Kit, microfluidic device and method for performing biotin assays

Abstract

Kits and microfluidic devices containing chemiluminescent detection systems and methods for determining the concentration of biotin in a sample are disclosed. The kits, microfluidic devices and methods utilize a sensitizer capable of generating singlet oxygen in its excited state and having an avidin or analog thereof directly or indirectly bound thereto, and a singlet oxygen activatable chemiluminescent compound having a biotin or analog thereof directly or indirectly bound thereto.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 24, 2019, application number 201980049810.7, and invention name “Kit, microfluidic device and method for biotin determination”.

[0002] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 62 / 711,694, filed on July 30, 2018. The entire contents of the above-referenced patent applications are hereby expressly incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of bioassay, and in particular to a kit and a microfluidic device containing a chemiluminescent detection system and a method for determining the concentration of biotin in a sample. Background Art

[0004] Biotin is often used in conjugates with antibodies or other small drug molecules for use as assay reagents. The tight binding of biotin to large protein molecules (e.g., avidin and streptavidin) coated on solid supports provides a convenient method for immobilizing antibodies or drugs on solid supports.

[0005] Biotin is well known in the art due to its use as a food supplement; for example, biotin is used to promote healthy hair and nail growth and treat various disease 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., to coat solid supports), high levels of biotin in a test sample can interfere with the assay signal in any assay that employs biotinylated assay components. Because interference is observed in various assays at different biotin concentrations, the level of biotin present in a patient sample should be quantified to determine whether the sample is suitable for use in a particular assay. However, most currently available biotin assays have a narrow range (i.e., 0-50 ng / ml), which is not suitable for detecting the wide dynamic range of biotin concentrations typically found in actual patient samples (i.e., 0-1500 ng / ml).

[0006] The only method for dealing with the narrow detection range of currently available biotin assays is to perform serial dilutions of the test sample in hopes of reaching the desired assay range; however, this is a cumbersome process that wastes test sample as well as reagents and also reduces turnaround time.

[0007] The field of medical diagnosis utilizes many different forms of assay techniques. An example of commercial assay is luminescent oxygen channel assay (LOCI®) technology. LOCI® advanced chemiluminescence assay is described in, for example, U.S. Patent No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference. Currently available LOCI® technology has high sensitivity and uses several reagents. Specifically, LOCI® assay requires that two of these reagents (referred to as "sensibead" and "chemibead") be maintained in the following manner by other specific binding partner assay reagents, wherein sensibead and chemibead are closely approached to each other to achieve signal. After being exposed to light of a specific wavelength, sensibead releases singlet oxygen, and if two beads are closely approached, singlet oxygen is transferred to chemibead; this causes a chemical reaction, which causes the chemibead to emit light, and the light can be measured at different wavelengths.

[0008] However, there is no biotin assay available in the LOCI® format.

[0009] Therefore, there is a need in the art for new and improved biotin assays that overcome the shortcomings and deficiencies of the prior art. The present disclosure relates to such assays, as well as kits and microfluidic devices containing the same and methods of use thereof. Summary of the invention

[0010] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it should be understood that the present disclosure is not limited to the application of the details of the construction and arrangement of the components set forth in the following description. The present 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.

[0011] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. The aforementioned techniques and procedures are generally implemented according to conventional methods well known in the art and as described in 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 medicinal and pharmaceutical chemistry as described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0012] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the technical levels of those skilled in the art to which the present 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 same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0013] In view of the present disclosure, all articles, compositions, kits and / or methods disclosed herein can be prepared and performed without undue experimentation. Although the articles, compositions, kits and / or methods have been described in terms of specific embodiments, it will be apparent to those skilled in the art that changes may be applied to the articles, compositions, kits and / or methods, and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present disclosure. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined in the appended claims.

[0014] As utilized in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings: When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Thus, the terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" may refer to one or more compounds, 2 or more compounds, 3 or more compounds, 4 or more compounds, or a greater number of compounds. The term "multiple" means "two or more".

[0015] The use of the term "at least one" will be understood to include one and any number more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to as many as 100 or 1000 or more, depending on the term to which it is attached; in addition, the number of 100 / 1000 is not considered restrictive, because higher limits can also produce satisfactory results. In addition, the use of the term "at least one of X, Y and Z" will be understood to include X alone, Y alone and Z alone, and any combination of X, Y and Z. The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is only for the purpose of distinguishing two or more items, and is not meant to imply, for example, any order or sequence or importance of one item relative to another, or any order of addition.

[0016] The term "or" used in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfies 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).

[0017] 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 conjunction with the embodiment is included in at least one embodiment. For example, the phrases "in some embodiments" or "an example" appearing in various places in the specification do not necessarily all refer to the same embodiment. In addition, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[0018] Throughout the application, the term "about" is used to indicate that a value includes the inherent variation of error of the composition / instrument / device, method used to determine the value, or the variation that exists between study subjects. For example, but not limited to, when the term "about" is utilized, the specified value may vary from the recited value 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%, as such variations are applicable to performing the disclosed methods and are understood by those of ordinary skill in the art.

[0019] As used in this specification and in one or more claims, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, 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, unrecited elements or method steps.

[0020] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important 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, and the like. Those skilled in the art will understand that, unless apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0021] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely or that the subsequently described event or circumstance occurs to a large extent or degree. For example, when related to a specific event or environment, the term "substantially" means that the subsequently described event or circumstance 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 in close proximity to each other, but are not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item, but is in close proximity to the other item.

[0022] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to each other and indirect association / binding of two moieties to each other. Non-limiting examples of association / binding include covalently binding one moiety to another, e.g., through a direct bond or through a spacer group, non-covalently binding one moiety to another directly or via a specific binding pair member bound to the moieties, incorporating one moiety into another, such as by dissolving one moiety in the other moiety or by synthesis, and coating one moiety on another moiety.

[0023] The terms "analog" and "derivative" are used interchangeably herein and refer to substances that contain in their structure the same basic carbon skeleton and carbon functionality as a given compound, but may also contain one or more substitutions thereto. As used herein, the term "substitution" will be understood to refer to the replacement of at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R may include H, hydroxyl, thiol, a halide selected from fluoride, chloride, bromide or iodite, a C1-C4 compound selected from one of the following: an optionally substituted straight, branched or cyclic alkyl, and a straight, branched or cyclic alkenyl, wherein the optional substituent is selected from one or more alkenyl alkyl, alkynyl alkyl, cycloalkyl, cycloalkenyl alkyl, aryl alkyl, heteroaryl alkyl, heterocycloalkyl, optionally substituted heterocycloalkenyl alkyl, aryl cycloalkyl and arylheterocycloalkyl, each of which is optionally substituted, wherein the optional substituents are selected from one or more of the following: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl and arylheterocycloalkyl, phenyl, cyano, hydroxy, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), carboxy, and -C(O))-alkyl.

[0024] The term "sample" as used herein will be understood to include any type of biological sample that can be utilized according to the present disclosure. Examples of fluid biological samples that can be utilized include, but are not limited to, whole blood or any portion 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, combinations thereof, and the like.

[0025] Certain non-limiting embodiments of the present disclosure relate to biotin assay compositions and kits containing them and methods of use thereof. In some assay embodiments, a signal generating system (SPS) member includes a sensitizer, such as, for example, a photosensitizer and a chemiluminescent composition; in these assay embodiments, the product generated by the activation of the sensitizer activates the chemiluminescent composition, thereby generating a detectable signal, which is related to the amount of bound and / or unbound biotin detected. An exemplary (but non-limiting) embodiment of the assay platform on which the present disclosure can be based is the luminescent oxygen channel assay (LOCI®; Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The LOCI® assay is described in, for example, U.S. Patent No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0026] Certain non-limiting embodiments of the present disclosure relate to a kit containing a chemiluminescent detection system for determining the concentration of biotin in a sample. The kit includes: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or an analog thereof (such as but not limited to 4'-hydroxyazobenzene-2-carboxylic acid (HABA)) directly or indirectly bound thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or an analog thereof (such as but not limited to streptavidin or traptavidin) directly or indirectly bound thereto, wherein the avidin / avidin analog is capable of binding to biotin in the sample or binding to (a).

[0027] A chemiluminescent compound (chemiluminescent agent) is a compound that is chemically activatable and, as a result of such activation, emits light of a specific wavelength. By way of illustration and not limitation, examples of chemiluminescent agents include, for example: olefins that can react with singlet oxygen or peroxides to form hydroperoxides or dioxetanes that can decompose into ketones or carboxylic acid derivatives; stable dioxetanes that can decompose by the action of light; alkynes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azocarbonyl compounds, such as (but not limited to) luminol; and aromatic compounds that can form endoperoxides. As a result of the activation reaction, the chemiluminescent agent directly or indirectly causes luminescence.

[0028] In certain embodiments, the singlet oxygen activatable chemiluminescent compound can be a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate substance that can decompose and simultaneously or subsequently emit light. The composition comprising the chemiluminescent compound can be directly excited by the activated chemiluminescent compound; alternatively, the composition can further comprise at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

[0029] 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 embodiments, the sensitizer is a photosensitizer. By way of example and not limitation, other sensitizers that can be chemically activated (by, for example, enzymes and metal salts) include other substances and compositions that can generate singlet oxygen with or without the activation of 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 the alkaline earth metals Ca, Sr, and Ba; d 0 Derivatives of elements of groups 3A, 4A, SA and 6A in configuration; oxides of actinides and lanthanides; and oxidants CIO - BrO - 、Au 3+ IO3 - and IO4 - ; and specifically, molybdate, peroxymolybdate, tungstate and peroxytungstate ions, and acetonitrile. The following references, which are expressly incorporated by reference in their entirety, provide further disclosure of sensitizer materials and compositions that also fall within the scope of the presently disclosed and claimed inventive concepts: 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 entire contents of each of which are expressly incorporated herein by reference.

[0030] Also included within the scope of photosensitizers are compounds that are not true sensitizers, but will release singlet oxygen molecules under the stimulation of heat, light, ionizing radiation or chemical activation. Members of this class of compounds include, for example (but not limited to), endoperoxides, such as 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. These compounds are heated or directly absorb light to release singlet oxygen.

[0031] Photosensitizers are sensitizers used to activate photoactive compounds by, for example, generating singlet oxygen through excitation with light. Photosensitizers are photoactivatable and include, for example, dyes and aromatic compounds, and are generally compounds composed of covalently bonded atoms, generally with 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) in the range of about 300 nm to about 1,000 nm or in the range of about 450 nm to 950 nm, and have an extinction coefficient of greater than 500 M at the maximum absorbance at the excitation wavelength. -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 may not react effectively with singlet oxygen. Examples of photosensitizers include, by way of illustration and not limitation, acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metalloporphyrins such as (but not limited to) hematoporphyrin; phthalocyanine; chlorophyll; rose bengal; and buckminsterfullerene and derivatives of these compounds.

[0032] Therefore, certain non-limiting embodiments of the present disclosure relate to a kit containing a chemiluminescent detection system for determining the concentration of biotin in a sample, wherein the kit includes: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or an analog thereof bound directly or indirectly thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having traptavidin bound directly or indirectly thereto, wherein the traptavidin is capable of binding to biotin in the sample or to (a). In other non-limiting embodiments, the kit includes: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having a biotin analogue bound directly or indirectly thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or an analog thereof bound directly or indirectly thereto, wherein the avidin or an analog thereof is capable of binding to biotin in the sample or to (a). In other non-limiting embodiments, the kit includes: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having 4'-hydroxyazobenzene-2-carboxylic acid (HABA) directly or indirectly bound thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having traptavidin directly or indirectly bound thereto, wherein the traptavidin is capable of binding to biotin in a sample or to (a).

[0033] Specific, non-limiting examples of compositions comprising chemiluminescent compounds and / or compositions comprising sensitizers that can be utilized in accordance with the present disclosure are described in U.S. Pat. Nos. 5,340,716; 5,538,834; 5,545,834; 5,578,498; 5,618,732; 5,672,478; 5,709,994; 5,811,311; 5,780,646; 5,929,049; 5,936,070; 6,002,000; 6,143,514; 6,180 ,354; 6,251,581; 6,340,599; 6,406,913; 6,406,667; 6,489,309; 6,692,975; 6,703,248; 6,797,481; 6,916,667; 6,949,524; 7,022,529; 7,033,775; 7,101,682; 7,179,660; 7,229,842; and 7,709,273; the entire contents of each of which are hereby expressly incorporated herein by reference.

[0034] In a specific (but non-limiting) embodiment, the composition of (a) and (b) is a particle having the other listed components coupled thereto and / or disposed therein, such as (but not limited to) a bead, a sphere, a spheroid, or a liposome. A specific (but non-limiting) example of a particle that can be utilized in accordance with the present disclosure is a latex particle, such as (but not limited to) a latex bead. Other non-limiting examples of particle types that can be used as the composition of (a) and (b) are disclosed in the patents listed in the above paragraphs.

[0035] Any biotin analog known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure, provided that the biotin analog: (1) is capable of associating with a singlet oxygen-activatable chemiluminescent compound; and (2) is capable of binding to avidin or an analog thereof associated with a sensitizer and thereby functioning as a competitor for any biotin present in the sample. Examples of biotin analogs that may be utilized in accordance with the present disclosure include, but are not limited to, those described in Athappilly et al. ( Protein Science (1997) 6:1338-1342), Torreggiani et al. Biospectroscopy (1998) 4:197-208), Levert et al. J Biol Chem (2002) 277:16347-16350), Yamamoto et al. Chem Asian J , (2015) 10:1071-8) (each of which is expressly incorporated herein by reference in its entirety). Specific non-limiting examples of biotin analogs include 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin (such as but not limited to 2-imino-biotin (IMBio)), biotin carbonate, biotin carbamate, biotin methyl ester (MEBio), desthiobiotin (DEBio) (such as but not limited to d -Desthiobiotin, dl -Desthiobiotin, dl -Desthiobiotin methyl ester and dl -Desthiobiotinol ( dl -Desthiobiotinol)), diaminobiotin (DABio), chloroacetylated biotin, biotin sulfone, thiobiotin (such as but not limited to 2-thiobiotin), methoxycarbonyl biotin methyl ester (1'- N -Methoxycarbonylbiotin methyl ester and 3'- N -methoxycarbonyl biotin methyl ester), bibiotin (such as but not limited to bibiotin phosphate), tetrabiotin, esters, salts and / or derivatives of any of the above, etc. In a specific (but non-limiting) example, the biotin analog is HABA or iminobiotin.

[0036] Any avidin analog known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure, so long as the avidin or avidin analog: (1) is capable of associating with a sensitizer; and (2) is capable of binding any biotin present in the sample; and (3) is also capable of binding biotin or an analog thereof associated with a singlet oxygen-activatable chemiluminescent compound. Non-limiting examples of avidin analogs that may be utilized in accordance with the present disclosure include Kang et al. ( J Drug Target (1995) 3:159-65) (the entire contents of which are expressly incorporated herein by reference). Specific non-limiting examples of avidin analogs include avidin, streptavidin, traptavidin, neutravidin, 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, and the like.

[0037] The reagents of the composition / kit / method can be provided in any form that allows them to function according to the disclosed concepts. For example, but not limiting, each reagent can be provided in liquid form and arranged in the kit in bulk and / or in a single aliquot. Alternatively, in a specific (but non-limiting) embodiment, one or more reagents can be arranged in the kit in the form of 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.

[0038] In addition to the reagents described in detail above, the kit may further contain other reagents for carrying out any specific assay described herein or otherwise contemplated. The nature of these additional reagents will depend on the specific assay format, and their identification is fully within the skill of those of ordinary skill in the art; therefore, it is not considered necessary to describe it separately. In addition, the components / reagents present in the kit may each be in a separate container / compartment, or the various components / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. In addition, the kit may include a microfluidic device in which the components / reagents are arranged.

[0039] The relative amounts of the various components / reagents in the kit can vary widely to provide a concentration of the components / reagents that substantially optimizes the reaction that needs to occur during the assay method, and further substantially optimizes the sensitivity of the assay. Where appropriate, one or more components / reagents in the kit can be provided as dry powders, such as lyophilized powders, and the kit can further include an excipient for dissolving the dry reagents; in this way, a reagent solution having an appropriate concentration for carrying out the method or assay according to the present disclosure can be obtained from these components. The kit can also include positive and / or negative controls. In addition, the kit can further include a set of written instructions explaining how to use the kit. The kit of this nature can be used for any method described herein or otherwise considered.

[0040] Certain additional non-limiting embodiments of the present disclosure relate to a microfluidic device comprising the components of any of the kits described above. Specifically, certain non-limiting embodiments include a microfluidic device for determining the concentration of biotin in a sample, wherein the microfluidic device comprises: (a) an inlet channel through which a sample is applied; (b) at least one first compartment capable of being fluidically connected to the inlet channel, the first compartment containing a sensitizer capable of generating singlet oxygen in its excited state and having any avidin or its analogue bound directly or indirectly thereto; and (c) at least one second compartment capable of being fluidically connected to the first compartment, the second compartment containing a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or its analogue bound directly or indirectly thereto.

[0041] Any of the sensitizers, avidin or analogs thereof, singlet oxygen activatable chemiluminescent compounds, and biotin or analogs thereof described in detail above or otherwise contemplated herein may be utilized in the microfluidic devices of the present disclosure.

[0042] In certain specific (but non-limiting) embodiments, the sensitizer of (b) has an avidin analog directly or indirectly bound thereto. Specific non-limiting examples of avidin analogs that can be used include avidin, streptavidin, traptavidin, neutravidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin and other forms of modified or genetically engineered avidin and any ester, salt or derivative of any of the above analogs.

[0043] In certain specific (but non-limiting) embodiments, the singlet oxygen-activatable chemiluminescent compound of (c) has a biotin analog directly or indirectly bound thereto. Non-limiting examples of biotin analogs that can be used include 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, bibiotin and tetrabiotin and any ester, salt or derivative of any of the above analogs. A specific non-limiting example of a biotin analog that can be used is 4'-hydroxyazobenzene-2-carboxylic acid (HABA).

[0044] In one non-limiting embodiment, the sensitizer of (b) has traptavidin bound directly or indirectly thereto, and / or the singlet oxygen activatable chemiluminescent compound of (c) has HABA bound directly or indirectly thereto.

[0045] The device may be provided with any arrangement of compartments and distribution of the two components therebetween that allows the device to function in accordance with the present disclosure.

[0046] Any compartment of a microfluidic device may be sealed so as to maintain reagents disposed therein in a substantially airtight environment until they are used; for example, a compartment containing a lyophilized reagent may be sealed to prevent any unintentional reconstitution of the reagent. An inlet channel and a compartment, as well as two compartments may be described as "capable of being in fluid communication with each other"; this phrase indicates that the compartments may each still be sealed, but the two compartments are capable of having fluid flow therebetween after piercing a seal formed therein or therebetween.

[0047] The microfluidic device of the present disclosure may be provided with any other desired features known in the art or otherwise considered herein. For example, but not limiting, the microfluidic device of the present 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, diluting solutions, excipients, interfering solutions, positive controls, negative controls, quality controls, etc. These additional compartments may be in fluid communication with one or more of the 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 dry reagents, and the 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 diluting solutions, and the compartments may be in fluid communication with any other compartment of the device.

[0048] In addition, any kit / microfluidic device described or otherwise contemplated herein may include multiple assays multiplexed in a single kit / device. When multiple assays are present, each of the assays may be constructed and function as described herein. Alternatively, the assays as described herein may be multiplexed with any other assay known in the art that may be included in the kit / microfluidic device of the present disclosure. Non-limiting examples of other assays that may be multiplexed 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.

[0049] When multiple assays are present in a single microfluidic device, multiple inlet channels can be connected to the sample application chamber. In certain embodiments, a portion of the sample can pass from the sample application chamber to multiple inlet channels without considering its contents. Alternatively, a structure can be present in the sample application chamber, the inlet channels, and / or the connection therebetween that allows certain components to be separated from the entire sample and delivered to different assays. Non-limiting examples of sample distribution devices that can be utilized according to the present disclosure are 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.

[0050] Certain non-limiting embodiments also relate to methods for detecting the presence and / or concentration of biotin in a sample. The method comprises the following steps: (a) combining simultaneously or in whole or in part: (1) a sample suspected of containing biotin; (2) a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or a biotin analog directly or indirectly bound thereto; and (3) an excess composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or avidin analog directly or indirectly bound thereto; (b) allowing (3) to bind to biotin present in the sample or to (2), thereby sensitizing the sample in the absence of biotin. (c) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes activation of the chemiluminescent compound present in the complex; (d) determining the amount of chemiluminescence generated by the activated chemiluminescent compound; (e) optionally repeating steps (b)-(d); and (f) detecting the presence and / or concentration of biotin by analyzing the amount of chemiluminescence thus generated, wherein the amount of chemiluminescence is inversely proportional to the amount of biotin in the sample.

[0051] Any sample for which the presence of biotin can be determined with the expectation can be used as a sample according to the methods of the present disclosure. Non-limiting examples of samples include biological samples such as, but not limited to, whole blood or any portion 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 washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0052] Any of the sensitizers, avidin or analogs thereof, singlet oxygen activatable chemiluminescent compounds, and biotin or analogs thereof described in detail above or otherwise contemplated herein may be utilized in the microfluidic devices of the present disclosure.

[0053] In certain specific (but non-limiting) embodiments, the singlet oxygen-activatable chemiluminescent compound of step (a)(2) has a biotin analog directly or indirectly bound thereto. Non-limiting examples of biotin analogs that can be used include 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, bibiotin and tetrabiotin and any ester, salt or derivative of any of the above analogs. A specific non-limiting example of a biotin analog that can be used is 4'-hydroxyazobenzene-2-carboxylic acid (HABA).

[0054] In certain specific (but non-limiting) embodiments, the sensitizer of step (a)(3) has an avidin analog directly or indirectly bound thereto. Specific non-limiting examples of avidin analogs that can be used include avidin, streptavidin, traptavidin, neutravidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin and other forms of modified or genetically engineered avidin and any ester, salt or derivative of any of the foregoing analogs.

[0055] In one non-limiting embodiment, the singlet oxygen activatable chemiluminescent compound of step (a)(2) has HABA directly or indirectly bound thereto, and / or the sensitizer of step (a)(3) has traptavidin directly or indirectly bound thereto.

[0056] When biotin is not present in the sample, a complex of the sensitizer and the chemiluminescent compound is formed and a signal is generated. In contrast, if biotin is present in the sample, it binds to the avidin / avidin analog that is bound to the sensitizer and prevents the formation of a complex, thereby preventing the generation of a signal. Therefore, the amount of biotin present in the sample is inversely proportional to the amount of signal generated.

[0057] As mentioned above, the various components of the method are provided in combination (simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition of the components can be changed; a person of ordinary skill in the art can determine the specific desired order of adding different components to the assay. The simplest order of addition is of course to add all substances at the same time and measure the signal generated by them. Alternatively, each component, or group of components, can be combined sequentially. In certain embodiments, an incubation step can be included after each addition as discussed above.

[0058] In an alternative (but non-limiting) embodiment, step (a) of the method comprises first combining and incubating the sample with a composition comprising a sensitizer having an avidin / avidin analog bound thereto, and then adding a composition comprising a singlet oxygen-activatable chemiluminescent compound having a biotin / biotin analog bound thereto.

[0059] In any non-limiting embodiment of the kit, microfluidic device or method described or otherwise contemplated herein, the sensitizer may be a photosensitizer. Thus, activation of the photosensitizer will include irradiation with light.

[0060] In any non-limiting embodiments of the kits, microfluidic devices, or methods described or otherwise contemplated herein, the singlet oxygen activatable chemiluminescent compound can be one that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose and simultaneously or subsequently emit light.

[0061] In addition, in any non-limiting embodiment of the kit, microfluidic device or method described herein or otherwise contemplated, the composition comprising a chemiluminescent compound may further comprise at least one fluorescent molecule excited by the activated chemiluminescent compound. In this case, the method described herein or otherwise contemplated may further comprise a step of measuring the amount of light emitted by the fluorescent molecule to determine the amount of analyte in the sample.

[0062] This application provides the following specific implementation methods of the project numbers: 1. A kit containing a chemiluminescent detection system for determining the concentration of biotin in a sample, the kit comprising: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or an analog thereof bound directly or indirectly thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having traptavidin bound directly or indirectly thereto.

[0063] 2. The kit of claim 1, wherein the composition of (a) has a biotin analog directly or indirectly bound to the singlet oxygen-activatable chemiluminescent compound, and wherein the biotin analog is selected from 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, bibiotin, tetrabiotin, and any ester, salt or derivative of any of the foregoing analogs.

[0064] 3. The kit of item 2, wherein the biotin analogue is HABA or iminobiotin.

[0065] 4. The kit of item 1, wherein the singlet oxygen activatable chemiluminescent compound undergoes a chemical reaction with singlet oxygen to form a metastable intermediate substance, which can decompose and simultaneously or subsequently emit light.

[0066] 5. The kit of item 1, wherein the composition comprising the chemiluminescent compound further comprises at least one fluorescent molecule excited by the activated chemiluminescent compound.

[0067] 6. The kit of item 1, wherein the sensitizer is a photosensitizer.

[0068] 7. A kit containing a chemiluminescent detection system for determining the concentration of biotin in a sample, the kit comprising: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having a biotin analog directly or indirectly bound thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or its analog directly or indirectly bound thereto, wherein the avidin or its analog is capable of binding to biotin in the sample or to (a).

[0069] 8. The kit of item 7, wherein the biotin analog is selected from 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, bibiotin, tetrabiotin and any ester, salt or derivative of any of the above analogs.

[0070] 9. The kit of claim 8, wherein the biotin analog is HABA or iminobiotin.

[0071] 10. The kit of claim 7, wherein the composition of (b) has an avidin analog directly or indirectly bound to the sensitizer, and wherein the avidin analog is selected from avidin, streptavidin, traptavidin, neutravidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, and any ester, salt, or derivative thereof of any of the foregoing analogs.

[0072] 11. The kit of item 7, wherein the singlet oxygen activatable chemiluminescent compound undergoes a chemical reaction with singlet oxygen to form a metastable intermediate substance, which can decompose and simultaneously or subsequently emit light.

[0073] 12. The kit of item 7, wherein the composition comprising the chemiluminescent compound further comprises at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

[0074] 13. The kit of item 7, wherein the sensitizer is a photosensitizer.

[0075] 14. A kit containing a chemiluminescent detection system for determining the concentration of biotin in a sample, the kit comprising: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having 4'-hydroxyazobenzene-2-carboxylic acid (HABA) directly or indirectly bound thereto; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having traptavidin directly or indirectly bound thereto.

[0076] 15. A microfluidic device for determining the concentration of biotin in a sample, the microfluidic device comprising: (a) an inlet channel through which a sample is applied; (b) at least one first compartment capable of being fluidically connected to the inlet channel, the first compartment containing a sensitizer capable of generating singlet oxygen in its excited state and having avidin or its analogue directly or indirectly bound thereto; and (c) at least one second compartment capable of being fluidically connected to the first compartment, the second compartment containing a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or its analogue directly or indirectly bound thereto.

[0077] 16. The microfluidic device of claim 15, wherein the sensitizer of (b) has an avidin analog directly or indirectly bound thereto, and wherein the avidin analog is selected from avidin, streptavidin, traptavidin, neutravidin, neutralite avidin, neutravidin, lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, and any ester, salt, or derivative thereof of any of the foregoing analogs.

[0078] 17. The microfluidic device of claim 15, wherein the singlet oxygen-activatable chemiluminescent compound of (c) has a biotin analog directly or indirectly bound thereto, and wherein the biotin analog is selected from 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, dibiotin, tetrabiotin, and any ester, salt or derivative of any of the foregoing analogs.

[0079] 18. The microfluidic device of item 15, wherein at least one of the following is satisfied: the sensitizer of (b) has traptavidin directly or indirectly bound thereto; and the singlet oxygen-activatable chemiluminescent compound of (c) has HABA directly or indirectly bound thereto.

[0080] 19. The microfluidic device of claim 15, wherein the singlet oxygen activatable chemiluminescent compound of (c) undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species, which can decompose and simultaneously or subsequently emit light.

[0081] 20. The microfluidic device of item 15, wherein the composition comprising a chemiluminescent compound of (c) further comprises at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

[0082] 21. The microfluidic device of claim 15, wherein the sensitizer is a photosensitizer.

[0083] 22. A method for detecting the presence and / or concentration of biotin in a sample, the method comprising the following steps: (a) combining simultaneously or in whole or in part sequentially: (1) a sample suspected of containing biotin; (2) a composition comprising a singlet oxygen activatable chemiluminescent compound having biotin or a biotin analog directly or indirectly bound thereto; and (3) an excess of a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or an avidin analog directly or indirectly bound thereto; (b) allowing (3) to bind to biotin present in the sample or to (2), thereby forming a complex between (2) and (3) in the absence of biotin, and bringing the sensitizer into close proximity with the chemiluminescent compound; (c) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes activation of the chemiluminescent compound present in the complex; (d) determining the amount of chemiluminescence generated by the activated chemiluminescent compound; (e) optionally repeating steps (b)-(d); and (f) The presence and / or concentration of biotin is detected by analyzing the amount of chemiluminescence so produced, wherein the amount of chemiluminescence is inversely proportional to the amount of biotin in the sample.

[0084] 23. The method of item 22, wherein the singlet oxygen-activatable chemiluminescent compound of step (a)(2) has a biotin analog directly or indirectly bound thereto, and wherein the biotin analog is selected from 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, dibiotin, tetrabiotin and any ester, salt or derivative of any of the above analogs.

[0085] 24. The method of claim 22, wherein the sensitizer of step (a)(3) has an avidin analog directly or indirectly bound thereto, and wherein the avidin analog is selected from avidin, streptavidin, traptavidin, neutravidin, neutralite avidin, neutravidin, lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, and any ester, salt, or derivative thereof of any of the foregoing analogs.

[0086] 25. The method of item 22, wherein at least one of the following is satisfied: the singlet oxygen-activatable chemiluminescent compound of step (a)(2) has HABA directly or indirectly bound thereto; and the sensitizer of step (a)(3) has traptavidin directly or indirectly bound thereto.

[0087] 26. The method of item 22, wherein the singlet oxygen activatable chemiluminescent compound undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species, which can decompose and simultaneously or subsequently emit light.

[0088] 27. The method of item 22, wherein the sensitizer is a photosensitizer, and the activation of the sensitizer in step (c) comprises irradiation with light.

[0089] 28. The method of item 22, wherein the sample is a biological sample.

[0090] 29. The method of item 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 washing fluid, semen, feces, pleural fluid, nasopharyngeal fluid and combinations thereof.

[0091] 30. The method of item 28, wherein the biological sample comprises at least one of lysed whole blood cells and lysed red blood cells.

[0092] 31. The method of item 22, wherein the composition comprising a chemiluminescent compound further comprises at least one fluorescent molecule excited by the activated chemiluminescent compound.

[0093] 32. The method of item 31, further comprising the step of measuring the amount of light emitted by the fluorescent molecule to determine the amount of analyte in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 The schematic depicts one non-limiting embodiment of a Biotin LOCI® assay constructed in accordance with the present disclosure.

[0095] Figure 2 The graphic depicts a standard curve for one non-limiting embodiment of a biotin LOCI® assay constructed in accordance with the present disclosure, wherein the assay utilizes biotin-coated chemibeads and streptavidin-coated sensibeads.

[0096] Figure 3 Graph depicting a standard curve for another non-limiting embodiment of a biotin LOCI® assay constructed in accordance with the present disclosure, wherein the assay utilizes biotin-coated chemibeads and traptavidin-coated sensibeads.

[0097] Figure 4 Illustration depicting Figure 2 and 3 Comparison with the standard curve.

[0098] Figure 5 The graphic depicts a standard curve for yet another non-limiting embodiment of a biotin LOCI® assay constructed in accordance with the present disclosure, wherein the assay utilizes 4'-hydroxyazobenzene-2-carboxylic acid (HABA)-coated chemibeads and streptavidin-coated sensibeads.

[0099] Figure 6 The diagram depicts yet another non-limiting embodiment of a biotin LOCI® assay constructed in accordance with the present disclosure, wherein the assay utilizes iminobiotin-coated chemibeads and avidin-coated sensibeads. DETAILED DESCRIPTION

[0100] Examples are provided below. However, it should be understood that the present disclosure and its application are not limited to the specific experiments, results and laboratory procedures disclosed herein. Instead, this example is provided only as one of the various embodiments and is intended to be exemplary, not exhaustive.

[0101] Example 1 The present disclosure relates to a robust biotin LOCI® assay with a wide assay range. The sample amount used in certain non-limiting embodiments of the present example is about 20 μl; however, by using a smaller sample amount (such as, but not limited to, about 3 μl), a biotin assay with a wider assay range (e.g., up to 1,500 ng / ml or even 40,000 ng / ml) can be achieved. However, these sample amounts should not be construed as limitations on the present disclosure; the use of any sample amount that can be assayed falls within the scope of the present disclosure. For example (but not limiting), the sample amount can fall within the range of about 0.1 μl to about 1,000 μl, or within the range of about 0.5 μl to about 100 μl, or within the range of about 1 μl to about 50 μl, or any integer or non-integer percentage value falling within any of the above ranges.

[0102] Biotin LOCI® assay diagram depicted in Figure 1 and described in detail below.

[0103] The biotin LOCI® assay includes: (i) chemibeads (CB) coated with biotin or a biotin analog (such as, but not limited to, 4'hydroxyazobenzene-2-carboxylic acid (HABA) or any other biotin analog disclosed or otherwise contemplated herein); and (ii) sensibeads (SB) coated with avidin or an avidin analog (such as, but not limited to, streptavidin, traptavidin, or any other avidin analog disclosed or otherwise contemplated herein). The biotin / biotin analog coated chemibeads compete with free biotin in the patient sample for binding to avidin / avidin analog coated sensibeads. The higher the free biotin concentration in the patient, the less binding of chemibeads and sensibeads is observed, which produces an inverse curve. As described in this and subsequent examples, the feasibility of this biotin LOCI® assay has been demonstrated using biotin / biotin analog coated chemibeads and avidin / avidin analog coated sensibeads.

[0104] Based on the above, a biotin assay has been developed (using the biotin LOCI® assay as an example). The first step of the assay involves incubating an excess of avidin / avidin analog coated sensibeads (such as, but not limited to, traptavidin coated sensibeads) with the sample so that any free biotin present in the sample can be "locked-in" by the sensibeads. Biotin / biotin analog coated chemibeads (such as, but not limited to, biotin coated or HABA coated chemibeads) are then added to clear the remaining binding sites from the biotin-avidin binding. If biotin is not present in the sample, the avidin / avidin analog coated sensibeads bind to the biotin / biotin analog coated chemibeads and generate a signal ( Figure 1 If biotin is present in the sample, the sample biotin binds to the avidin / avidin analog coated sensibeads and the sensibeads are no longer able to bind to the biotin / biotin analog coated chemibeads ( Figure 1 Thus, the higher the free biotin concentration in the patient sample, the less binding of chemibeads and sensibeads is observed, which generates an inverse standard curve.

[0105] Example 2 In this example, the biotin LOCI® assay described in Example 1 was performed using chemibeads (CB) coated with biotin and sensibeads (SB) coated with streptavidin. The sample volume used was 20 μl. The total reaction volume was 218 μl and the time to first result was 16 minutes.

[0106] The data obtained from this Biotin LOCI® assay are shown in Figure 2 and Table 1. As can be seen, standard curves were obtained over a much wider range of biotin concentrations than any currently available biotin assay.

[0107] Table 1: Precision of the Biotin LOCI® Assay using Biotin-CB and Streptavidin-SB repeat 0 ng / ml 10 ng / ml 35 ng / ml 80 ng / ml 200 ng / mL average value 0 9.9 36 78 186 SD 0.19 0.56 1.16 2.84 7.11 CV -- 5.7% 3.2% 3.7% 3.9%

[0108] Example 3 In addition to the biotin LOCI® assay embodiments described in Examples 1 and 2, some additional non-limiting embodiments of the biotin LOCI® assay include the use of biotin analogs and / or avidin analogs. For example (but not limiting), in the biotin LOCI® assay, the use of HABA-coated chemibeads and / or traptavidin-coated sensibeads provides multiple advantages over the prior art. First, HABA is a dye that is weakly bound to avidin (or streptavidin or traptavidin), and the binding of HABA to avidin or its analogs is weaker than biotin; as a result, the use of HABA provides higher sensitivity for biotin assays, because HABA acts as a scavenger (rather than a competitor) for the binding of biotin to sensibead. Secondly, due to its unique characteristics, traptavidin binds free biotin with half the binding rate and 1 / 10 of the dissociation rate. Therefore, the use of traptavidin provides a "locking" mechanism for free biotin, because once biotin is bound to traptavidin, it is almost not dissociated from the binding site.

[0109] When using traptavidin-coated sensibeads, the biotin-traptavidin binding "locks" the sample biotin in place with minimal biotin dissociation, and thus the added chemibeads do not compete with the sample biotin for the binding of traptavidin; therefore, the use of traptavidin-coated sensibeads produces a hapten assay with more robust precision. Additionally, since there is no competition from sample biotin, the assay signal is more linearly proportional to the free biotin concentration over a wider dynamic range, resulting in an extended assay range, when compared to conventional competition assays.

[0110] Thus, in certain non-limiting embodiments, the unique characteristics of traptavidin (alone or in combination with the use of HABA or another biotin analog) are exploited to generate a biotin assay with robust precision and extended dynamic range.

[0111] Example 4 In this example, the biotin LOCI® assay described in Example 3 was performed using chemibeads (CB) coated with biotin and sensibeads (SB) coated with traptavidin. The data obtained from this biotin LOCI® assay are shown in Figure 3 and Table 2. As can be seen, standard curves were obtained over a much wider range of biotin concentrations than any currently available biotin assay.

[0112] Table 2: Precision of the Biotin LOCI® Assay Using Biotin-CB and Traptavidin-SB repeat 0 ng / ml 10 ng / ml 35 ng / ml 80 ng / ml 200 ng / mL average value 0 9.4 35 84 205 SD 0.296 0.54 0.98 2.34 5.39 CV -- 5.7% 2.8% 2.8% 2.6%

[0113] Overall, except for 0 ng / mL, sensibeads coated with traptavidin showed significantly improved precision over streptavidin-coated sensibeads due to the ten-fold slower off-rate of traptavidin, which locks (or "traps") the bound sample biotin into place on the sensibead surface (see Figure 4 and Tables 1 and 2). In addition, the curve for traptavidin-SB is steeper than that for streptavidin-SB ( Figure 4 ).

[0114] Example 5 In this example, the biotin LOCI® assay described in Example 3 was performed using chemibeads (CB) coated with HABA and sensibeads (SB) coated with streptavidin. The data obtained from this biotin LOCI® assay are shown in Figure 5 As can be seen, a standard curve was obtained over a much wider range of biotin concentrations than any currently available biotin assay.

[0115] Example 6 Figure 6 Another non-limiting embodiment of the biotin LOCI® assay is depicted, wherein chemibeads coated with iminobiotin are utilized. In this embodiment, the chemibeads are coated with iminobiotin, and the sensibeads are coated with streptavidin or trapavidin. Iminobiotin has a pH-dependent binding to avidin; iminobiotin has a higher affinity to avidin at higher pHs, and dissociates from avidin when the pH is low. Utilizing this feature, iminobiotin-coated CBs can be incubated with SB and samples at lower pH to ensure that biotin binds to SB. After the binding of SB to biotin is complete, the pH is raised to enable the CB and SB to bind, thus generating a LOCI signal.

[0116] Therefore, according to the present disclosure, compositions, kits and devices and methods of producing and using the same that fully satisfy the objects and advantages described above have been provided. Although the present disclosure has been described in conjunction with the specific drawings, experiments, results and language set forth above, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such substitutions, modifications and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A kit containing a chemiluminescent detection system for determining the concentration of biotin in a sample, the kit comprising: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having a biotin analog directly or indirectly bound thereto; and (b) A composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or its analogue directly or indirectly bound thereto, wherein the avidin or its analogue is capable of binding to biotin in a sample or to (a).

2. The kit of claim 1, wherein the biotin analog is selected from the group consisting of 4'-hydroxyazobenzene-2-carboxylic acid (HABA), iminobiotin, biotin carbonate, biotin carbamate, biotin methyl ester, desthiobiotin, diaminobiotin, chloroacetylated biotin, biotin sulfone, thiobiotin, methoxycarbonylbiotin methyl ester, bibiotin, tetrabiotin, and any ester, salt or derivative of any of the foregoing analogs.

3. The kit of claim 2, wherein the biotin analog is HABA or iminobiotin.

4. The kit of claim 1, wherein the composition of (b) has an avidin analog directly or indirectly bound to the sensitizer, and wherein the avidin analog is selected from the group consisting of avidin, streptavidin, traptavidin, neutravidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, and any ester, salt, or derivative thereof of any of the foregoing analogs.

5. The kit of claim 1, wherein the singlet oxygen activatable chemiluminescent compound undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose and simultaneously or subsequently emit light.

6. The kit of claim 1, wherein the composition comprising a chemiluminescent compound further comprises at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

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

8. A microfluidic device for determining the concentration of biotin in a sample, the microfluidic device comprising: (a) an inlet channel through which a sample is applied; (b) at least one first compartment capable of being in fluid communication with the inlet channel, the first compartment containing a sensitizer capable of generating singlet oxygen in its excited state and having avidin or an analog thereof directly or indirectly bound thereto; and (c) at least one second compartment capable of being in fluid communication with the first compartment, the second compartment containing a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or an analog thereof directly or indirectly bound thereto.

9. The microfluidic device of claim 8, wherein the sensitizer of (b) has an avidin analog directly or indirectly bound thereto, and wherein the avidin analog is selected from the group consisting of avidin, streptavidin, traptavidin, neutravidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, and any ester, salt, or derivative thereof of any of the foregoing analogs.

10. A method for detecting the presence and / or concentration of biotin in a sample, the method comprising the steps of: (a) either simultaneously or in whole or in part in succession: (1) Samples suspected of containing biotin; (2) a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or a biotin analog directly or indirectly bound thereto; and (3) an excess of a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having avidin or an avidin analog directly or indirectly bound thereto; (b) allowing (3) to bind to biotin present in the sample or to (2), thereby forming a complex between (2) and (3) in the absence of biotin and bringing the sensitizer into close proximity with the chemiluminescent compound; (c) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes activation of the chemiluminescent compound present in the complex; (d) measuring the amount of chemiluminescence generated by the activated chemiluminescent compound; (e) optionally repeating steps (b)-(d); and (f) detecting the presence and / or concentration of biotin by analyzing the amount of chemiluminescence so generated, wherein the amount of chemiluminescence is inversely proportional to the amount of biotin in the sample.

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