Patent Application
20160025736
Immunoassay technologies are widely used in the field of medical diagnostics. One example of a commercially used immunoassay is the induced luminescence immunoassay (LOCI®) technology. The induced luminescence immunoassay is described in U.S. Pat. No. 5,340,716 (Ullman), the entire contents of which are expressly incorporated herein by reference. The currently available LOCI® technology involves a homogeneous assay (i.e., no wash steps involved) that has high sensitivity, and the assay uses several reagents and requires that two of these reagents (referred to as a “sensibead” and a “chemibead”) held by other immunoassay reagents to be in close proximity to achieve a signal. Upon exposure to light at a certain wavelength, the sensibead releases singlet oxygen, and if the two beads are in close proximity, the singlet oxygen is transferred to the chemibead; this causes a chemical reaction that results in the chemibead giving off light that can be measured at a different wavelength.
However, there are obstacles that exist for this technology. There are multiple factors that can contribute to background signal, such as but not limited to, (1) the nonspecifically binding of two beads to one another, and (2) the presence of two unattached beads that are simply in close proximity to one another. For these reasons, the final reaction mixture is diluted prior to light exposure to dissociate nonspecifically bound beads and to increase the mean particle distance between unbound beads. In addition, as the assay is homogeneous, plasma separation is required, and thus whole blood cannot be directly used in this diagnostic platform.
The presently disclosed and claimed inventive concept(s) is directed to new and improved compositions, assays, and methods of production and use thereof; this technology provides a heterogeneous assay format in which background signal is reduced and plasma separation is not required.
Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation, and/or results. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, 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. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Unless otherwise defined herein, scientific, and technical terms used in connection with the presently disclosed and claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.
All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed and claimed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated 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.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this presently disclosed and claimed inventive concept(s) have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the presently disclosed and claimed inventive concept(s). All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the appended claims.
As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. For example but not by way of limitation, when the term “about” is utilized, the designated value may vary by plus or minus twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent. The use of the term “at least one” will be understood to include one as well as any quantity 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” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may 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, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.
As used in this specification and claim(s), the words “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.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding 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 if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, the phrase “associated with” includes covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.
The term “purified” as used herein means at least one order of magnitude of purification is achieved compared to the starting material or of the natural material, for example but not by way of limitation, two, three, four, or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term “purified” as utilized herein does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.
Throughout the specification and claims, unless the context requires otherwise, the terms “substantially” and “about” will be understood to not be limited to the specific terms qualified by these adjectives/adverbs, but allow for minor variations and/or deviations that do not result in a significant impact thereto. For example, in certain instances the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value and/or the variation that exists among study subjects. Similarly, the term “substantially” may also relate to 80% or higher, such as 85% or higher, or 90% or higher, or 95% or higher, or 99% or higher, and the like.
The terms “analog” and “derivative” are used herein interchangeably and refer to a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound, but can also contain one or more substitutions thereto. The term “substitution” as used herein 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, halogenid selected from fluoride, chloride bromide or iodite, a C1-C4 compound selected one of the following: linear, branched or cyclic alkyl, optionally substituted, and linear branched or cyclic alkenyl, wherein the optional substitutents are selected from one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted wherein the optional substitutents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cyclalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocyclalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, —NH (alkyl), —NH(cycloalkyl)2, carboxy, and —C(O))-alkyl.
In particular embodiments, the term “analog” as used herein refers to a compound that binds to the same binding partner (i.e., antibody) as a target analyte but that is chemically different from the target analyte. For example but not by way of limitation, when the target analyte is a peptide, polypeptide, or protein, the target analyte may possess an epitope to which a binding partner binds (i.e., for indirect association of the solid phase, singlet oxygen-activatable chemiluminescent composition, and/or sensitizer with the target analyte). In this example, an analog of the target analyte possesses an epitope that is identical to the epitope of the target analyte that is recognized by the binding partner; therefore, the analog is capable of binding to the binding partner to which the target analyte binds, even through the analyte may have a different amino acid sequence than the target analyte and thus be less than 100% identical thereto.
The term “sample” as used herein will be understood to include any type of biological sample that may be utilized in accordance with the presently disclosed and claimed inventive concept(s). Examples of biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof, plasma, serum, saliva, sputum, cerebrospinal fluid (CSF), skin, interstitial fluids, tears, mucus, urine, swabs, and the like.
The term “binding partner” as used herein will be understood to refer to any molecule capable of associating with another molecule. For example but not by way of limitation, the binding partner may be an antibody (including polyclonal or monoclonal antibodies), antibody fragments (such as but not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody), a receptor, a ligand, aptamers, antibody substitute proteins or peptides (i.e., engineered binding proteins/peptides), molecular imprinted polymers (i.e., inorganic matrices), combinations or derivatives thereof, as well as any other molecules capable of specific binding to the analyte (or analog thereof).
Turning now to particular embodiments of the presently claimed and disclosed inventive concept(s), assay compositions as well as kits containing same and methods of use thereof are disclosed. In some assay embodiments, signal producing system (sps) members comprise a sensitizer such as, for example, a photosensitizer, and a chemiluminescent composition where activation of the sensitizer results in a product that activates the chemiluminescent composition. One sps member usually generates a detectable signal that relates to the amount of bound and/or unbound sps member, i.e., the amount of sps member bound or not bound to the analyte being detected or to an agent that reflects the amount of the analyte to be detected. An exemplary embodiment of an assay platform on which the presently disclosed and claimed inventive concept(s) is based is the induced luminescence immunoassay (LOCI®). The induced luminescence immunoassay is described in U.S. Pat. No. 5,340,716 (Ullman), the entire contents of which are expressly incorporated herein by reference.
The presently disclosed and claimed inventive concept(s) includes a composition containing a chemiluminescent detection system. In certain embodiments, the composition includes at least three components: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound capable of directly or indirectly binding to the target analyte; (b) a sensitizer capable of directly or indirectly binding to a target analyte and capable of generating singlet oxygen in its excited slate; and (c) a binding partner associated with a solid phase, wherein the binding partner is specific for at least one of (a), (b), and the target analyte (and/or a portion of a sandwich complex formed therefrom), and is thereby capable of attaching the sandwich complex to the solid phase. In other embodiments, the composition includes at least two components: (a) a composition capable of directly or indirectly binding to a target analyte, the composition comprising a singlet oxygen-activatable chemiluminescent compound and a sensitizer capable of generating singlet oxygen in its excited state; and (b) a binding partner associated with a solid phase, wherein the binding partner is specific for the target analyte such that the binding partner is capable of capturing target analyte bound to the composition of (a) on the solid phase. In this embodiment, the singlet oxygen-activatable chemiluminescent compound and the sensitizer are applied together in a single composition; this composition may be in the form of a unibead or similar formulation.
In certain embodiments, the composition includes at least three components: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound capable of directly or indirectly binding to the target analyte; (b) a sensitizer capable of directly or indirectly binding to a target analyte and capable of generating singlet oxygen in its excited state; and (c) a binding partner associated with a solid phase, wherein the binding partner is specific for a portion of the sandwich complex formed of (a), (b), and the target analyte, and is thereby capable of attaching the sandwich complex to the solid phase.
In other embodiments, only one of the sensitizer and activatable chemiluminescent composition is capable of directly or indirectly binding to the target analyte, and the other component binds to the component capable of binding to the target analyte. For example, the composition comprising the singlet oxygen-activatable chemiluminescent compound may be capable of directly or indirectly binding to the target analyte, while the sensitizer may be capable of binding to the composition comprising the singlet oxygen-activatable chemiluminescent compound. Alternatively, the sensitizer may be capable of directly or indirectly binding to the target analyte, while the composition comprising the singlet oxygen-activatable chemiluminescent compound is capable of binding to the sensitizer. In either of these examples, the binding partner associated with the solid phase is capable of binding to the target analyte and thus attaching the sandwich complex formed of the two components and target analyte to the solid phase.
In other embodiments, the composition includes at least two components: (a) a composition capable of directly or indirectly binding to a target analyte, the composition comprising a singlet oxygen-activatable chemiluminescent compound and a sensitizer capable of generating singlet oxygen in its excited state; and (b) a binding partner associated with a solid phase, wherein the binding partner is specific for the target analyte such that the binding partner is capable of capturing target analyte bound to the composition of (a) on the solid phase. In this embodiment, the singlet oxygen-activatable chemiluminescent compound and the sensitizer are applied together in a single composition; this composition may be in the form of a unibead or similar formulation.
In other embodiments, the composition contains a competitive chemiluminescent detection system. In this embodiment, the binding partner associated with the solid phase has target analyte or an analog thereof bound thereto. When a single composition containing both singlet oxygen-activatable chemiluminescent compound and sensitizer is utilized, the single composition is capable of directly or indirectly binding to the target analyte (or an analog thereof) bound to the binding partner or to target analyte present in a sample. When these two reagents are present in separate compositions, then each reagent (i.e., the composition containing the singlet oxygen-activatable chemiluminescent compound and the sensitizer) is capable of directly or indirectly binding to the target analyte (or an analog thereof) bound to the binding partner or to target analyte present in a sample.
In another embodiment of a competitive chemiluminescent detection system, target analyte or an analog thereof is bound to either the sensitizer or the composition comprising the singlet oxygen-activatable chemiluminescent compound. In this embodiment, the other two reagents are capable of directly or indirectly binding to the target analyte or analog thereof bound to the sensitizer/chemiluminescent composition or to target analyte present in a sample.
In yet another embodiment of the competitive chemiluminescent detection system, the single composition containing both singlet oxygen-activatable chemiluminescent compound and sensitizer is utilized and has target analyte or an analog thereof bound thereto. The binding partner associated with the solid phase is then capable of directly or indirectly binding to the target analyte or an analog thereof bound to the single composition or to target analyte present in a sample.
Any of the compositions described above or otherwise contemplated herein may further include a wash solution. In addition, any of the compositions described herein above or otherwise contemplated herein may also include a microfluidics device in which one or more of the above-described components are applied.
A sensitizer is a molecule, usually a compound, for generation of a reactive intermediate such as, for example, singlet oxygen, for activation of a chemiluminescent compound. In some embodiments, the sensitizer is a photosensitizer. Other sensitizers that can be chemi-activated (by, e.g., enzymes and metal salts) include, by way of example and not limitation, other substances and compositions that can produce 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 the alkaline earth metals Ca, Sr, and Ba; derivatives of elements of groups 3A, 4A, 5A, and 6A in d0 configuration; oxides of actinides and lanthanides; and oxidizers CIO−, BrO−, Au3+, IO3−, and IO4; and in particular, molybdate, peroxomolybdate, tungstate, and peroxotungstate ions, and acetonitrile. The following references, which are hereby expressly incorporated by reference in their entirety, provide further disclosure regarding sensitizer substances and compositions that also fall within the scope of the presently disclosed and claimed inventive concept: 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 Boutterny, J. Am. Chem. Soc., 119:5286-5294 (1997); and Almeida et al., Anal. Chim. Acta, 482:99-104 (2003).
Also included within the scope of photosensitizers are compounds that are not true sensitizers but which on excitation by heat, light, ionizing radiation, or chemical activation will release a molecule of singlet oxygen. Members of this class of compounds include, for example, the endoperoxides such as 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, and 5,6,11,12-tetraphenyl naphthalene 5,12-endoperoxide. Heating or direct absorption of light by these compounds releases singlet oxygen.
A photosensitizer is a sensitizer for activation of a photoactive compound, for example, by generation of singlet oxygen by excitation with light. The photosensitizers are photoactivatable and include, e.g., dyes and aromatic compounds, and are usually compounds comprised of covalently bonded atoms, usually with multiple conjugated double or triple bonds. The compounds should absorb light in the wavelength range of 200 to 1,100 nm, or 300 to 1,000 nm, or 450 to 950 nm, with an extinction coefficient at its absorbance maximum greater than 500 M−1 cm−1, or greater than 5,000 M−1 cm−1, or greater than 50,000 M−1cm−1, at the excitation wavelength. Photosensitizers should be relatively photostable and may not react efficiently with singlet oxygen. Examples of photosensitizers, by way of illustration and not limitation, include acetone, benzophenone, 9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue, metallo-porphyrins, such as hematoporphyrin, phthalocyanines, chlorophylls, rose bengal, and buckminsterfullerene, for example, and derivatives of these compounds.
A chemiluminescent compound (chemiluminescer) is a compound that is chemically activatable and, as a result of such activation, emits light at a certain wavelength. Examples of chemiluminescers, by way of illustration and not limitation, include olefins capable of reacting with singlet oxygen or a peroxide to form hydroperoxides or dioxetanes, which can decompose to ketones or carboxylic acid derivatives; stable dioxetanes which can decompose by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azo carbonyls such as luminol; and aromatic compounds that can form endoperoxides, for example. As a consequence of the activation reaction, the chemiluminescers directly or indirectly cause the emission of light.
In certain embodiments, the singlet oxygen-activatable chemiluminescent compound may be a substance that undergoes a chemical reaction with singlet oxygen to form a metastabile intermediate species that can decompose with the simultaneous or subsequent emission of light. The composition comprising the singlet oxygen-activatable chemiluminescent compound may associate with the target analyte (or analog thereof) by any method known in the art; for example but not by way of limitation, the composition may have a second analyte-specific binding partner associated therewith that allows for the indirect association of the chemiluminescent compound to the target analyte. The composition comprising the chemiluminescent compound may be directly excited by the activated chemiluminescent compound; alternatively, the composition may further comprise at least one fluorescent molecule that is excited by the activated chemiluminescent compound.
Particular, non-limiting examples of chemiluminescent compounds and photosensitizers that may be utilized in accordance with the presently disclosed and claimed inventive concept(s) are set forth in U.S. Pat. No 5,340,716 (Ullman, et al.), the entire contents of which are hereby expressly incorporated herein by reference.
Sensitizers utilized in accordance with the presently disclosed and claimed inventive concept(s) may be capable of indirectly binding to the target analyte (or analog thereof) via an association with streptavidin. In this manner, biotin is associated with a first analyte-specific binding partner, and the binding of streptavidin and biotin, in combination with the binding of the first analyte-specific binding partner to the target analyte (or analog thereof), results in the indirect association of the sensitizer to the target analyte (or analog thereof). In one non-limiting example, the sensitizer may be a photosensitizer, such that the sensitizer is activated by irradiation with light.
Any solid phase known in the art or otherwise contemplated herein may be utilized in the claimed compositions/kits/methods. The solid phase may possess any structure and shape that allows the solid phase to function in accordance with the presently disclosed and claimed inventive concept(s). Examples of solid phase structures include, but are not limited to, particulate, including beads and particles (including but not limited to, magnetic particles), film, membrane, tube, well, strip, rod, and planar surfaces such as, e.g., plate (i.e., ELISA plate). Depending on the type of assay, the solid support may or may not be suspendable in the medium in which it is employed. Non-limiting examples of a suspendable solid support include polymeric materials such as latex particles and magnetic particles. Other solid support compositions include but are not limited to, polymers, such as poly(vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly-(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate) nylon, and poly(vinyl butyrate); these compositions may be used alone, in combination with one another and/or in conjunction with other materials.
In certain embodiments, the solid phase may be a particle. The particles generally have an average diameter in a range of from about 0.02 to about 100 microns, or from about 0.05 to about 100 microns, or from about 0.1 to about 100 microns, or from about 0.5 to about 100 microns, or from about 0.02 to about 50 microns, or from about 0.05 to about 50 microns, or from about 0.1 to about 50 microns, or from about 0.5 to about 50 microns, or from about 0.02 to about 20 microns, or from about 0.05 to about 20 microns, or from about 0.1 to about 20 microns, or from about 0.5 to about 20 microns, or from about 0.3 microns to about 10 microns, or about 0.3 microns to about 5 microns. In certain embodiments, the particles may be latex particles or chromium dioxide, iron oxide, or other magnetic particles.
A latex particle is a particulate water suspendable, water insoluble polymeric material. In certain embodiments, the latex is a substituted polyethylene such as polystyrene-butadiene, polyacrylamide polystyrene, polystyrene with amino groups, poly-acrylic acid, polymethacrylic acid, acrylonitrile-butadiene, styrene copolymers, polyvinyl acetate-acrylate, polyvinyl pyridine, vinyl-chloride acrylate copolymers, and the like.
Polymeric particles can be formed from addition or condensation polymers. The particles will be readily dispersible in an aqueous medium and can be functionalizable so as to permit conjugation to one or more members. The particles can also be derived from naturally occurring materials, naturally occurring materials that are synthetically modified, and synthetic materials. In some embodiments the particles have, either naturally occurring or synthetically introduced, a reactive functionality such as, for example, amine groups, which are reactive with a corresponding reactive functionality such as, for example, aldehyde groups.
The manner of association of the solid phase with the binding partner depends on one or more of the properties of the solid phase, the properties of the binding partner/reagent, the surface area and porosity of the solid phase, the nature of any solvent employed, etc. The association may be by adsorption of the binding partner by the solid phase, covalent bonding of the binding partner to the solid phase, dissolution or dispersion of the binding partner in the solid phase, non-covalent bonding of the binding partner to the solid phase by means of binding pair members (e.g., avidin-biotin and digoxin-antibody for digoxin), for example. In this manner the binding partner is “associated with” the solid phase.
Association of a binding partner with latex particles may involve incorporation during formation of the particles by polymerization, or incorporation into preformed particles, e.g., by non-covalent dissolution into the particles, for example. In some approaches a solution containing the binding partner may be employed. Solvents that may be utilized include, for example, alcohols, including, e.g., ethanol, ethoxyethanol, methoxyethanol, ethylene glycol, and benzyl alcohol; amides such as, e.g., dimethyl formamide, formamide, acetamide, and tetramethyl urea; sulfoxides such as, e.g., dimethyl sulfoxide and sulfolane; and ethers such as, e.g., carbitol, ethyl carbitol, and dimethoxy ethane; and water; and mixtures of two or more of the above. The use of solvents having high boiling points in which the particles are insoluble permits the use of elevated temperatures to facilitate dissolution of the compounds into the particles and are particularly suitable. The solvents may be used singly or in combination. A solvent should be selected that does not interfere with the signal producing ability of the reagent because of its intrinsic properties or ability to be removed from the particles. In some embodiments aromatic solvents may be employed such as, for example, dibutylphthalate, benzonitrile, naphthonitrile, dioctylterephthalate, dichlorobenzene, diphenylether, and dimethoxybenzene.
The reagents of the compositions/kits/methods may be provided in any form that allows them to function in accordance with the presently disclosed and claimed inventive concept(s). For example, but not by way of limitation, the reagents may be applied in the form of single aliquot lyophilized reagents. The use of dried reagents in microfluidics devices is described in detail in co-pending application U.S. Ser. No. 61/562,677, the entire contents of which are hereby expressly incorporated herein by reference.
The presently disclosed and claimed inventive concept(s) further includes kits useful for conveniently performing an assay for the determination of an analyte; the kit may contain any combination of the above-described components/reagents (including any of the embodiments of compositions described herein above); in addition, the kit may further contain other reagent(s) for conducting any of the particular assays described or otherwise contemplated herein. The nature of these additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art.
The components/reagents may each be in separate containers/compartments, or various components/reagents can be combined in one or more containers/compartments, depending on the cross-reactivity and stability of the components/reagents. The kit can further include other separately packaged reagents for conducting an assay, such as additional sbp members, sps members, and ancillary reagents, for example. In addition, the kit may include a microfluidics device in which the components/reagents are applied.
The relative amounts of the various components/reagents in the kits can vary widely to provide for concentrations of the components/reagents that substantially optimize the reactions that need to occur during the assay methods and further to optimize substantially the sensitivity of an assay. Under appropriate circumstances one or more of the components/reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the presently disclosed and claimed inventive concept(s) can be obtained from these components. Positive and/or negative controls may be included with the kit. The kit can further include a set of written instructions explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.
The presently disclosed and claimed inventive concept(s) is further directed to a microfluidics device that includes a sample application chamber in which a sample may be applied and an inlet channel in fluidic communication therewith that is also in fluidic communication with one or more compartments containing the three components described herein above (i.e., sensitizer, composition comprising singlet oxygen-activatable chemiluminescent compound (or a composition comprising both sensitizer and singlet oxygen-activatable chemiluminescent compound), solid phase with binding partner associated therewith, and any of the above having target analyte or an analog thereof bound thereto for use in a competitive assay format). The device may be provided with any number of compartments, any arrangement of compartments, and any distribution of the three components there between, so long as the device is able to function in accordance with the presently disclosed and claimed inventive concept(s); non-limiting examples of device structure are provided in the Figures for illustrative purposes only.
Any of the compartments of the microfluidics device may be sealed to maintain reagent(s) applied therein in a substantially air tight environment until use thereof; for example, compartments containing lyophilized reagent(s) may be sealed to prevent any unintentional reconstitution of the reagent. The inlet channel and a compartment, as well as two compartments, may be described as being “capable of being in fluidic communication” with one another; this phrase indicates that the compartment(s) may still be sealed, but the two compartments are capable of having fluid flow there between upon puncture of a seal formed therein or there between.
The microfluidics devices of the presently disclosed and claimed inventive concept(s) may be provided with any other desired features known in the art or otherwise contemplated herein. For example but not by way of limitation, the microfluidics devices of the presently disclosed and claimed inventive concept(s) may further include a read chamber; the read chamber may be the compartment containing the solid phase having a binding partner associated therewith, or the read chamber may be in fluidic communication with said compartment. The microfluidics device may further include one or more compartments containing other solutions, such as but not limited to, wash solutions, dilution solutions, excipients, interference solutions, positive controls, negative controls, quality controls, and the like. For example, the microfluidics device may include one or more compartments containing a wash solution, and these compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device. In another example, the microfluidics device may further include one or more compartments containing at least one excipient for dissolution of one or more dried reagents, and the compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device. Further, the microfluidics device may further include one or more compartments containing a dilution solution, and the compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device.
In addition, any of the kits/microfluidics devices described or otherwise contemplated herein may include multiple assays multiplexed in a single kit/device. When multiple assays are present, both of the assays may be constructed and function as described herein. Alternatively, an assay as described herein may be multiplexed with any other assay known in the art that is capable of being contained within the kits/microfluidics devices of the presently disclosed and claimed inventive concept(s). 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, fr4, T4, PSA, fPSA, and cPSA, and combinations thereof.
When multiple assays are present in a single microfluidics device, multiple inlet channels may be connected to the sample application chamber. In certain embodiments, a portion of the sample may be passed from the sample application chamber to the multiple inlet channels without regard for the content thereof. Alternatively, structure(s) may be present in the sample application chamber, the inlet channels, and/or the connection there between that allow for separation of certain components from the whole sample and delivery of said components to the different assays. A non-limiting example of a sample distribution device that may be utilized in accordance with the presently disclosed and claimed inventive concept(s) is described in detail in Provisional Application No. 61/790,580, filed Mar. 15, 2013, entitled “Microfluidic Distributing Device.”
The presently disclosed and claimed inventive concept(s) is further directed to a method for detecting the presence and/or concentration of a target analyte in a sample (such as but not limited to, whole blood, lysed whole blood cells, or red blood cells). In one embodiment, the method includes the steps of combining, either simultaneously or wholly or partially sequentially: a sample suspected of containing the target analyte with the sensitizer, composition comprising the singlet oxygen-activatable chemiluminescent compound, and the binding partner associated with the solid phase as described herein above (wherein the binding partner, the sensitizer, and the composition comprising the singlet oxygen-activatable chemiluminescent compound are capable of directly or indirectly binding to target analyte and/or to each other, as described herein above). The composition comprising the chemiluminescent compound, the sensitizer and/or the binding partner are allowed to bind to any target analyte present in the Sample (and/or to each other), whereby a sandwich complex associated with the solid phase is formed and the sensitizer is brought into close proximity to the chemiluminescent compound. The sensitizer is then activated to generate singlet oxygen, wherein activation of the sensitizer present in the sandwich complex causes the activation of the chemiluminescent compound present in the sandwich complex. The amount of chemiluminescence generated by the activated chemiluminescent compound is then determined, and the binding, activating, and/or determining steps may optionally be repeated for a desired number of times. The presence and/or concentration of the target analyte are detected by analyzing the amount of chemiluminescence so produced, wherein the amount of chemiluminescence is directly proportional to the amount of target analyte in the sample.
In a similar embodiment of the method, the sample and binding partner are combined as described herein above with a single composition that contains both singlet oxygen-activatable chemiluminescent compound and sensitizer; in this manner, the binding partner is specific for the target analyte such that the binding partner is capable of capturing target analyte bound to the single composition on the solid phase. The single composition and binding partner are allowed to bind to target analyte present in the sample, and the single composition becomes associated with the solid phase. The solid phase is then washed to substantially remove unbound or non-specifically bound sample or single composition, and the sensitizer is activated to generate singlet oxygen, wherein activation of the sensitizer causes activation of the chemiluminescent compound. The amount of chemiluminescence generated by the activated chemiluminescent compound is determined, and the binding, activating, and/or determining steps may optionally be repeated for a desired number of times. The presence and/or concentration of the target analyte are detected by analyzing the amount of chemiluminescence so produced, wherein the amount of chemiluminescence is directly proportional to the amount of target analyte in the sample.
In certain other embodiments, the method involves a competitive assay format, in which a target analyte or an analog thereof is attached to the binding partner associated with the solid phase, wherein any target analyte present in the sample competes with the solid phase-attached analyte or analog thereof for binding to the sensitizer/singlet oxygen-activatable chemiluminescent compound. In these embodiments, the sample is combined as described above with the binding partner having target analyte or analog thereof bound thereto and either a single composition comprising both sensitizer/singlet oxygen-activatable chemiluminescent compound, or with sensitizer and a separate composition comprising singlet oxygen-activatable chemiluminescent compound. When the single composition is utilized, the single composition is capable of directly or indirectly binding to the target analyte or analog thereof bound to the binding partner or to target analyte present in a sample. Likewise, when separate reagents are utilized, each of the reagents is capable of directly or indirectly binding to the target analyte or analog thereof bound to the binding partner or to target analyte present in the sample. The above listed reagents are allowed to bind either to target analyte or analog thereof bound to the solid phase or to target analyte present in the sample; when two reagents are utilized, the binding thereof to target analyte or analog thereof bound to the binding partner forms a sandwich complex associated with the solid phase such that the sensitizer is brought into close proximity to the chemiluminescent compound, and whereby binding of the two reagents to target analyte present in the sample forms a sandwich complex that is not associated with the solid phase. In a similar manner, when a single composition is utilized, the binding thereof to target analyte or analog thereof bound to the binding partner associates the single composition with the solid phase, whereas the binding of the single composition to target analyte present in the sample prevents association of the single composition with the solid phase. In either embodiment, the solid phase is then washed to substantially remove unbound or non-specifically bound sample and reagents and to substantially remove any complexes formed by binding of the dual reagents (or single composition containing the two reagents) to target analyte present in the sample. The remaining steps of the method are conducted as described in the previous embodiments, with the exception that the amount of chemiluminescence is inversely proportional to the amount of target analyte present in the sample.
In another embodiment of the competitive assay format, a target analyte or an analog thereof is attached to either the sensitizer or to the composition comprising singlet oxygen-activatable chemiluminescent compound (or to the single composition containing both reagents), wherein any target analyte present in the sample competes with the sensitizer/chemiluminescent composition-bound analyte or analog thereof for binding to the binding partner associated with the solid phase and/or to the other component (when two separate reagents are utilized). In these embodiments, the sample, binding partner associated with the solid phase, and the sensitizer and chemiluminescent compositions (whether in single composition form or as separate compositions) are combined as described above and allowed to bind either to target analyte or analog thereof bound to one of the two components or to target analyte present in the sample. Binding of the component containing target analyte or analog thereof to the binding partner (and the other component when present in two separate compositions) forms a sandwich complex associated with the solid phase wherein the sensitizer is brought into close proximity to the chemiluminescent compound. In contrast, binding of the binding partner (and non-target analyte containing component if the two compositions are present as separate reagents) to target analyte present in the sample forms a sandwich complex that is associated with the solid phase but which does not contain one or both of the sensitizer and the chemiluminescent compound. The solid phase is then washed to substantially remove unbound or non-specifically bound sample and reagents. The remaining steps of the method are conducted as described in the previous embodiments, with the exception that the amount of chemiluminescence is inversely proportional to the amount of target analyte present in the sample.
When the composition comprising the chemiluminescent compound includes a fluorescent molecule that is excited by the activated chemiluminescent compound, the method may further include the step of measuring the amount of light emitted by the fluorescent molecules to determine the amount of analyte in the sample.
As mentioned above, the various components of the method are provided in combination (either simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition of the components may be varied; a person having ordinary skill in the art can determine the particular desired order of addition of the different components to the assay. The simplest order of addition, of course, is to add all the materials simultaneously and determine the signal produced therefrom. Alternatively, each of the components, or groups of components, can be combined sequentially. In certain embodiments, an incubation step may be involved subsequent to each addition as discussed above.
As described herein above, any of the embodiments of the presently disclosed and claimed inventive concept(s) may be provided in the form of a heterogeneous assay; that is, the method may further include one or more washing steps employed after an incubation step(s). When the reagents are added to the assay in a sequential format, the method may include multiple washing steps (i.e., after each reagent addition and incubation with the reaction). The washing steps function to reduce background signal and potentially increase analytical sensitivity. For example but not by way of limitation, one embodiment of the method may further include the step of substantially washing away unbound or non-specifically bound sample, sensitizer, and composition comprising the singlet oxygen-activatable chemiluminescent compound from the solid phase having the sandwich complex attached thereto, prior to activation of the sensitizer. Washing steps may also be utilized when a unibead or other similar single composition containing dual reagents is used. In addition, washing steps may be included in competitive assay embodiments as described above, to remove reagents bound to target analyte present in the sample.
Turning now to the Drawings,
While the sensitizer 20 and composition 22 including the singlet oxygen-activatable chemiluminescent compound are depicted in
The inlet channel 16 may simply transfer a portion of the sample to the first compartment 18, or the inlet channel 16 may contain structure(s) that allow for separation of certain components from the whole sample (i.e., separation filter(s) that provide for separation of plasma or red blood cells from a whole blood sample applied in the sample application chamber 14) and/or detection of degradation (such as but not limited to, hemolysis) in the sample.
Any of the microfluidics devices described or otherwise contemplated herein may be provided with additional compartments containing other reagents/solutions. For example,
Any of the compartments of any of the microfluidics devices described or otherwise contemplated herein may be sealed to maintain reagent(s) applied therein in a substantially air tight and/or substantially light tight environment until use thereof; for example, compartments containing lyophilized reagent(s) may be sealed to prevent any unintentional reconstitution of the reagent and/or exposure of any of the reagents to light. The inlet channel and a first compartment, as well as two compartments, may be described as being “capable of fluidic communication” with one another; this phrase indicates that the compartment(s) may still be sealed, but are capable of having fluid flow there between upon puncture of a seal formed therein.
In addition, it is to be understood that any of the microfluidics devices described or otherwise contemplated herein may further be provided with additional chambers and/or other fluidic circuits. For example, but not by way of limitation, any of the microfluidics devices may additionally contain mixing chamber(s) and/or fluidic circuit(s) that are applied between two reagent chambers.
The microfluidics device 50 includes a housing 52 that includes a sample application chamber 53, an inlet channel 54, a first compartment 56, a second compartment 58, and a waste compartment 60. A sample (such as, but not limited to, a blood sample) may be applied to the sample application chamber 53, which is in (or is capable of being in) fluidic communication with the inlet channel 54. The inlet channel 54 is in (or capable of being in) fluidic communication with the first compartment 56. The first compartment 56 contains a predetermined amount of sensitizer 62 and a predetermined amount of a composition 64 that includes a singlet oxygen-activatable chemiluminescent compound. The second compartment 58 is in (or is capable of being in) fluidic communication with the first compartment 56; the second compartment 58 contains a predetermined amount of a composition 66 that includes a solid phase having the binding partner associated therewith. The second compartment 58 may further be defined as a read chamber and is in (or is capable of being in) fluidic communication with the waste compartment 60.
While the sensitizer 62 and composition 64 including the singlet oxygen-activatable chemiluminescent compound are depicted in
The order of distribution of the reagents 62, 64, and 66 in the compartments 56 and 58 is for the purposes of example only and should not be construed as limiting. The reagents 62, 64, and 66 may be applied in the compartments 56 and 58 in any desired order. For example, the predetermined amount of sensitizer 62 may be applied in the second compartment 58 along with the composition 66. The microfluidics device 50 may further be provided with one or more additional compartments containing wash solution and/or excipient (as described above with respect to
The microfluidics device 150 includes a housing 152 that includes a sample application chamber 153, an inlet channel 154, a first compartment 156, a second compartment 158, a third compartment 160, and a waste compartment 162. A sample (such as, but not limited to, a blood sample) may be applied to the sample application chamber 153, which is in (or is capable of being in) fluidic communication with the inlet channel 154. The inlet channel 154 is in (or capable of being in) fluidic communication with the first compartment 156. The first compartment 156 contains a predetermined amount of a composition 164 that includes a singlet oxygen-activatable chemiluminescent compound. The second compartment 158 is in (or is capable of being in) fluidic communication with the first compartment 156; the second compartment 158 contains a predetermined amount of sensitizer 166. The third compartment 160 is in (or is capable of being in) fluidic communication with the second compartment 158; the third compartment 160 contains a predetermined amount of a composition 168 that includes a solid phase having the binding partner associated therewith. The third compartment 160 may further be defined as a read chamber and is in (or is capable of being in) fluidic communication with the waste compartment 162. When the microfluidics device 150 is utilized in a competitive assay format, it will be understood that one of the composition 164, the sensitizer 166, and the composition 168 may further include target analyte or an analog thereof bound thereto.
The order of distribution of the reagents 164, 166, and 168 in the compartments 156, 158, and 160 is for the purposes of example only and should not be construed as limiting. The reagents 164, 166, and 168 may be applied in the compartments 156, 158, and 160 in any desired order.
The microfluidics device 150 is also illustrated as containing a fourth compartment 170 that contains a predetermined amount of wash solution 172. The fourth compartment 170 is illustrated as being in (or capable of being in) fluidic communication with the inlet channel 154 and/or the first compartment 156; however, it is to be understood that the fourth compartment 170 may be in (or may be capable of being in) fluidic communication with any of the compartments 156, 158, and/or 160. The presence of wash solution 172 is for the purposes of example only; it is to be understood that the solution present in the fourth compartment may be excipient, or the microfluidics device may contain a fifth compartment containing excipient, as described in detail herein above. In addition, the presence of the fourth compartment 170 in the microfluidics device 150 is for purposes of example only, and it is to be understood that the microfluidics device 150 may be produced without said compartment if desired.
As stated herein above, any of the assay structures described herein above may be multiplexed with additional assay(s) in a single microfluidics device.
Thus, in accordance with the presently disclosed and claimed inventive concept(s), there has been provided compositions comprising a chemiluminescent system, as well as kits and microfluidics devices containing same and methods of use thereof, that fully satisfy the objectives and advantages set forth herein above. Although the presently disclosed and claimed inventive concept(s) has been described in conjunction with the specific drawings, experimentation, results and language set forth herein above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the presently disclosed and claimed inventive concept(s).
This application claims benefit of provisional application U.S. Ser. No. 61/787,735, filed Mar. 15, 2013. The entire contents of the before-referenced application are expressly incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/026053 | 3/13/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61787735 | Mar 2013 | US |
