biotin assay uses a competitive form and requires the use of a biotin- (or biotin analog-) coated chemibead (Biotin-CB). Ab1-CB and Biotin-CB each contain different dyes, allowing for the separate detection of complexes containing the two chemibeads in a single reaction. Furthermore, both assays use the same streptavidin-coated sensibead (SB).

[0117] although Figure 4 One order of addition for this multiplex assay is described, but this order should not be considered a limitation of this disclosure; in fact, the various assay components can be added in any order, and the two assays can be performed in any desired order. Therefore, Figure 4 The order and sequence of additions of the measurements shown are not limiting of the scope of this disclosure.

[0118] By performing appropriate calibration using a standard curve, the biotin signal obtained from the above biotin assay can be converted into the sample biotin concentration ([biotin] ng / ml), such as... Figure 5 As shown (and described in detail in U.S. Serial No. 62 / 711,694 (filed July 30, 2018)). In some non-limiting embodiments, this concentration can be obtained prior to the determination of the target analyte. The calculated biotin concentration can be utilized in one of four different ways:

[0119] (1) If the biotin signal or the calculated biotin concentration is below the threshold (it is known that above the threshold, biotin begins to interfere with the determination of the target analyte), the target analyte determination result is reported as normal.

[0120] (2) If the biotin signal or the calculated biotin concentration is above the threshold level (i.e., above the level at which biotin is known to begin to interfere with the determination of the target analyte), the target analyte result is marked as unreliable and not reported.

[0121] (3) If the biotin signal or calculated biotin concentration is higher than the threshold level, the biotin concentration or signal value is used to correct the target analyte determination results.

[0122] (4) This option utilizes a combination of (1), (2), and (3) – that is, when biotin levels are higher than the threshold level affecting target analyte determination, but not overwhelmingly exceeding the threshold level, the affected target analyte determination results are corrected. If the detected biotin signal / calculated concentration is low (i.e., below the threshold level, so that biotin does not interfere with the target analyte results), the target analyte results are reported as normal. If the detected biotin signal / calculated concentration is too high (i.e., above the maximum threshold level) and cannot be corrected, it should be flagged, and the target analyte results should be marked as unreliable.

[0123] Figure 6 The diagram illustrates various scenarios related to biotin interference with target analyte signals and their possible corrections. The top curve indicates the measurements obtained in the absence of biotin. The curve directly below the top curve represents a low biotin concentration with minimal impact on the target analyte signal; in this case, no correction is needed for the target analyte results. In the next curve, the biotin concentration / signal exceeds a threshold, indicating biotin interference with the target analyte measurement; in this case, correction is required based on the detected amount of biotin interference. That is, the target analyte measurement results are corrected using either the biotin concentration or signal value. In the bottom curve, the biotin concentration or signal value is significantly higher than the threshold and exceeds a maximum threshold, making it impossible to correct the target analyte concentration; therefore, the amount of biotin interference present necessitates labeling to indicate that the target analyte results are unreliable.

[0124] Regarding the components measured, when biotin is absent or present in minimal amounts in the sample, all biotin-chemibead is present. Figure 4 In the "Rxn stage 1", both bind to sensibead; in this case, the target analyte signal is not interfered with by biotin. When the sample biotin level is sufficiently high, some of the sample biotin and biotin-chemibead do not bind to sensibead. Figure 4 The sensibead added in “Rxn stage 1” binds and therefore continues to exist until Figure 4The sensibead binding reaction in “Rxn stage 2”. Sufficient amounts of sample biotin and biotin-chemibead spill-over will affect Figure 4 The formation of the sensibead / biotin-antibody 1 / target analyte / antibody 2-chemibead complex in "Rxn stage 2" (in Figure 4 The description is "SB::Biotin-Ab1::PrX::Ab2-CB"), which leads to a reduction in the target analyte signal, such as... Figure 6 As shown in the diagram.

[0125] Figure 7 The table above depicts hypothetical scenarios of target analyte (PrX) signals (in thousands) obtained by multiple determination according to this disclosure, both in the absence and presence of sample biotin. It can be seen that at any minimum sample biotin concentration (10 ng / ml), no effect on the target analyte signal is observed at any tested target analyte concentration. Once a threshold biotin concentration is exceeded (e.g., any value between 100 ng / ml and 500 ng / ml), the effect of sample biotin concentration on the PrX signal can be observed, and the PrX value can subsequently be corrected based on the amount of biotin interference present. However, once the maximum threshold level is reached (e.g., any value between 3000 ng / ml), the effect of biotin interference on the PrX signal becomes so large that it is no longer possible to correct for the biotin interference, and therefore the PrX signal must be marked as unreliable. Please note that any value of 3000 ng / ml disclosed is for illustrative purposes only; the scope of this disclosure includes any threshold calculated as described herein or otherwise considered, and includes thresholds much below 3000 ng / ml as well as thresholds much above 3000 ng / ml.

[0126] Figure 7 The table above depicts the detected signals of the actual target analyte, while at the same time Figure 7 The table below depicts the percentage of interference of sample biotin on the PrX signal (based on the values ​​in the table above). It can be seen that even when the sample biotin concentration remains constant, different percentages of interference are observed at different PrX concentrations.

[0127] Figure 8 and 9 Two methods are illustrated, which can be used to correct the target analyte signal based on the detected sample biotin signal / concentration. Figure 9 The method illustrated in the example uses Figure 7 All the data that exists in it, and Figure 8 The method illustrated in the example utilizes from Figure 7A subset of the data. However, it should be understood that these two embodiments are provided for illustrative purposes and any method that can be used to correct the target analyte signal based on the sample biotin signal / concentration will be apparent to those skilled in the art based on this disclosure.

[0128] exist Figure 8 In the middle, using the shaded rectangle in the table above (from Figure 7 (Simply reproduce), using the sample biotin signal / concentration to correct the PrX signal, to provide a more localized correction of the PrX signal. For example... Figure 8 As shown in the table below, the values ​​from the shaded rectangles are rearranged and then input into statistical software, which performs regression based on the data from the shaded rectangles to obtain a correction equation. The regression correction equation obtained in this embodiment is shown below. Figure 8 The bottom.

[0129] This step can be performed during co-calibration using calibrators containing both PrX and biotin at various known concentrations (either during manufacturing or via customer calibration). Co-calibration of the two analytes creates a surface equation separate from the individual calibrations of biotin and PrX. Biotin or PrX single-analyte calibration is performed in the absence of other analytes. However, this co-calibration is performed when both analytes are present at various concentrations. The purpose of co-calibration is to obtain a surface equation in which the PrX concentration (or the PrX signal at 0 ng / ml biotin) acts as a function of the PrX signal in the presence of biotin interference.

[0130] exist Figure 8 The reason for using a subset of data in this method is to ensure more accurate regression for correction at a more local level. Therefore, if the PrX signal and biotin signal / concentration (ng / ml) are known, the accurate PrX concentration (ng / ml) can be calculated.

[0131] Alternatively, the corrected PrX concentration can be calculated by performing a similar regression using the entire obtained table or the entire assay range (biotin and PrX), such as... Figure 9 As shown. In Figure 9 On the left, from Figure 7 The data has been simply rearranged into the table shown. Then, regression analysis was performed as described above to obtain the regression equation, as follows: Figure 9 As shown on the right.

[0132] Regardless of the specific method used Figure 8 and 9 The methods all generate regression equations to predict the concentration of the target analyte (PrX) in the presence of biotin interference.

[0133] Therefore, compositions, kits, and devices, as well as methods of their manufacture and use, have been provided according to this disclosure to fully satisfy the purposes and advantages described above. Although this disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A kit for performing multiplex assays using a chemiluminescence detection system for determining the concentrations of biotin and at least one additional target analyte in a sample, the kit comprising: (a) A composition comprising: Singlet oxygen can activate chemiluminescent compounds having biotin directly or indirectly bound to it, wherein the biotin is capable of binding to a biotin-specific binding protein; and Fluorescent molecules are excited by activated chemiluminescent compounds; (b) A composition comprising: Singlet oxygen can activate chemiluminescent compounds that have a first analyte-specific binding partner for the target analyte, which binds to it directly or indirectly; and A fluorescent molecule that is excited by an activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a); (c) A biotinylated second analyte-specific binding pair of a target analyte, wherein the second analyte-specific binding pair binds an epitope of the target analyte that is different from that of the first analyte-specific binding pair in (b); and (d) A composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner that binds directly or indirectly thereto.

2. The kit of claim 1, wherein the singlet oxygen-activated chemiluminescent compound is a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate, the metastable intermediate being capable of decomposing and emitting light simultaneously or subsequently.

3. The kit of claim 1, wherein the sensitizer is a photosensitizer.

4. The kit of claim 1, wherein the first and second analyte-specific binding couplers of (b) and (c) are each antibodies against the target analyte.

5. The kit of claim 1, wherein the biotin-specific binding partner of (d) is selected from avidin, streptavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, and succinylated avidin.

6. The kit of claim 1, wherein the biotin-specific binding coupler in (d) is an antibody against biotin.

7. The kit of claim 1, wherein the singlet oxygen-activated chemiluminescent compound of (a) and / or (b) has a hapten that binds directly or indirectly thereto.

8. The kit of claim 1, wherein the fluorescent molecules of (a) and (b) are each independently selected from terbium, uranium, samarium, europium, gadolinium and dysprosium.

9. The kit of claim 8, wherein: Terbium emits light with a wavelength of approximately 545 nm; Uranium emits light with a wavelength of approximately 612 nm; and Samarium emits light with a wavelength of approximately 645 nm.

10. The kit of claim 1, further comprising: (e) A composition comprising: Singlet oxygen can activate chemiluminescent compounds that have a first analyte-specific binding partner that binds directly or indirectly to a second target analyte. and Fluorescent molecules, which are excited by activated chemiluminescent compounds, wherein the fluorescent molecules are different from the fluorescent molecules of (a) and (b) and emit light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) A biotinylated second analyte-specific binding coupler of a second target analyte, wherein the second analyte-specific binding coupler binds an epitope of the second target analyte that is different from the first analyte-specific binding coupler of (e).

11. The kit of claim 10, wherein the fluorescent molecule in (e) is selected from terbium, uranium, samarium, europium, gadolinium and dysprosium.

12. A microfluidic device for determining the concentrations of biotin and at least one additional target analyte in a sample, said microfluidic device comprising: (i) Through its inlet channel for applying the sample; (ii) at least a first compartment, which is in fluid communication with the inlet passage and contains: (a) At least a first composition comprising a singlet oxygen-activated chemiluminescent compound and having biotin directly or indirectly bound thereto, the biotin being capable of binding a biotin-specific binding protein, the first composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound. (b) At least a second composition comprising a singlet oxygen-activated chemiluminescent compound and having a first analyte-specific binding partner for a target analyte that binds directly or indirectly thereto, the second composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a). and (c) A biotinylated second analyte-specific binding pair of a target analyte, wherein the second analyte-specific binding pair binds an epitope of the target analyte that is different from that of the first analyte-specific binding pair in (b); and (d) A composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner that binds directly or indirectly thereto.

13. The microfluidic device of claim 12, wherein (a)-(d) exist in the same compartment.

14. The microfluidic device of claim 12, wherein (a)-(d) are separated between two or more compartments.

15. The microfluidic device of claim 12, wherein the singlet oxygen-activated chemiluminescent compounds of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate, said metastable intermediate being capable of decomposing and emitting light simultaneously or subsequently.

16. The microfluidic device of claim 12, wherein the sensitizer is a photosensitizer.

17. The microfluidic device of claim 12, wherein the first and second analyte-specific binding couplers of (b) and (c) are each antibodies against the target analyte.

18. The microfluidic device of claim 12, wherein the biotin-specific binding partner of (iii) is selected from avidin, streptavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, and succinylated avidin.

19. The microfluidic device of claim 12, wherein the biotin-specific binding partner of (iii) is an antibody against biotin.

20. The microfluidic device of claim 12, wherein the singlet oxygen-activated chemiluminescent compound of (a) and / or (b) has a hapten that binds directly or indirectly thereto.

21. The microfluidic device of claim 12, wherein the fluorescent molecules of (a) and (b) are each independently selected from terbium, uranium, samarium, europium, gadolinium and dysprosium.

22. The microfluidic device of claim 21, wherein: Terbium emits light with a wavelength of approximately 545 nm; Uranium emits light with a wavelength of approximately 612 nm; and Samarium emits light with a wavelength of approximately 645 nm.

23. The microfluidic device of claim 12, wherein (ii) further comprises: (e) At least a third composition comprising a singlet oxygen-activated chemiluminescent compound and having a first analyte-specific binding pair of its second target analyte, which is directly or indirectly bound thereto, the composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) A biotinylated second analyte-specific binding coupler of a second target analyte, wherein the second analyte-specific binding coupler binds an epitope of the second target analyte that is different from the first analyte-specific binding coupler of (e).

24. The microfluidic device of claim 23, wherein the fluorescent molecule of the third composition is selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium.

25. A method for detecting the presence and / or concentration of biotin and at least one additional target analyte in a sample, the method comprising the steps of: (1) Combine samples suspected of containing biotin and at least one additional target analyte with the following in sequence, either all or some of them: (a) At least a first composition comprising a singlet oxygen-activated chemiluminescent compound and having biotin directly or indirectly bound thereto, the biotin being capable of binding a biotin-specific binding protein, the first composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound. (b) At least a second composition comprising a singlet oxygen-activated chemiluminescent compound and having a first analyte-specific binding partner for a target analyte that binds directly or indirectly thereto, the second composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a). (c) A biotinylated second analyte-specific binding pair of a target analyte, wherein the second analyte-specific binding pair binds an epitope of the target analyte that is different from that of the first analyte-specific binding pair in (b); and (d) An excess of the composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner that binds directly or indirectly thereto; (2) Allowing (b) and (c) to bind to the target analyte present in the sample and (c) to bind to (d), wherein the indirect binding of (b) and (d) (via (c) and the target analyte) results in the formation of a target analyte complex, wherein the sensitizer is in close proximity to the chemiluminescent compound, and wherein (a) binds to (d) in the absence of biotin in the sample and results in the formation of a biotin complex, wherein the sensitizer is in close proximity to the chemiluminescent compound, and (3) Activate the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the biotin complex and the target analyte complex causes activation of the chemiluminescent compound present in each complex; (4) The amount of chemiluminescence generated by the chemiluminescent compound activated in the biotin complex is determined by measuring the amount of light emitted by the fluorescent molecules in (a), wherein the amount of biotin in the sample is inversely proportional to the amount of light emitted. (5) The amount of chemiluminescence generated by the chemiluminescent compound activated in the target analyte complex is determined by measuring the amount of light emitted by the fluorescent molecules in (b) to determine the amount of target analyte in the sample; (6) Optionally repeat steps (2)-(5); and (7) Correct the result of step (5) based on any biotin interference detected in step (4), or mark the result of step (5) as unreliable if the biotin concentration detected in step (4) is higher than the maximum threshold level.

26. The method of claim 25, wherein the singlet oxygen-activated chemiluminescent compound is a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate, the metastable intermediate being decomposed and emitting light simultaneously or subsequently.

27. The method of claim 25, wherein the sensitizer is a photosensitizer, and the activation of the sensitizer in step (3) includes irradiation with light.

28. The method of claim 25, wherein the sample is a biological sample.

29. The method of claim 28, wherein the biological sample is selected from whole blood or any part thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

30. The method of claim 25, wherein the first and second analyte-specific binding couplers in (b) and (c) are each antibodies against the target analyte.

31. The method of claim 25, wherein the biotin-specific binding partner in (d) is selected from avidin, streptavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, and succinylated avidin.

32. The method of claim 25, wherein the biotin-specific binding partner in (d) is an antibody against biotin.

33. The method of claim 25, wherein the singlet oxygen of (a) and / or (b) is chemiluminescent compound having a hapten that binds directly or indirectly thereto.

34. The method of claim 25, wherein the fluorescent molecules of (a) and (b) are each independently selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium.

35. The method of claim 34, wherein: Terbium emits light with a wavelength of approximately 545 nm; Uranium emits light with a wavelength of approximately 612 nm; and Samarium emits light with a wavelength of approximately 645 nm.

36. The method of claim 25, wherein step (1) further comprises combining with (a)-(d): (e) at least a third composition comprising a singlet oxygen-activated chemiluminescent compound having a first analyte-specific binding pair with which it is directly or indirectly bound, the composition further comprising a fluorescent molecule excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) A biotinylated second analyte-specific binding coupler of a second target analyte, wherein the second analyte-specific binding coupler binds an epitope of the second target analyte that is different from the first analyte-specific binding coupler of (e).

37. The method of claim 36, wherein the method further comprises the following steps: (8) The amount of chemiluminescence generated by the chemiluminescent compound activated in the second target analyte complex is determined by measuring the amount of light emitted by the fluorescent molecules of (e) to determine the amount of target analyte in the sample; and (9) Correct the result of step (8) based on any biotin interference detected in step (4), or mark the result of step (8) as unreliable if the biotin concentration detected in step (4) is higher than the maximum threshold level.

38. The method of claim 36, wherein the fluorescent molecule in (e) is selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium.

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