Nanoparticles comprising polymeric gamma globulin and methods of production and use thereof

Abstract

A diagnostic immunoassay reagent composition comprising nanoparticles formed from polymeric gamma globulin is disclosed. The nanoparticle has at least one functional group on its surface, and at least one analyte-specific binding partner is attached to the nanoparticle through the at least one functional group. Also disclosed are kits, devices, and systems comprising the diagnostic immunoassay reagent compositions, as well as methods of producing and using the diagnostic immunoassay reagent compositions.

Description

[0001] Citation of Related Applications

[0002] not applicable.

[0003] Federally funded research or development

[0004] not applicable. Background Art

[0005] In the field of medicine and clinical chemistry, many studies and determinations of physiologically reactive substances or analytes are carried out by utilizing the interaction between specific binding pairs. For example, the target analyte in a patient sample can be a member of the specific binding pair, and the target analyte can be detected by using the corresponding member of the specific binding pair fixed on a solid support. For example (but not limiting), the immobilized binding pair member can be an antigen for detecting a target antibody in a sample, or vice versa (i.e., the immobilized binding pair member can be an antibody for detecting a target analyte in a sample), or the immobilized binding pair member can be a ligand for detecting a target receptor in a sample, or vice versa (i.e., the immobilized binding pair member can be a receptor for detecting a target ligand in a sample). Various supports or surface materials have been developed for these applications, and various bonding or "functionalization" techniques are needed to fix the specific binding pair member on a support or surface.

[0006] Surfaces functionalized with binders are often used as a basic test framework for substance detection. Key issues encountered in the process of functionalizing surfaces with binders include the large amount of materials and resources required and a large amount of time, especially for "delicate" systems. In addition, the coupling process and the resulting waste materials can be harmful, even for some of the surfaces used. The management of the corresponding processes and waste is usually also associated with extensive safety measures and high costs, and may even have to be stopped for regulatory reasons, with significant consequences for the entire business.

[0007] For example, non-magnetic latex particles (NMLPs) are widely used as surfaces (also referred to as "solid phases") in homogeneous immunoassays. Binding agents such as antigens or antibodies are attached to these latex beads and can be attached by chemical reactions. A non-limiting example of such attachment reactions involves the use of an azomethine reaction followed by a reduction process that can use sodium cyanoborohydride (NaBH3CN), a hazardous chemical compound that must be properly managed. Functionalized NMLPs bind to the target analyte in a multivalent manner to form aggregates that can be visualized or quantified using spectrophotometry, particularly (but not limiting) turbidimetry or nephelometry. Quantifying the change in this turbidity (or haziness) or scattered light of the reaction mixture as a function of analyte concentration is the basis of homogeneous immunoagglutination assays.

[0008] In addition to the hazardous materials used in their production, these latex beads used in immunoassays ultimately become waste. The waste disposal costs associated with these immunoassay reagents containing latex beads will continue to be a challenge, and the costs are likely to increase significantly in the future.

[0009] Therefore, there is a need in the art for new and improved diagnostic immunoassay reagents that overcome the deficiencies and shortcomings of the prior art. The present disclosure relates to such reagents, as well as kits and microfluidic devices containing such reagents, and methods of producing and using the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A photograph of one non-limiting embodiment of a protein nanoparticle constructed according to the present disclosure, wherein the protein nanoparticle is formed from polymeric goat gamma globulin (pGGG). The protein nanoparticles shown in this figure are present in solution at a concentration of 10 mg / ml.

[0011] Figure 2 Photographs of solutions containing pGGG protein nanoparticles (1 mg / ml) that have been functionalized on their surface with biotin (left) or fluorescein (right).

[0012] Figure 3 The figure depicts the UV-visible chromatograms of pGGG nanoparticles at 280, 340, and 600 nm. When pGGG nanoparticles were injected into a Sepharose CL-2B column (1.0 x 30 cm), high molecular weight peaks were detected simultaneously at 280, 340, and 600 nm [with the main peak (approximately 100 nm) being excluded at approximately 7 ml]. The absorbance at 340 nm is typically used in typical solution agglutination immunoassays. This chromatographic behavior mimics that of the environmentally unfriendly microplastic polystyrene beads used in the prior art.

[0013] Figure 4 The graph depicts the aggregation of pGGG nanoparticles at A340nm over time. When biotinylated protein nanoparticles (1 mg) and avidin (1 mg) were mixed together, aggregation occurred over time. The buffer was 25 mM sodium phosphate, 75 mM sodium chloride, and 0.05% Tween 20, pH 7.4. When avidin was mixed with protein nanoparticles that did not contain biotin on their surface, no aggregation occurred. This shows that protein nanoparticles can be manipulated similarly to latex beads and liposomes to cause aggregation by turbidimetric methods.

[0014] Figure 5 The graph depicts the binding curve of agglutination at A340nm as a function of avidin (in μg). The binding curve was generated when 20 μg of avidin was gradually added to 1 mg of biotinylated protein nanoparticles.

[0015] Figure 6 Included are photographs illustrating biotinylated pGGG nanoparticles (1 mg / ml) in a test cuvette before (left) and after (right) addition of avidin, illustrating visual detection of agglutination by turbidimetry.

[0016] Figure 7 The figure depicts the dynamic light scattering curve of biotinylated protein nanoparticles (average approximately 100 nm) and the dynamic light scattering curve after an agglutination event in the presence of avidin. The size of the nanoparticles after agglutination is currently approximately 1000 nm.

[0017] Figure 8 The figure depicts the dynamic light scattering curves of unbiotinylated protein nanoparticles and after the addition of avidin. The size remains unchanged. This demonstrates that aggregation can only occur when the nanoparticles are appropriately functionalized.

[0018] Figure 9 The figure depicts the dynamic light scattering curve of polymerized bovine gamma globulin (pBGG). The pBGG nanoparticles shown here have a size range of 100-500 nm. Detailed Description of the Invention

[0020] Before explaining at least one embodiment of the present disclosure in detail through exemplary statements and results, it should be understood that the present disclosure is not limited in its application to the details of the construction and arrangement of the components set forth in the following description. The present disclosure is capable of other embodiments or can be practiced or implemented in various ways. Therefore, the statements used herein are intended to be given the broadest possible scope and meaning; and the embodiments are intended to be exemplary rather than exhaustive. Furthermore, it should be understood that the phraseology and terminology used herein are for descriptive purposes only and should not be considered as limiting.

[0021] Unless otherwise limited herein, the 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. In addition, 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 according to conventional methods well known in the art, and are carried out as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature and laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry as described herein are those well known and commonly used in the art. Chemical synthesis and chemical analysis adopt standard techniques.

[0022] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the levels of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the same extent as if each individual patent or publication were specifically and individually indicated to be incorporated by reference.

[0023] According to the present disclosure, all articles, compositions, kits and / or methods disclosed herein can be made and performed without excessive experimentation. Although these 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 various modifications may be made to the articles, compositions, kits and / or methods described herein and the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the present disclosure. It will be apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope and concept of the present disclosure as defined in the appended claims.

[0024] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0025] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the terms "a" or "an" can mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Therefore, unless the context clearly indicates otherwise, the terms "a", "an", and "the" include plural referents. Thus, for example, reference to "a compound" can mean one or more compounds, two or more compounds, three or more compounds, four or more compounds, or an even greater number of compounds. The term "plurality" means "two or more".

[0026] 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 be extended to 100 or 1000 or more, depending on the term to which it is connected; furthermore, numbers of 100 / 1000 should not 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 terms (i.e., "first," "second," "third," "fourth," etc.) is only used to distinguish between two or more items and is not meant to, for example, imply any order or sequence or the importance of one item over another or any order of addition.

[0027] The use of the term "or" in the claims is intended to mean inclusive "and / or" unless explicitly stated to mean only alternatives, or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: 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).

[0028] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" indicates that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. For example, the appearances of the phrases "in some embodiments" or "an example" in different places in the specification do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as non-limiting to the claims.

[0029] Throughout this application, the term "about" is used to indicate that a value includes inherent error variations in the composition / instrument / device, the method used to determine the value, or differences between the subjects of study. For example, and not limitation, when the term "about" is used, the specified value may vary from the specified 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 suitable for practicing the disclosed methods and are understood by those skilled in the art.

[0030] As used in this specification and claims, 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 “contain” (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.

[0031] 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 if order is important in the particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, specifically included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. It will be understood by those skilled in the art that, unless otherwise apparent from the context, there is generally no limit on the number of items or terms in any combination.

[0032] The term "substantially" as used herein refers to that the event or situation of subsequent description occurs completely, or that the event or situation of subsequent description occurs in a very large range or to an extent. For example, when relevant to a particular event or situation, the term "substantially" refers to that the event or situation of subsequent description 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 refer to that two projects are 100% adjacent to each other, or that two projects are next to each other but not 100% adjacent to each other, or that a part for one of two projects is not 100% adjacent to another project, but is next to another project.

[0033] As used herein, the phrases "associated with" and "coupled with" include direct association / binding of two moieties to one another, as well as indirect association / binding of two moieties to one another. Non-limiting examples of association / coupling include, for example, covalent binding of one moiety to another through a direct bond or through a spacer group, non-covalent binding of one moiety to another directly or via a specific binding pair member bound to the moiety, incorporation of one moiety into another by, for example, dissolving one moiety in the other moiety or by synthesis, and coating one moiety onto another moiety.

[0034] 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. The term "substituted" as used herein is understood to mean replacing at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R may include H, hydroxyl, thiol, a halogen selected from fluorine, chlorine, bromine or iodine, a C1-C4 compound selected from one of the following: optionally substituted linear, branched or cyclic alkyl groups, and linear, branched or cyclic alkenyl groups, wherein the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl 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, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), carboxyl and -C(O)-alkyl.

[0035] As used herein, the term "sample" should be understood to include any type of biological sample that can be used in accordance with the present disclosure. Examples of useful biological samples 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 washings, semen, stool, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0036] The term "specific binding partner," particularly (but not limiting) as used herein in the term "target analyte-specific binding partner," should be understood to refer to any molecule capable of specifically associating with a target analyte. For example, but not limiting, a binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic or organic matrix), combinations or derivatives thereof, and any other molecule capable of specifically binding to a target analyte.

[0037] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity for analyte binding (e.g., but not limited to Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates that retain at least a portion of the variable region of an intact antibody), antibody surrogate proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0038] "Analyte" refers to a macromolecule that is recognized by an analyte-specific binding partner, such as, but not limited to, an antibody. Both analytes and haptens contain at least one antigenic determinant, or "epitope," which is a region on an antigen or hapten to which an analyte-specific binding partner (i.e., an antibody) binds. Typically, an epitope on a hapten is the entire molecule.

[0039] Turning now to the inventive concepts, certain non-limiting embodiments of the present disclosure relate to a diagnostic reagent composition (e.g., but not limited to, a diagnostic immunoassay reagent composition) for detecting a target analyte in a biological sample. The diagnostic reagent composition comprises a protein nanoparticle comprising polymeric gamma globulin and at least one analyte-specific binding partner associated therewith.

[0040] Any gamma globulin known in the art or otherwise contemplated herein that is capable of polymerizing to form protein nanoparticles, which naturally has at least one functional group disposed thereon, can be used in accordance with the present disclosure. Non-limiting examples of useful gamma globulins include mammalian gamma globulins (such as, but not limited to, goat gamma globulin (GGG) and bovine gamma globulin (BGG)), as well as non-mammalian gamma globulins.

[0041] The protein nanoparticles may have disposed thereon any functional group naturally present in gamma globulin, non-limiting examples of which include carboxyl, amine, and / or thiol groups.

[0042] In certain specific (but non-limiting) embodiments, the protein nanoparticles may comprise one or more proteins other than gamma globulin. A non-limiting example of an additional protein that may be included is serum albumin. However, it should be understood that any additional protein may be included so long as the protein nanoparticles are able to function as described herein.

[0043] Any analyte-specific binding partner known in the art or otherwise contemplated herein that is capable of specifically binding to an analyte to be detected and thus useful in diagnostic detection of the analyte may be used in accordance with the present disclosure. Non-limiting examples of types of analyte-specific binding partners useful for diagnostic purposes in accordance with the present disclosure include receptors, ligands, antigens, antibodies, aptamers, molecularly imprinted polymers, and the like, as well as derivatives and variants thereof, and any combination thereof.

[0044] The target analyte can be any molecule present in a biological sample that needs to be detected and / or quantified. For example (but not limitation), when the target analyte is an antigen, the target analyte-specific binding partner can be an antibody or fragment thereof that specifically binds thereto; when the target analyte is an antibody, the target analyte-specific binding partner can be the antigen that the antibody specifically binds to; when the target analyte is a ligand, the target analyte-specific binding partner can be a receptor (or a portion or derivative thereof) that specifically binds thereto; and when the target analyte is a receptor, the target analyte-specific binding partner can be a ligand that specifically binds thereto.

[0045] Nanoparticles made from polymerized gamma globulin can be provided in any size, shape, and dimension, so long as the nanoparticles are capable of functioning according to the present disclosure. Non-limiting examples of nanoparticle sizes that can be utilized in accordance with the present disclosure include about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 75 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 125 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 175 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 225 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 275 nm, about 280 nm, about 290 nm, about 300 nm, about 325 nm, about 350 nm, about and greater, and ranges formed by any of the foregoing values ​​(i.e., a range from about 20 nm to about 1000 nm, a range from about 50 nm to about 1000 nm, a range from about 50 nm to about 500 nm, a range from about 100 nm to about 500 nm, a range from about 100 nm to about 200 nm, etc.).

[0046] In certain specific (but non-limiting) embodiments, a diagnostic reagent composition can be provided, which has any (one or more) elements or features that allow detection of a complex of (one or more) nanoparticles and an analyte bound thereto. In certain specific (but non-limiting) embodiments, the diagnostic reagent composition can have at least one dye associated therewith to facilitate detection of the bound analyte. For example (but non-limiting), the nanoparticles can have at least one dye incorporated into or otherwise associated therewith. The dye can be used for (for example but non-limiting) spectrophotometric luminescence detection (i.e., chemiluminescence or fluorescence detection). Non-limiting examples include fluorescein, rhodamine, nitrobenzofurazan (NBD), etc.

[0047] In certain specific (but non-limiting) embodiments, the nanoparticles may be biotinylated. Biotinylation of the nanoparticles allows for indirect attachment of another biotinylated protein or target molecule to the particle via tetrameric streptavidin.

[0048] Certain non-limiting embodiments of the present disclosure relate to kits comprising one or more of any diagnostic reagent compositions disclosed herein or otherwise contemplated. In certain specific (but non-limiting) embodiments, the kit further comprises at least one additional assay reagent that interacts with the diagnostic reagent composition for detecting the presence and / or concentration of a target analyte in a biological sample.

[0049] In a specific (but non-limiting) embodiment, the kit comprises two or more diagnostic reagent compositions. The two or more diagnostic reagent compositions can be arranged in separate compositions, or the compositions can be arranged together in a single composition for multiple determinations of two different target analytes. When arranged together in a single composition, the first and second diagnostic reagent compositions provided have different dyes associated therewith (or other types of detection mechanisms different from each other), which allows detection of two target analytes in a single reaction.

[0050] The compositions / reagents in the kit can be provided in any form that allows them to function according to the present disclosure. For example, but not limiting, each reagent can be provided in liquid form and placed in the kit in bulk and / or in individual portions. Alternatively, in a specific (but non-limiting) embodiment, one or more reagents can be placed in the kit in the form of individual lyophilized reagents. The use of dried reagents in kits / microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.

[0051] In addition to the compositions / reagents described in detail above, the kit may also include (one or more) other reagents for performing 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 well within the skill of those skilled in the art; therefore, further description thereof is considered unnecessary. In addition, the compositions / reagents present in the kit may each be located in a separate container / compartment, or the various compositions / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the compositions / reagents. In addition, the kit may include a microfluidic device in which the compositions / reagents are provided.

[0052] The relative amounts of the various compositions / reagents in the kit can vary widely to provide a composition / reagent concentration that significantly optimizes the reaction that needs to occur during the assay method and further significantly optimizes the sensitivity and selectivity of the assay. Where appropriate, one or more compositions / reagents in the kit can be provided in the form of a dry powder (e.g., a lyophilized powder), and the kit can also include (one or more) excipients for dissolving the dry reagents; in this way, a reagent solution having a concentration suitable for carrying out the method or assay according to the present disclosure can be obtained from these compositions. The kit can also include positive and / or negative controls. In addition, the kit can also include a set of written instructions for explaining how to use the kit. Kits of this nature can be used for any method described herein or otherwise envisioned.

[0053] Certain additional non-limiting embodiments of the present disclosure relate to a microfluidic device comprising one or more of any diagnostic reagent compositions described above or otherwise contemplated herein. Specifically, certain non-limiting embodiments include a microfluidic device for determining the concentration of at least one target analyte in a sample. The microfluidic device comprises: (i) an inlet channel through which the sample is applied; and (ii) at least one first compartment capable of being in fluid communication with the inlet channel and comprising at least one of any diagnostic reagent compositions disclosed herein or otherwise contemplated. The (one or more) compartments in (ii) may further comprise any additional reagents required to perform an assay for detecting the target analyte. Any assay reagent disclosed or contemplated herein or otherwise known in the art may be used in the microfluidic device of the present disclosure.

[0054] Microfluidic devices can be provided with any arrangement of compartments and the distribution of various compositions / reagents therebetween that allow the device to function in accordance with the present disclosure. That is, when a diagnostic reagent composition is used in combination with a second assay reagent, the two reagents can be placed in the same compartment or in different compartments. When the two reagents are separated into two compartments, the diagnostic reagent composition can be placed in a first compartment in fluid communication with the inlet channel, and at least one additional assay reagent can be placed in a second compartment in fluid communication with the first compartment.

[0055] In a specific (but non-limiting) embodiment, microfluidic device comprises two or more any diagnostic reagent compositions disclosed herein or envisioned in addition, wherein two or more diagnostic reagent compositions are placed in microfluidic device together so that the multiple determination of two different target analytes can be carried out in microfluidic device. In this way, first and second diagnostic reagent compositions (and any additional diagnostic reagent compositions, if present) are provided, which have different dyes associated therewith (or other types of detection mechanisms different from each other), which allow detection of two target analytes in a single reaction. Two or more diagnostic reagent compositions can be placed in the same or separated compartment of microfluidic device.

[0056] Alternatively, the microfluidic device may comprise two or more of any diagnostic reagent compositions disclosed or otherwise contemplated herein, wherein the two or more diagnostic reagent compositions are disposed in separate compartments within the microfluidic device and have separate reading chambers for their detection, thereby allowing for the detection of two different target analytes within the microfluidic device.

[0057] The microfluidic device of the present disclosure may be of any design or configuration known in the art or otherwise contemplated herein for use in diagnostic analyte assays (e.g., but not limited to, diagnostic immunoassays). In certain specific (but non-limiting) embodiments, the microfluidic device may be in the form of a kit configured to be inserted into an automated diagnostic test instrument system that performs a diagnostic assay. Alternatively, the microfluidic device may be a stand-alone product that can be read without the need for a diagnostic test instrument system. For example, but not limiting, the microfluidic device may be in the form of a lateral flow device that can be implemented at a point-of-care (POC) location.

[0058] Any compartment of a microfluidic device can be sealed to maintain the reagent(s) disposed therein in a substantially airtight environment prior to use; for example, a compartment containing lyophilized reagent(s) can be sealed to prevent any accidental reconstitution of the reagents. An inlet channel and a compartment, as well as two compartments, can be described as being "capable of fluid communication" with each other; this phrase indicates that each compartment can still be sealed, but that fluid flow can occur between the two compartments after a seal formed therein or therebetween is broken.

[0059] The microfluidic device of the present disclosure can be provided, which has any other desired features known in the art or otherwise contemplated herein. For example, but not limiting, the microfluidic device of the present disclosure may also include a reading chamber; the reading chamber can be any compartment containing the above-mentioned (one or more) reagents, or the reading chamber can be in fluid communication with the compartment. The microfluidic device may also include one or more additional compartments containing other solutions, such as (but not limited to) washing solutions, diluents, excipients, interfering solutions, positive controls, negative controls, quality controls, etc. These additional compartments can be in fluid communication with one or more other compartments. For example, the microfluidic device may also include one or more compartments containing washing solutions, and these compartments can be in fluid communication with any other compartment of the device. In another example, the microfluidic device may also include one or more compartments containing excipients for dissolving one or more dry reagents, and these compartments can be in fluid communication with any other compartment of the device. In yet another example, the microfluidic device may include one or more compartments containing diluents, and these compartments can be in fluid communication with any other compartment of the device.

[0060] Certain non-limiting embodiments of the present disclosure relate to a method of preparing any diagnostic reagent composition disclosed herein or otherwise contemplated. In one specific (but non-limiting) embodiment, the method comprises the steps of: polymerizing gamma globulin (GG) to form polymeric GG (pGG) nanoparticles; and attaching at least one analyte-specific binding partner to the pGG nanoparticles via at least one naturally occurring functional group disposed on the surface of the nanoparticles.

[0061] The polymerization and attachment reactions can be carried out by any method known in the art or otherwise contemplated herein. For example (but not limiting), pGG nanoparticles can be formed by a technique selected from solvent evaporation, nanoprecipitation, salting out, and emulsification techniques. In one non-limiting example, pGG nanoparticles are formed by heating GG to a temperature in the range of about 65° C. to about 67° C. for a period of time.

[0062] Furthermore, in certain non-limiting embodiments, at least one additional element (such as, but not limited to, a dye) can be incorporated into the diagnostic reagent composition during the production process.

[0063] Certain non-limiting embodiments of the present disclosure relate to a method for determining the presence and / or concentration of at least one target analyte in a biological sample. In this method, the biological sample is combined with at least one diagnostic reagent composition disclosed herein or otherwise contemplated under conditions that allow the target analyte present in the biological sample to substantially bind to the analyte-specific binding partner extending from the outer surface of the nanoparticles of the diagnostic reagent composition, thereby forming a complex; then, the presence and / or concentration of the target analyte is determined based on any complex formed. The determining step can be performed using any assay method known in the art.

[0064] In certain specific (but non-limiting) embodiments, the method employs a homogeneous assay format. For example (but not limiting), the binding of the target analyte to the diagnostic reagent composition results in agglutination, and thus no additional reagents are required for detecting the target analyte. The results of the agglutination assay can be detected manually or automatically (i.e., visually or spectrophotometrically), and can be detected using turbidimetry or nephelometry.

[0065] Alternatively, the method may employ a heterogeneous assay format, where additional reagents must be used in combination with the diagnostic reagent composition for detecting the target analyte in the biological sample. For example (but not limiting), the method may employ a heterogeneous format, such as a sandwich assay. When a second reagent must be used, the biological sample may be contacted with the diagnostic reagent composition and the second reagent simultaneously or in whole or in part sequentially. In addition, the detection step of the method will involve detecting a complex comprising the diagnostic reagent composition / target analyte / second assay reagent.

[0066] Certain non-limiting embodiments of the present disclosure relate to a method for determining the presence and / or concentration of at least two target analytes in a biological sample. In this method, the biological sample is combined with at least two of any diagnostic reagent compositions disclosed herein or otherwise contemplated (i.e., a first diagnostic reagent composition and a second diagnostic reagent composition) under conditions that allow: (1) the first target analyte present in the biological sample to substantially bind to the analyte-specific binding partner extending from the outer surface of the nanoparticles of the first diagnostic reagent composition, thereby forming a first complex; and (2) the second target analyte present in the biological sample to substantially bind to the analyte-specific binding partner extending from the outer surface of the nanoparticles of the second diagnostic reagent composition, thereby forming a second complex. Then, based on any first complex and second complex formed, the presence and / or concentration of each of the first and second target analytes is determined. The determining step can be performed using any assay method known in the art.

[0067] Non-limiting examples of biological samples that can be used according to the various methods of the present disclosure include 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, stool, pleural fluid, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0068] As described above, when a diagnostic reagent composition is used in combination with a second reagent, the two compositions can be added simultaneously or sequentially. In addition, when two or more diagnostic reagent compositions are used in the same reaction, the two diagnostic reagent compositions can be added simultaneously or sequentially. When the various compositions used in the method are added sequentially, the order in which the compositions are added can be varied; one of ordinary skill in the art can determine the specific desired order in which the different compositions are added to the assay. Of course, the simplest order of addition is to add all materials simultaneously and measure the signals generated thereby. Alternatively, each composition, or group of compositions, can be combined sequentially. In certain embodiments, an incubation step may be involved after one or more additions. Example

[0069] Examples are provided below. However, the present disclosure should be understood not to limit its application to the specific experiments, results, and experimental procedures disclosed herein. Instead, these examples are simply provided as one of various embodiments and are intended to be illustrative, not exhaustive.

[0070] Example 1

[0071] Non-magnetic latex particles (NMLPs) have been widely used in the diagnostic industry, where antigens or antibodies are immobilized on the particles for use in competitive or sandwich assay formats. The target analyte aggregates the particles together, forming a turbid or cloudy solution that can be quantified spectrophotometrically. However, these plastic beads are toxic and environmentally unfriendly, and have significant waste disposal costs associated with them, which will rise significantly in the future. In addition, certain procedures for functionalizing the surface can require large amounts of materials, a lot of time, and resources. In addition, the coupling process and waste materials can also be hazardous. Managing the various processes and waste is often associated with extensive safety measures and high costs, and may even have to be stopped for regulatory reasons.

[0072] The present disclosure addresses these challenges by using biodegradable protein nanoparticles that naturally contain functional groups (such as, but not limited to, carboxyl, amine, or thiol moieties) and attaching antibodies or antigens (or other analyte-specific binding partners) to the surface of the nanoparticles by conventional chemical methods. Gamma-globulin (GG), such as, but not limited to, goat gamma-globulin (GGG) and bovine gamma-globulin (BGG)) is polymerized (such as, but not limited to, by heat treatment) to form polymerized gamma-globulin (pGG) nanoparticles. This method of producing pGG as biodegradable protein nanoparticles reduces the amount of material required and is environmentally neutral and, therefore, sustainable.

[0073] These functionalized beads bind to the target analyte in a multivalent manner, thereby forming aggregates that can be visualized or quantified using spectrophotometry (particularly turbidimetry or scattered light). Quantifying the turbidity (or turbidity) of the reaction mixture based on this change in analyte concentration is the basis of homogeneous immunoagglutination assays. In contrast, latex beads in the prior art will eventually become waste, and their waste disposal costs will continue to be a challenge that may rise significantly in the future. The present disclosure replaces these currently used toxic beads with environmentally friendly and biodegradable protein nanoparticles.

[0074] The present disclosure is based on the use of biodegradable protein nanoparticles that are about 20 nm to about 1000 nm (typically <500 nm) and contain amino acid residues with reactive groups (e.g., COOH, NH3, SH, etc.) that are easily attached to the surface by conventional chemical methods such as biotin, fluorescein, antibodies, antigens, or ligands. These protein nanoparticles are colloids and are well suspended in buffers and solutions commonly used in diagnostic reagents. They can be manufactured by various methods known in the art (e.g., solvent evaporation, nanoprecipitation, salting out, emulsification, etc.). For this example, a 10 mg / ml solution of goat gamma globin (GGG) was conveniently heated at 65-67°C until its transmittance at 340 nm reached about 30%. Similar to latex beads, the polymerized GGG (pGGG) product is white and milky in appearance ( Figure 1 ).

[0075] These protein nanoparticles can be modified by attaching to them specific binding partners for the target analyte. For example, Figure 2 The left figure shows the functionalization of protein nanoparticles to include biotin on their surface. In addition, protein nanoparticles can also be produced (or modified after production to contain) dyes associated with them; Figure 2 The right image shows protein nanoparticles functionalized with fluorescein on their surface.

[0076] The chromatographic behavior of pGGG nanoparticles is similar to that of NMLPs. Figure 3 As shown, pGGG nanoparticles were normally excluded by a size exclusion column (e.g., CL-2B with an exclusion limit of approximately 100 nm) and could be detected at 280, 340, and 600 nm. Based on these data, it was determined that these pGGG particles could be used in agglutination assays. Figure 4 As shown, when biotinylated nanoparticles (1 mg) were exposed to avidin (1 mg), aggregation occurred almost immediately, and this resulted in an increase in absorbance readings at 340 nm over time. When there was no biotin on the particle surface, the absorbance readings remained unchanged. This data indicates that pGGG protein nanoparticles have similar performance to latex beads and liposomes in terms of aggregation by turbidimetry.

[0077] Next, a calibration curve (A340nm vs. μg-avidin) was formed when increasing amounts of avidin were added to a solution containing biotinylated pGGG nanoparticles ( Figure 5 Thus, quantitative analysis of avidin can be achieved based on this homogeneous agglutination assay format using functionalized biotinylated pGGG nanoparticles. Figure 6 Images of the test cuvette before (left) and after (right) the addition of avidin at the end of the reaction. Figure 7 As shown in Figure 2, this agglutination event is also easily observed on a dynamic light scattering (DLS) detector. In this case, agglutinated particles as large as about 1 μm are seen on DLS. In contrast, when there is no biotin on the surface of pGGG nanoparticles, the particle size remains unchanged even in the presence of avidin, as shown in Figure 2. Figure 8 shown.

[0078] The functionalized protein nanoparticles (pGG) disclosed herein can replace the "toxic" plastic beads currently used in homogeneous immunoassays. This replacement avoids the need for expensive waste disposal units and provides an alternative to NMLP, which is particularly important in the event that NMLP is banned in the future. The main features of the protein pGG nanoparticles disclosed herein are their biodegradability, suitable size (e.g., 20-1000 nm), ease of attachment of functionalized molecules or proteins, and behavioral similarity to traditional NMLP. The pGG nanoparticles disclosed herein can also be used in immunoassays in a variety of ways. Non-limiting examples of other uses include as nonspecific interference blockers, signal amplifiers and enhancers, colloidal stationary phases that help stabilize proteins, functionalized dye carriers in multiplex formats (e.g., flow cytometry immunoassays), and the like.

[0079] Example 2

[0080] Example 1 describes the use of goat gamma globulin to produce polymeric protein nanoparticles (pGGG nanoparticles) for use in preparing diagnostic reagent compositions to which target analyte-specific binding partners can be attached. However, the present disclosure is not limited to the use of goat gamma globulin to produce nanoparticles; it should be understood that other gamma globulins can also be used in a similar manner, and thus the present disclosure encompasses the use of other gamma globulins in the production of diagnostic reagent compositions according to the present disclosure.

[0081] For example, Figure 9 The diagram shows the production of polymeric bovine gamma globulin (BGG) (or pBGG) protein nanoparticles. The pBGG protein nanoparticles were produced using a heating technique similar to that described in Example 1 above.

[0082] These pBGG protein nanoparticles were attached to target analyte-specific binding partners in a manner similar to that described in Example 1 above, and the diagnostic reagent composition containing the pBGG protein nanoparticles was incorporated into an agglutination immunoassay to detect analytes bound to the target analyte-specific binding partners.

[0083] Non-limiting illustrative embodiments

[0084] The following is a list of non-limiting illustrative embodiments disclosed herein:

[0085] Illustrative embodiments 1. A diagnostic immunoassay reagent composition for detecting a target analyte in a biological sample, comprising: biodegradable protein nanoparticles comprising polymeric gamma globulin (pGG) and having at least one functional group thereon; and at least one analyte-specific binding partner attached to the biodegradable protein nanoparticles via the at least one functional group.

[0086] Illustrative Embodiment 2. The composition of Illustrative Embodiment 1, wherein the at least one functional group is selected from a carboxyl group, an amine group, or a thiol group.

[0087] Illustrative Embodiment 3. The composition of Illustrative Embodiment 1 or 2, wherein the nanoparticles have a diameter in the range of about 20 nm to about 1000 nm.

[0088] Illustrative Embodiment 4. The composition of any of Illustrative Embodiments 1-3, wherein the analyte-specific binding partner comprises an antibody or fragment thereof that specifically binds to the target analyte.

[0089] Illustrative Embodiment 5. The composition of any of Illustrative Embodiments 1-4, wherein the analyte-specific binding partner comprises an antigen to which the target analyte specifically binds.

[0090] Illustrative Embodiment 6. The composition of any of Illustrative Embodiments 1-5, wherein the at least one analyte-specific binding partner comprises at least one receptor, or a portion or derivative thereof, that specifically binds to the target analyte.

[0091] Illustrative Embodiment 7. The composition of any of Illustrative Embodiments 1-6, wherein at least one analyte-specific binding partner comprises a ligand for the target analyte.

[0092] Illustrative Embodiment 8. The composition of any one of Illustrative Embodiments 1-7, wherein the nanoparticles are biotinylated.

[0093] Illustrative embodiment 9. The composition of any of illustrative embodiments 1-8, wherein the nanoparticles have at least one dye attached thereto.

[0094] Illustrative Embodiment 10. The composition of Illustrative Embodiment 9, wherein the dye comprises fluorescein.

[0095] Illustrative Embodiment 11. The composition of any one of Illustrative Embodiments 1-10, wherein the gamma globulin is goat gamma globulin.

[0096] Illustrative Embodiment 12. The composition of any one of Illustrative Embodiments 1-11, wherein the gamma globulin is bovine gamma globulin.

[0097] Illustrative embodiment 13. A kit comprising: at least one diagnostic reagent composition of any one of illustrative embodiments 1-12.

[0098] Illustrative Embodiment 14. The kit of Illustrative Embodiment 13, further comprising at least one additional reagent for a diagnostic immunoassay.

[0099] Illustrative embodiment 15. The kit of illustrative embodiment 13 or 14, further comprising at least two diagnostic reagent compositions of any one of illustrative embodiments 1-12.

[0100] Illustrative Embodiment 15A. The kit of Illustrative Embodiment 15, wherein the kit is for use in a multiplex assay.

[0101] Illustrative embodiment 16. A microfluidic device comprising: (i) an inlet channel through which a sample is applied; and (ii) at least one compartment capable of being in fluid communication with the inlet channel, wherein the at least one compartment comprises at least one diagnostic reagent composition of any one of illustrative embodiments 1-12.

[0102] Illustrative Embodiment 17. The microfluidic device of Illustrative Embodiment 16, further defined as a microfluidic device for performing multiplexed assays, and wherein (ii) comprises at least two diagnostic reagent compositions.

[0103] Illustrative embodiment 18. A method for producing a diagnostic reagent composition for detecting a target analyte in a biological sample, the method comprising the steps of: polymerizing gamma globulin (GG) to form polymeric GG (pGG) nanoparticles; and attaching at least one analyte-specific binding partner to the pGG nanoparticles.

[0104] Illustrative embodiment 19. The method of illustrative embodiment 18, wherein the pGG nanoparticles are formed by heating the GG to a temperature in the range of about 65°C to about 67°C for a period of time.

[0105] Illustrative embodiment 20. The method of illustrative embodiment 18 or 19, wherein the pGG nanoparticles are formed by a technique selected from the group consisting of solvent evaporation, nanoprecipitation, salting out, and emulsification techniques.

[0106] Illustrative Embodiment 21. The method of any of Illustrative Embodiments 18-20, wherein at least one functional group is selected from a carboxyl group, an amine group, or a thiol group.

[0107] Illustrative Embodiment 22. The method of any of Illustrative Embodiments 18-21, wherein the nanoparticles have a diameter in the range of about 20 nm to about 1000 nm.

[0108] Illustrative Embodiment 23. The method of any of Illustrative Embodiments 18-22, wherein the analyte-specific binding partner comprises an antibody or fragment thereof that specifically binds to the target analyte.

[0109] Illustrative Embodiment 24. The method of any one of Illustrative Embodiments 18-23, wherein the analyte-specific binding partner comprises an antigen to which the target analyte specifically binds.

[0110] Illustrative Embodiment 25. The method of any of Illustrative Embodiments 18-24, wherein the at least one analyte-specific binding partner comprises at least one receptor, or a portion or derivative thereof, that specifically binds to the target analyte.

[0111] Illustrative Embodiment 26. The method of any of Illustrative Embodiments 18-25, wherein at least one analyte-specific binding partner comprises a ligand for the target analyte.

[0112] Illustrative Embodiment 27. The method of any one of Illustrative Embodiments 18-26, wherein the nanoparticles are biotinylated.

[0113] Illustrative Embodiment 28. The method of any of Illustrative Embodiments 18-27, wherein the nanoparticles have at least one dye attached thereto.

[0114] Illustrative Embodiment 29. The method of Illustrative Embodiment 28, wherein the dye comprises fluorescein.

[0115] Illustrative embodiment 30. The method of any one of illustrative embodiments 18-29, wherein the gamma globulin is goat gamma globulin.

[0116] Illustrative Embodiment 31. The method of any one of Illustrative Embodiments 18-29, wherein the gamma globulin is bovine gamma globulin.

[0117] Illustrative embodiment 32. A method for determining the presence and / or concentration of a target analyte in a biological sample, the method comprising the steps of: combining the biological sample with at least one diagnostic reagent composition of any one of illustrative embodiments 1-12 under conditions that allow at least one analyte-specific binding partner to bind to the target analyte present in the sample to form a complex; and determining the presence and / or concentration of the target analyte based on any complex formed.

[0118] Illustrative embodiment 33. The method of illustrative embodiment 32, wherein the method comprises a homogeneous assay format.

[0119] Illustrative embodiment 34. The method of illustrative embodiment 33, wherein the assay format comprises an agglutination assay.

[0120] Illustrative embodiment 35. The method of illustrative embodiment 34, wherein the formation of aggregates is detected visually or spectrophotometrically.

[0121] Illustrative Embodiment 36. The method of Illustrative Embodiment 35, wherein the detecting is performed by turbidimetry or nephelometry.

[0122] Illustrative embodiment 37. The method of illustrative embodiment 32, wherein the method comprises a heterogeneous assay format.

[0123] Illustrative Embodiment 38. The method of Illustrative Embodiment 37, wherein the assay format comprises a sandwich assay.

[0124] Illustrative embodiment 39. A method for determining the presence and / or concentration of at least two target analytes in a biological sample, the method comprising the steps of: combining the biological sample with a first diagnostic reagent composition of any one of illustrative embodiments 1-12 and a second diagnostic reagent composition of any one of illustrative embodiments 1-12 under conditions that allow the analyte-specific binding partner of the first diagnostic reagent composition to bind to the first target analyte present in the sample to form a first complex, and allow the analyte-specific binding partner of the second diagnostic reagent composition to bind to the second target analyte present in the sample to form a second complex; determining the presence and / or concentration of the first target analyte based on any first complexes formed; and determining the presence and / or concentration of the second target analyte based on any second complexes formed.

[0125] Illustrative embodiment 40. The method of any of illustrative embodiments 32-39, wherein the biological sample is selected from whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0126] Thus, according to the present disclosure, compositions, kits, and devices, as well as methods for producing and using the same, are provided that fully satisfy the purposes and advantages set forth above. Although the present disclosure has been described in conjunction with the specific figures, experiments, results, and statements set forth above, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A diagnostic immunoassay reagent composition for detecting a target analyte in a biological sample, comprising: Biodegradable protein nanoparticles comprising polymeric gamma globulin (pGG) and having at least one functional group thereon; and At least one analyte-specific binding partner is attached to the biodegradable protein nanoparticle via the at least one functional group.

2. The composition of claim 1, wherein the at least one functional group is selected from a carboxyl group, an amine group, or a thiol group.

3. The composition of claim 1, wherein the nanoparticles have a diameter in the range of about 20 nm to about 1000 nm.

4. The composition of claim 1, wherein the analyte-specific binding partner comprises an antibody or fragment thereof that specifically binds to a target analyte.

5. The composition of claim 1, wherein the analyte-specific binding partner comprises an antigen to which the target analyte specifically binds.

6. The composition of claim 1, wherein the at least one analyte-specific binding partner comprises a receptor, or a portion or derivative thereof, that specifically binds to a target analyte.

7. The composition of claim 1, wherein the at least one analyte-specific binding partner comprises a ligand for a target analyte.

8. The composition of claim 1, wherein the nanoparticles have at least one dye attached thereto.

9. The composition of claim 1, wherein the gamma globulin is goat gamma globulin.

10. The composition of claim 1, wherein the gamma globulin is bovine gamma globulin.

11. A kit comprising: at least one diagnostic reagent composition according to any one of claims 1 to 10.

12. The kit of claim 11, further comprising at least one additional reagent for a diagnostic immunoassay.

13. The kit according to claim 11, further comprising at least two diagnostic reagent compositions according to any one of claims 1 to 10.

14. A microfluidic device comprising: (i) an inlet channel through which the sample is applied; and (ii) at least one compartment capable of being in fluid communication with the inlet channel, wherein the at least one compartment comprises at least one diagnostic reagent composition according to any one of claims 1 to 10.

15. A method for producing a diagnostic reagent composition for detecting a target analyte in a biological sample, the method comprising the steps of: polymerizing gamma globulin (GG) to form polymerized GG nanoparticles (pGG); and At least one analyte-specific binding partner is attached to the pGG nanoparticles.

16. The method of claim 15, wherein the pGG nanoparticles are formed by heating the GG to a temperature in the range of about 65°C to about 67°C for a period of time.

17. The method of claim 15, wherein the pGG nanoparticles are formed by a technique selected from the group consisting of solvent evaporation, nanoprecipitation, salting out, and emulsification techniques.

18. A method for determining the presence and / or concentration of a target analyte in a biological sample, the method comprising the steps of: combining the biological sample with at least one diagnostic reagent composition according to any one of claims 1 to 10 under conditions that allow at least one analyte-specific binding partner to bind to a target analyte present in the sample to form a complex; and The presence and / or concentration of the target analyte is determined based on any complexes formed.

19. The method of claim 18, wherein the method comprises a homogeneous assay format, and wherein the assay format comprises an agglutination assay.

20. The method of claim 18, wherein the biological sample is selected from whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural fluid, nasopharyngeal fluid, and combinations thereof.

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