Colorimetric insulin immunosensor and methods of producing and using same

Abstract

Compositions, reagents, kits, and systems are disclosed for performing an immunoassay for insulin. The immunoassay utilizes a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label. The capture antibody and the detection antibody specifically bind to non-overlapping epitopes of insulin so that a sandwich is formed.

Description

COLORIMETRIC INSULIN IMMUNOSENSOR AND METHODS OF PRODUCING AND USING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 594,502, filed October 31, 2023. The entire contents of the above-referenced patent application(s) are hereby expressly incorporated herein by reference.BACKGROUND

[0002] Nanoparticle-based colorimetric sensors offer a straightforward, sensitive, and user-friendly approach to detection, requiring minimal sample preparation and no sophisticated equipment. A sensitive and selective enzymatic assay based on the peroxidase- like activity of magnetic greigite (Fe3S4) utilizing sulfur vacancies to function as charge trapping and short-range scattering centers by introducing localized gap states. This helps the adsorption of peroxide on the Fe3S4 surface, facilitating glucose detection in serum with a linear range of 0.5-150 pM and a detection limit as low as 0.1 pM2. Antonio et al. reported a rapid assay for detecting C-reactive protein using citrate-capped gold nanoparticles. Platinum nanoparticles for the detection of cardiac troponin have also been reported. Other analytes such as cancer cells, proteins, and bacteria have also been detected utilizing magnetic, platinum-gold, and copper nanoparticles, they were able to detect as few as 2.17xl02colony forming unit / ml (CFU / ml) of listeria monocytogenes bacteria that cause foodborne illness, 10 cells / mL of cancer cells and 1.95 pg / mL of alpha-fetoprotein. Furthermore, a recent study has explored the use of graphene oxide / gold nanoparticles / Triton X-100 nanocomposites for insulin detection. While promising, this approach lacks selective and specific interactions. The inherent visual nature of colorimetric sensing allows for quick, convenient measurements, making it particularly suitable for point-of-care (POC) tests in remote settings.

[0003] Magnetic particle-based colorimetric immunosensors have garnered increased attention during the pandemic era due to their significant advantages such as negligible interference with biological matrix, lower signal-to-noise ratio, highly stable physical properties, faster separation, and low cost. Utilizing magnetic particles (MPs) to immobilize biomolecules such as antibodies, proteins, enzymes, and nucleotides enables the simple,rapid, cost-effective, and efficient capture of target biomolecules from complex real sample matrices. These biosensing strategies are not only biocompatible and environmentally friendly but also cost-effective. Additionally, MP-based sensors offer reduced interference because biological samples typically exhibit little to no magnetic signals. The high surface-to- volume ratio, ease of dispersibility, and ability to conjugate with a variety of target-specific receptor molecules make MPs highly effective for sensitive detection in minimally processed samples. Magnetic particles have been versatile in developing assays to detect CA125 antigen, hepatitis B antigen, and various other analytes.

[0004] Compared to the millimolar concentrations of blood glucose, insulin, a hormone regulating glucose, is present at picomolar levels in fasting serum samples of both normal individuals and those with juvenile diabetes (type 1). In patients with insulin-resistant diabetes, known as type 2 diabetes, fasting insulin levels typically exceed 70 pM. While HbAlC tests are the most common clinical method for diagnosing diabetes, frequent monitoring of insulin is also crucial, particularly for individuals with malignant insulinoma, which can lead to frequent hypoglycemic episodes, in cases of pancreatic disorders or damage, helping diagnose impaired insulin production. Monitoring insulin levels is also necessary for understanding unexplained factitious hypoglycemia in non-diabetic individuals, assisting athletes with type 1 diabetes in achieving peak performance, and in the management of artificial pancreatic systems.

[0005] In immunoassays, using selective antibodies for target proteins ensures high specificity in detecting desired molecules. A sandwich-type electrochemical immunoassay has been reported for detecting serum insulin with a detection limit of 50 fM (Sun et al., Microchimica Acta (2019), 186(1):6) and in saliva samples with a detection limit of 41 pM (Vargas et al., Angewandte Chemie International Edition (2019) 58(19):6376-6379). Liu et al. demonstrated a fluorescence quenching method for insulin detection with a detection limit of 0.048 U / mL (Liu et al., Taianta (2019) 199:596-602). While effective, this approach requires multiple DNA probes, increasing cost and complexity. G-Quadruplex systems and aptamerbased assays, known for their sensitivity due to multiple binding sites, can detect various analytes. Despite their potential, they pose design challenges (Cao et al., Inorg Chem Front (2017) 4(l):10— 32). Chemiluminescence has been used with a detection limit of 1.6 pM but involves multiple steps (Sun et al., Anal Chim Acta (2019) 1089:152-164). Interferometric reflectance spectroscopy offers label-free detection of biological molecules, yet it is costly(Chhasatia et al., Sens Actuators B Chem (2018) 273:1313-1322). Chromatography-based techniques, while documented for insulin detection, often require lengthy preparation and running times, making them less ideal for immediate application needs. Given these limitations, especially in resource-constrained or remote settings, there is a critical need for new methods that are user-friendly, cost-effective, and independent of complex instrumentation to detect molecules at ultra-low concentrations (Shen et al., Analyst (2019) 144(14):4139-4148).BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 graphically illustrates one non-limiting embodiment of a colorimetric immunosensor and methods of producing and using same for insulin detection in biofluids, in accordance with the present disclosure.

[0007] FIG. 2 illustrates a colorimetric insulin sandwich immunoassay in 10 mM phosphate buffered saline using magnetic particle-capture antibody conjugates and various spiked insulin concentrations completing the immunoassembly by HRP-labeled sandwich detection antibody: A) Increase in color intensity with spiked insulin concentration in 10 mM PBS buffer (a-g represents unspiked, 10 pM, 100 pM, 250 pM, 500 pM, 1 nM, and 10 nM insulin concentrations spiked in 10 mM phosphate buffered saline, respectively, and the corresponding B) 3D representation (Imaged software) and C) UV-visible absorption spectra correlating with the relative color intensity at the TMB oxidation wavelength maximum of 652 nm. D) The graph presents the relationship between absorbance and insulin concentration, and the inset is a linear semi-logarithmic concentration plot (R2= 0.98). Mean ± STDEV is shown for N=3.

[0008] FIG. 3 illustrates a colorimetric insulin sandwich immunoassay in 2-fold diluted serum using magnetic particle-capture antibody conjugates and various spiked insulin concentrations completing the immunoassembly by HRP-labeled sandwich detection antibody: A) Increase in color intensity with spiked insulin concentration in 2-fold diluted serum: a. represents insulin-unspiked control serum added with the magnetic particles- antibody conjugate, and b-f. correspond to 10 pM, 100 pM, 500 pM, 1 nM, and 10 nM spiked insulin concentrations in serum, and B) 3D representation (Imaged software) and C) UV-visible absorption spectra correlating with the relative color intensity at the TMB oxidation wavelength maximum of 652 nm. D) Semi-logarithmic linear concentration plot illustratingthe relationship between absorbance and insulin concentration (mean ± STDEV is shown for two replicates). E) Correlation chart of the ELISA method performed by an independent lab with the magnetic colorimetric sensor of the present disclosure for type-1 and healthy serum insulin samples.

[0009] FIG. 4 illustrates colorimetric sandwich immunoassay response over the unspiked control matrix response for various artificial saliva dilutions. This figure presents data from the UV-visible absorption spectrum assessing background activity and interference in the artificial saliva matrix at various dilutions using 10 mM PBS. Solid curves represent 1 nM insulin spiked into different dilutions of artificial saliva, while dotted curves indicate the absorbance of artificial saliva without any spiked insulin at each corresponding dilution.

[0010] FIG. 5 illustrates a colorimetric insulin sandwich immunoassay in 20-fold diluted saliva using magnetic particle-capture antibody conjugates and various spiked insulin concentrations completing the immunoassembly by HRP-labeled sandwich detection antibody: A) Increase in color intensity with spiked insulin concentration in 20-fold diluted saliva: a. represents insulin-unspiked control saliva added with the magnetic particles- antibody conjugate, and b, c, d, e, f, g, and h represent the color intensity increase to 1 pM, 10 pM, 50 pM, 100 pM, 250 pM, 500 pM, and 1 nM insulin concentrations spiked into 20- times diluted saliva, respectively. B) 3D representation (ImageJ software) and C) UV-visible absorption spectra correlating with the relative color intensity at the TMB oxidation wavelength maximum of 652 nm. D) Semi-logarithmic linear concentration plot illustrating the relationship between absorbance and insulin concentration (mean ± STDEV is shown for three replicates).

[0011] FIG. 6 illustrates a colorimetric insulin sandwich immunoassay in 20-times diluted A) human type-1 diabetic fasting saliva and B) 20-times diluted human healthy saliva using magnetic particle-capture antibody conjugates and various spiked insulin concentrations completing the immunoassembly by HRP-labeled sandwich detection antibody (a. represents insulin-unspiked control saliva added with the magnetic particles-antibody conjugate, and b, c, and d represent 100 pM, 1 nM, and 10 nM insulin concentrations spiked in the saliva, respectively). C) and D) 3D representations (ImageJ software). E) and F) UV-visible absorption spectra correlating with the relative color intensity at the TMB oxidation wavelength maximum of 652 nm with spiked insulin concentration. G) Bar chart comparison of the percentage absorbance changes for insulin spiked at three different concentrations (100 pM,1 nM, and 10 nM) in 20-times diluted human healthy non-fasting saliva (HS), 20-times diluted type-1 diabetic fasting human saliva (HS1), and 20-times diluted artificial saliva (AS), (mean ± STDEV is shown for two replicates).

[0012] FIG. 7 illustrates a 3D Representation of SPRi pixel intensity changes. This figure visualizes the MPs-based colorimetric sandwich immunoassay response to various insulin concentrations in buffer solution using surface plasmon resonance imaging, a show the baseline response of magnetic particle-bound conjugates with buffer (no spiked insulin), and b, c, d, e, and f illustrate the responses to 10 pM, 100 pM, 1 nM, 100 nM, and 250 nM concentrations of insulin spiked in 10 mM phosphate-buffered saline, respectively.

[0013] FIG. 8 illustrates UV-visible absorption spectra of the colorimetric assay were performed over a period of 8 days with the covalently linked insulin-antibody magnetic particle conjugates stored as individually conjugated vials at 2-8 °C and used after different days of storage as labeled in each graph. The spiked insulin concentration was 1 nM in each case (solid curve) in 10 mM PBS buffer, and the magnetic particle-bound conjugates with buffer (no-insulin) response is shown as a dotted curve.DETAILED DESCRIPTION

[0014] Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary language and results, 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. 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.

[0015] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed 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. 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 specificreferences 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. Standard techniques are used for chemical syntheses and chemical analyses.

[0016] 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 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.

[0017] All of the compositions and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the 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 methods described herein without departing from the concept, spirit, and scope of the inventive concept(s). All such similar substitutions 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.

[0018] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0019] The use of the term "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." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0020] 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 towhich 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.

[0021] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0022] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0023] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / 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 twenty percent, or fifteen percent, or 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 from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0024] 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.

[0025] 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, AAB, 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.

[0026] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0027] As used herein, the phrases "associated with" and "coupled to" include both 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 associations / couplings include 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.

[0028] The term "isolated" as used herein means that a biological material, such as but not limited to a nucleic acid or protein, has been removed from its original environment in which it is naturally present. For example, a polynucleotide present in a plant, mammal or animal is present in its natural state and is not considered to be isolated. The samepolynucleotide separated from the adjacent nucleic acid sequences in which it is naturally inserted in the genome of the plant or animal is considered as being "isolated".

[0029] The term "isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with the biological activity and which may be present, for example, due to incomplete purification, addition of stabilizers or mixtures with pharmaceutically acceptable excipients, and the like.

[0030] "Isolated polypeptide" or "isolated protein" as used herein means a polypeptide or protein which is substantially free of those compounds that are normally associated with the polypeptide or protein in a natural state, including but not limited to, other proteins or polypeptides, nucleic acids, carbohydrates, lipids and the like.

[0031] 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.

[0032] The term "patient" or "subject" as used herein includes human and veterinary subjects. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, nonhuman primates, and any other animal that has mammary tissue.

[0033] The term "sample" as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluidic biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0034] The term "antibody" is used in the broadest sense, and specifically (but not by way of limitation) covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, so long as they exhibit the desired biologicalactivity of analyte binding. Thus, the term "antibody" or "antibody peptide(s)" refers to a full- length immunoglobulin molecule (i.e., an intact antibody) or an antigen-binding fragment thereof that competes with the intact antibody for specific antigen binding. Antigen-binding fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®), and other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, e.g., Hudson et al. (Nature Med. (2003) 9:129-134). The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2).

[0035] The term "antigen binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigenbinding fragment" of an antibody include but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments are obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.

[0036] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0037] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0038] The terms "CDR," and its plural "CDRs," refer to a complementarity determining region (CDR) of an antibody or antibody fragment, which determine the binding character of an antibody or antibody fragment. In most instances, three CDRs are present in a light chainvariable region (CDRL1, CDRL2 and CDRL3) and three CDRs are present in a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. Among the various CDRs, the CDR3 sequences, and particularly CDRH3, are the most diverse and therefore have the strongest contribution to antibody specificity. There are at least two techniques for determining CDRs: (1) an approach based on crossspecies sequence variability ( i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety).

[0039] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitopic determinants usually include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three- dimensional structural characteristics (e.g., a "conformational epitope"), as well as specific charge characteristics.

[0040] An epitope is defined as "the same" as another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise epitopes that are the same. In certain embodiments, epitopes that are the same may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope.

[0041] An antibody "specifically binds" an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody comprises an antigen-binding site that specifically binds to a particular epitope. In certain such embodiments, the antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope or closely related epitopes. In certain instances, for example, homologous proteins from different species may comprise the same epitope. In certain embodiments, an antibody specifically binds to an antigen with a dissociation constant of no greater than 10-6 M, 10-7 M, 10-8 M, or 10-9 M. When an antibody specifically binds to a receptor or ligand (i.e., counterreceptor), it may substantiallyinhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand when an excess of antibody reduces the quantity of receptor bound to ligand by at least about 20%, 40%, 60%, 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0042] An "isolated" antibody is one which has been separated and / or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In certain embodiments, the antibody will be purified as measurable by at least three different methods: 1) to greater than 50% by weight of antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, such as at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, alternatively, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the environment in which the antibody is produced will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In addition, the "isolated antibody" is substantially free of other antibodies having different antigenic specificities. An isolated antibody may, however, have some crossreactivity to other, related antigens.

[0043] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that in one method of production they may be synthesized by a hybridoma culture, and thus are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production ofthe antibody by any particular method. For example, in one embodiment, the monoclonal antibodies produced in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0044] The monoclonal antibodies utilized in accordance with the present disclosure may be produced by any methodology known in the art including, but not limited to, a result of a deliberate immunization protocol; a result of an immune response that results in the production of antibodies naturally in the course of a disease or cancer; phage-derived antibodies; and the like. In addition to the hybridoma production method listed above, the monoclonal antibodies of the present disclosure may be produced by other various methods such as, but not limited to, recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567); isolation of antibody fragments from a phage display library (see, e.g., Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); as well as various other monoclonal antibody production techniques (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)). Further, many monoclonal antibodies that may be utilized in the conjugates and methods disclosed or otherwise contemplated herein are widely commercially available, and therefore no further description thereof is deemed necessary.

[0045] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Generally, a substantially pure composition will comprise more than about 50% percent of all macromolecular species present in the composition, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0046] An "analyte" is a molecule that is capable of being recognized by an analytespecific binding partner, such as (but not limited to) an antibody. An analyte comprises at least one antigenic determinant or "epitope," which is the region of the analyte which binds to the analyte-specific binding partner (i.e., antibody).

[0047] Turning now to non-limiting embodiments of the present disclosure, compositions, reagents, kits, systems, and immunoassays are disclosed for the colorimetric detection of insulin in a sandwich immunoassay, as well as methods of producing and usingsame. Biomarkers provide critical molecular insights into diseases and abnormal conditions. However, detecting them at ultra-low concentrations is a challenge, particularly in areas with limited resources and access to sophisticated instruments. The present disclosure is primarily focused on mitigating this challenge. Certain non-limiting embodiments of the present disclosure include a colorimetric immunosensor for detecting insulin, an essential hormone biomarker that regulates glucose metabolism, at picomolar concentrations using functionalized magnetic particles. This immunosensor utilizes a two-antibody sandwich immunoassay: one antibody is covalently conjugated to the nanoparticles to capture and isolate the target marker, while the other is labeled for colorimetric detection of insulin. The color intensity of the assay correlates with insulin concentration. The immunosensor can be utilized for insulin detection in (for example, but not by way of limitation) buffer, saliva, and serum samples. As shown in the Examples below, the immunosensor has detection limits in the picomolar range and can be utilized for the detection of various conditions, including type 1 and type 2 diabetes. The immunoassays and methods of the present disclosure enhance biomarker analysis in biofluids through an equipment-free colorimetric method, which is particularly relevant for point-of-need applications.

[0048] Certain non-limiting embodiments of the present disclosure are directed to a sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample. The immunoassay includes two components associated with two anti-insulin antibodies. A first component is a conjugate formed of a capture antibody attached to a functionalized magnetic nanoparticle, and the second component includes a detection antibody labeled with a colorimetric label. The capture antibody and the detection antibody specifically bind to non-overlapping epitopes of insulin so that a sandwich is formed, and the color intensity generated in the assay correlates with insulin concentration.

[0049] Certain non-limiting embodiments of the present disclosure are directed to a kit for performingthe sandwich immunoassay for detection of an insulin concentration in at least one fluidic biological sample. The kit includes the two components described in detail herein (i.e., capture antibody-magnetic nanoparticle conjugate and labeled detection antibody).

[0050] Certain non-limiting embodiments of the present disclosure include an immunoassembly that comprises the two components described in detail herein (i.e., capture antibody-magnetic nanoparticle conjugate and labeled detection antibody) attached to a single insulin molecule to form a sandwich.

[0051] Certain non-limiting embodiments of the present disclosure are directed to a sensor for performing a sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample. The sensor includes a housing comprising one or more compartments into which are disposed the two components described in detail herein (i.e., the capture anti-insulin antibody-magnetic nanoparticle conjugate and the labeled detection anti-insulin antibody).

[0052] Any anti-insulin antibodies known in the art or otherwise contemplated herein may be utilized as the capture and detection antibodies, so long as the two antibodies utilized bind to substantially non-overlapping epitopes of insulin so that the sandwich immunoassembly can be formed. Anti-insulin antibodies (as well as the epitopes to which they bind) are well known in the art and widely commercially available; non-limiting examples of commercially available anti-insulin antibodies include Novus Biologicals Product No. NBP3- 23494 - "Insulin Antibody (01)," and Product No. NB200-435H - "Insulin / Proinsulin Antibody (D3E7cc) [HRP]," (Fisher Scientific, Hampton, NH); Product No. ab253508 - "Human Insulin Antibody Pair - BSA and Azide free," (Abeam Inc., Cambridge, UK); Product No. LS-C85862 "Polyclonal Guinea pig anti-Human Insulin Antibody (IHC)" and Product No. LS-C318387 - "Polyclonal Rabbit anti-Human Insulin Antibody (HRP, WB)" (LSBio, Shirley, MA); Product No. 12018 "Monoclonal Anti-Insulin antibody produced in mouse" (Sigma-Aldrich, St. Louis, MO); and USBio's matched pair of Capture antibody Product No. 474290 and Detection antibody Product No. 397258 (US Bio, Salem, MA); and the like. Therefore, no further disclosure related to the anti-insulin antibodies utilized herein is deemed necessary.

[0053] The nanoparticles may be formed of any magnetic material known in the art or otherwise contemplated herein that responds to an external field and is capable of functioning in accordance with the various embodiments disclosed herein. Non-limiting examples of magnetic nanoparticles include gold nanoparticles, magnetite nanoparticles, iron (such as, but not limited to, iron, iron oxide, and ferrite) nanoparticles, non-iron-based nanoparticles (such as, but not limited to, cobalt, nickel, manganese, and their respective oxides), non-metallic magnetic nanoparticles, and the like, as well as any combinations thereof.

[0054] The magnetic nanoparticles may be provided with any size diameter that allows the particles to function in accordance with the present disclosure. Non-limiting examples of sizes include about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm,about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, about 1 micron, about 1.1 microns, about 1.2 microns, about 1.3 microns, about 1.4 microns, about 1.5 microns, about 1.6 microns, about 1.7 microns, about 1.8 microns, about 1.9 microns, about 2 microns, and the like, as well as a range formed from two of the above values (e.g., a range of from about 100 nm to about 2 microns).

[0055] In a particular (but non-limiting) embodiment, the functionalized magnetic nanoparticle has a size of about 200 nm.

[0056] The magnetic nanoparticles may be functionalized by any methods known in the art of otherwise disclosed herein that will allow for attachment of the capture antibody thereto. Non-limiting examples of functionalization methods that may be utilized in accordance with the present disclosure include carboxyl-functionalization, citrate- stabilization, all possible bioconjugation strategies, and the like, as well as any combinations thereof.

[0057] The detection antibody may be labeled with any colorimetric label that is capable of manual visual detection and provides sufficient color intensity for detection of a specific insulin concentration across a wide range. Non-limiting examples of colorimetric labels that may be utilized include enzymatic, non-enzymatic, chemical, optical, biological labels, reagents, and compounds, and the like, as well as any combinations thereof. In certain nonlimiting embodiments, the colorimetric label is an enzyme capable of generating a colored reaction product upon exposure to a substrate. Non-limiting examples of enzymes that may be utilized in accordance with the present disclosure include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.

[0058] In certain particular (but non-limiting) embodiments, the immunoassays / kits / immunoassemblies may further comprise one or more additional components / reagents. For example, but not by way of limitation, the immunoassays / kits / immu noassemblies may further include a substrate for the enzyme of the colorimetric label. Non-limiting examples of substrates that may be utilized when the colorimetric label is peroxidase include TMB (3,3',5,5'-tetramethylbenzidine), 3,3'-diaminobenzidine (DAB), luminol, ABTS (2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid]), all color-producing reagents and compounds, and the like. Non-limiting examples of substrates that may be utilized when the colorimetric label is ALP include pNPP (para- Nitrophenylphosphate), BCIP / NBT (5-Bromo-4-chloro-3-indolyl phosphate along with nitro blue tetrazolium), Fast Red TR / Naphthol AS-MX and TR phosphate, and the like. Non-limiting examples of substrates that may be utilized when the colorimetric label is beta-galactosidase include Bluo-gal, X-gal, and the like.

[0059] Other non-limiting examples of additional reagent(s) that may be present in the kit include at least one dilution fluid for dilution of the fluidic biological sample, at least one wash buffer, at least one quality control (of known insulin concentration), and the like, as well as any combinations thereof.

[0060] In certain particular (but non-limiting) embodiments, the immunoassays / kits / immunoassemblies may further comprise one or more additional components / devices. For example (but not by way of limitation), the immunoassays / kits / immunoassemblies may further include at least one magnetic device for capture of the sandwich and / or at least one receptacle in which the immunoassay is performed. Non-limiting examples of magnetic devices that may be utilized in accordance with the present disclosure include a magnetic rack in which the receptacles in which the assay is performed can be disposed, all magnetic responsive sources, and the like.

[0061] The kits of the present disclosure may be provided with one or more additional reagents or components that can be used in any of the methods of the present disclosure, as described herein above. The various components / reagents present in the kits may each be in separate containers / compartments, or various components / reagents can be combined in one or more containers / compartments, depending on the sterility, cross-reactivity, and stability of the components / reagents. In addition, the kit may be disposed in any packaging that allows the components present therein to function in accordance with the present disclosure. In certain non-limiting embodiments, the kit further comprises a sealed packaging in which the components are disposed. In addition, the kit can further include a set of written instructions explaining how to use one or more components of the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.

[0062] The immunoassay may be performed in any format and by any methods known in the art or otherwise disclosed herein. For example (but not by way of limitation), theY1immunoassay may be performed by combining reagents in a liquid suspension reaction within one or more receptacle(s). Alternatively (and / or in addition thereto), the reagents may be disposed at separate positions on a strip of absorbent pad or membrane (such as, but not limited to, a cellulose membrane) for performing a lateral flow test. In yet another alternative, a preformed disposable and / or multi-use sensor or cartridge may be provided in which the reagents are disposed and in which the immunoassay may be conducted.

[0063] When the reagents are disposed in a sensorthat includes a housing, the conjugate and the labeled detection antibody may be disposed in the same compartment or in different compartments. In addition, in certain particular (but non-limiting) embodiments, the sensor may further include the substrate for the colorimetric label; in these instances, the substrate is disposed in a different compartment from the labeled detection antibody to prevent interaction therebetween until the sandwich immunoassembly is formed.

[0064] In certain particular (but non-limiting) embodiments, the sensor further comprises at least one read compartment that is directly or indirectly in fluidic communication with the compartment(s) containing the conjugate and labeled detection antibody. The magnetic device for capturing the sandwich immunoassembly will be associated with the read compartment.

[0065] The composition, kits, systems, and methods of the present disclosure have a broad dynamic insulin detection range. In certain particular (but non-limiting) embodiments, the immunoassay detects an insulin concentration in a range of from about 10 pM to about 100 nM.

[0066] The results of the immunoassay (and the corresponding insulin concentration detected by the resulting color intensity) may be detected manually by visual inspection or may be detected automatically by an instrument / meter. When the result is detected automatically, the receptacle / lateral flow strip / sensor / cartridge is configured for insertion within an instrument for detection. When the result is detected manually via visual inspection, the kit / system may further include a standards chart of various color intensities and their corresponding insulin concentrations for comparison to the results of the colorimetric immunoassay.

[0067] Certain non-limiting embodiments of the present disclosure are directed to a method of detecting an insulin concentration in a fluidic biological sample. In the method, the fluidic biological sample is combined with any of the capture antibody-magnetic nanoparticleconjugates and any of the labeled detection antibodies disclosed or otherwise contemplated herein to form a mixture, and the mixture is incubated under conditions so that a sandwich is formed when the capture and detection antibodies bind to insulin. The mixture is then exposed to a magnetic device to capture the sandwich, and a substrate for the colorimetric label is added. The insulin concentration present in the fluidic biological sample is then determined based on a resulting color intensity in the mixture at a location to which the magnetic device is exposed, as the color intensity generated in the assay correlates with insulin concentration present in the fluidic biological sample.

[0068] Non-limiting examples of fluidic biological samples that may be tested in accordance with the present disclosure include urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and the like, as well as combinations thereof.

[0069] The methods of the present disclosure have a broad dynamic insulin detection range. In certain particular (but non-limiting) embodiments, the immunoassay detects an insulin concentration in a range of from about 10 pM to about 100 nM.

[0070] The results of the immunoassay (and the corresponding insulin concentration detected by the resulting color intensity) may be detected manually by visual inspection. For example (but not by way of limitation), a standards chart of various color intensities and their corresponding insulin concentrations may be provided for visual detection of the results of the colorimetric immunoassay. Alternatively, the results of the immunoassay (and the corresponding insulin concentration detected by the resulting color intensity) may be detected automatically by an instrument / meter. Automated methods of determining analyte concentration based on known color intensity changes in a colorimetric immunoassay are well known in the art, and thus no further description thereof is deemed necessary.EXAMPLES

[0071] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[0072] In this Example, a magnetic-particle-based colorimetric insulin sandwichimmunosensor was developed and tested for detecting ultra-low picomolar concentrations of insulin (FIG. 1). Tests were conducted in a simple buffer solution and then expanded to 2- times diluted serum, 20-times diluted artificial saliva, and human salivary samples. The goal was to achieve minimal dilution while maintaining sensor functionality, successfully enabled in a 2-fold diluted serum. However, greater dilution was necessary for saliva due to its frothy, viscous nature and to improve signal differentiation from the control saliva (no-insulin) samples at lower concentrations, as detailed in the Results and Discussion section. The color intensity of the assay correlates with insulin concentration, providing a reliable, cost-effective means to monitor insulin levels, particularly beneficial for point-of-care applications in resource-limited settings.

[0073] Experimental Section

[0074] Materials. Fluid MAG-CT (200 nm) citrate-functionalized magnetic particles (MPs) were acquired from Chemicell GmbH (Berlin, Germany). N-Hydroxysuccinimide (NHS), polyethylene glycol sorbitan monolaurate (TWEEN® 20), 2-aminoethanol (ethanolamine), disodium hydrogen phosphate (NazHPC ), 2-(N-Morpholino) ethane sulfonic acid hydrate (MES hydrate), a monoclonal anti-insulin antibody produced in mouse, insulin antigen, and human serum were purchased from Sigma-Aldrich (St. Louis, MO). Enhanced K-Blue® TMB (3,3',5,5'-tetramethylbenzidine) substrate was obtained from Neogen® (Lexington, KY). Sodium chloride (NaCI), potassium chloride (KCI), and sodium hydroxide (NaOH) were sourced from Millipore Sigma (Burlington, MA). l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and potassium dihydrogen phosphate (KH2PO4) were purchased from Thermo Fisher Scientific (Waltham, MA). DNase-free water was procured from Fisher Bioreagents. The insulin rabbit anti-human polyclonal (HRP) detection antibody was acquired from LS Bio (Shirley, MA). Artificial saliva (sodium carboxy methyl cellulose, potassium phosphate monobasic, potassium chloride, potassium phosphate dibasic, magnesium chloride hexahydrate, calcium chloride dehydrates, methyl-p-hydroxybenzoate) for medical and dental research was purchased from Pickering Laboratories (Mountain View, CA). Finally, a magnetic rack (6.73" x 2.24" x 2.13") for 100-250 microliter Eppendorf tubes (16 tubes) was obtained from Sergi Lab Supplies (Seattle, WA).

[0075] Methods. UV-Vis spectrophotometer was Varian Cary 100 Bio, Varian Inc., CA. The hydrodynamic size and zeta potential of the particles were measured using a ZetaPALS potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA), providinginsight into particle stability and interaction. Measurements were conducted at a 90° angle, with the sample diluted 5-fold in 10 mM PBS (pH 7.4, 25°C). To validate the color sensor results with increasing insulin concentration, the SPRimager®-ll array instrument (Horizon model, GWC Technologies, Madison, Wl, USA) was used, which operates at room temperature and uses a fixed SPRi source wavelength of 800 nm. Reflectivity changes at an optimally selected angle indicating surface modification or molecular binding events were recorded. A charge-coupled device (CCD) camera captured these changes as variations in pixel intensity. All experiments were performed in accordance with the Guidelines "Occupational Safety and Health Standards, OSHA Bloodborne Pathogen Standards, U. S. Department of Labor" and approved by the ethics committee at Oklahoma State University. Informed consent was obtained from human participants in this study.

[0076] Colorimetric sandwich immunoassay. In this Example, magnetic particles (MPs) with a hydrodynamic size of 200 nm were selected due to their rapid magnetic separation in biofluids, a result of enhanced magnetophoretic mobility (Witte et al., J Magn Magn Mater (2017) 427:320-324). The choice of 200 nm particles is due to their relatively lower surface area compared to particles smaller than 100 nm. This reduced surface area diminishes the contact area for the intrinsic peroxidase activity of the MPs, consequently lowering the background color in control samples that are not spiked with insulin. Each Eppendorf tube contained 12.5 pL of a 25 mg / mL citrate carboxylated MP (-COOH, sodium salt) suspension (0.31 mg, ~7xlO10magnetic particles). To activate the -COOH groups, 50 pL of a freshly prepared solution containing 0.4 M EDC and 0.1 M NHS in MES buffer (pH 6.5) was added and incubated for 15 minutes, converting them into reactive N-succinimidyl ester groups. A strong neodymium magnetic rack was utilized for efficient separation of the -COOH-activated MPs from the bulk unreacted EDC / NHS solution and washed them twice with 10 mM phosphate- buffered saline (PBS). These groups are known to form stable amide bonds with the amine groups on the lysine residues of the anti-insulin monoclonal antibody (Vargas et al., Biosens Bioelectron (2020) 167:112512). To accomplish this antibody conjugation, the MPs were incubated with the anti-insulin monoclonal antibody (50 pg / mL, 50 pL) for 30 minutes under ice-cold conditions to support the solution antibody protein stability while conjugating to the particles. After the immobilization, the antibody-attached MPs were magnetically separated and washed three times with 10 mM phosphate-buffered saline (PBS) to remove unbound antibodies with intermittent magnetic separation at each wash. To block any remaining activechemical sites on the -COOH-activated MPs and thus reduce non-specific binding in subsequent assay steps, 50 pL of 2.5 M ethanolamine was added to the MP-antibody conjugate and incubated for 15 minutes. Finally, a magnetic separation was performed, and the ethanolamine-blocked conjugates were washed once with 200 pL of 10 mM PBS.

[0077] Various concentrations of insulin solutions were prepared in 10 mM PBS buffer, 2- times diluted insulin-free healthy human serum, or 20-times diluted commercial artificial saliva. These solutions were incubated with the ethanolamine-blocked, insulin-antibody- modified MPs for 30 minutes, followed by washing with PBS buffer and intermittent magnetic separation to remove any unbound substances. Next, 50 pL of a 1 pg / mL horseradish peroxidase-labeled detection insulin antibody solution in PBS (pH 7.4) was added. This mixture was incubated for another 30 minutes in the ice-cold box with occasional stirring to allow binding to the insulin molecules on the monoclonal antibody MPs, forming a sandwich immuno-assembly. After washing the immunocomplex three times with PBS buffer, 20 pL of Enhanced K-blue TMB substrate solution (containing 3,3',5,5'-tetramethylbenzidine, and hydrogen peroxide) was added and incubated for 25-30 seconds to allow color development. The total assay time is approximately 2 hours. The blue color intensity, indicative of the bound insulin concentration, was generated from the peroxidase-labeled detection antibody- catalyzed TMB enzymatic reaction. Finally, the insulin immuno-assembly MPs were magnetically separated, and the absorbance was measured at 652 nm using a UV-Vis spectrophotometer to quantify the insulin levels. For visual documentation, photos of the sample tubes were captured using an iPhone 12 camera. Control samples included buffer two- times diluted serum and 20-times diluted artificial saliva.

[0078] I mageJ analysis. A smartphone camera captured images of the colorimetric change occurring in the sample Eppendorf tubes at the end of the assay. Then, the images corresponding to each insulin concentration were cropped into similar-sized snips. The pixel intensity increase with insulin concentration is represented using the ImageJ software, along with the 3D representation to easily visualize the color intensity changes that correlate with the UV-vis absorbance increase of the TMB oxidation catalyzed by the HRP-labeled detection antibody bound to the magnetic particle-capture antibody-insulin-immunoassembly.

[0079] Results and Discussion

[0080] Hydrodynamic size. Magnetic particles and magnetic particle-bound insulinantibody conjugates were characterized by measuring their size distribution in a liquidenvironment using dynamic light scattering (DLS), accounting for Brownian motion and hydrodynamic interactions. The measured hydrodynamic size of the as-received, unconjugated MPs from Chemicell Inc. was 202 ± 20 nm, which agrees with the manufacturer's specified average size of 200 nm. Following the immobilization of the capture monoclonal anti-insulin antibody onto the MPs, the hydrodynamic size increased to 372 ± 45 nm (N = 3 replicates). This increase is from the antibody immobilization and associated intermolecular and / or interparticle aggregation when attached to the nanoparticle surfaces, as noted in prior studies (Smith et al., Anal Biochem (2011) 410(l):124-132; and Khan et al., IntJ Nanomedicine (2014) 899). The size distribution was quantified using the polydispersity index (PDI), a dimensionless value ranging from 0 to 1, where values less than 0.1 indicate well-dispersed particles. The citrate-carboxylic acid functionalized MPs exhibited a PDI of 0.186, which decreased to 0.009 after the capture insulin-antibody was attached. This indicates a narrower size distribution of the aggregated antibody-MP conjugate compared to the original particles.

[0081] Zeta(^) potential. Initially, the carboxyl-functionalized magnetic particles exhibited a negative < potential of -36.47 ± 0.13 mV, attributed to the citrate (-COOH) surface groups. After the covalent immobilization of activated -COOH groups with the amine groups of lysine residues on the capture antibody to form stable amide bonds, the potential shifted to a less negative value of -15.27 ± 0.19 mV (N = 5 replicates). This shift towards a less negative zeta potential is indicative of the neutralization of the surface negative charge of the MPs due to antibody attachment and subsequent ethanolamine blocking. The remaining net negative charge is likely due to the negative charges on the antibody at pH 7.4, given its isoelectric point (pl) of 6.0-6.5 (Soderberg et al., Biochem Biophys Res Common (1998) 249(l):86-89), as well as from any residual free citrate groups of the MPs.

[0082] Assay in buffer (10 mM PBS, pH 7.4). The assay results indicate a clear correlation between increasing insulin concentrations (ranging from 10 pM to 10 nM) in the buffer and an increase in color intensity, as shown in FIG. 2, Panels A-D. Visually, color intensities for concentrations from 100 pM to 10 nM were notably distinct compared to the lower 10 pM concentrations, which closely resembled the background color of the buffer, a result of the intrinsic peroxidase activity of MPs. A UV-Vis spectral analysis was performed to validate the visual observations. The maximum absorbance at 652 nm corresponds to the formation of the oxidized TMB compound facilitated by the enzymatic activity of the HRP-labeled detectionantibody in the sandwich structure. The chromogenic activity of TMB is a result of its two easily oxidizable amino groups of benzidine, which are enhanced due to its sensitivity to HRP. In the presence of the horseradish peroxidase / F Ch system, TMB forms a blue one-electron oxidation product, perpetuating a redox cycle that facilitates the color development characteristic of this assay. The sensor demonstrated a robust linear, logarithmic response across the range of 10 pM to 1 nM.

[0083] Detection in two times diluted serum. The serum is a complex matrix containing approximately 20,000 different proteins, spanning a wide range of concentrations and molecular weights, which can significantly influence the analytical response of sensors. Detecting insulin in serum is particularly challenging due to its low molecular weight (~ 6 kDa). The sandwich assay method of the present disclosure addresses this issue by employing two specific antibodies to enhance selectivity. Additionally, to minimize background interference, the serum was diluted 2-fold with 10 mM PBS. In the serum analysis, there was a discernible increase in color intensity corresponding to the rise in spiked insulin concentration from 10 pM to 10 nM (FIG. 3, Panel A for visual color changes and FIG. 3, Panel B and FIG. 4, Panel D for 3D representation), as compared to the background color of the serum (no-insulin), which is attributable to the intrinsic peroxidase activity of the MPs (FIG. 3, Panel C, curve a). Gao et al. have shown that the peroxidase-like activity of MPs increases with increasing temperature, lowering pH (more acidic), and high molar hydrogen peroxide (H2O2) concentrations. MPs alone require a high peroxide concentration of 530 mM and a reaction time of over 100 seconds to exhibit significant peroxidase activity. To counteract this non-enzymatic background activity, the assay of this Example was conducted under ice-cold conditions using a 100 mM peroxide concentration at a physiological pH of 7.4. These measures effectively mitigated the intrinsic peroxidase activity of MPs. The optimized conditions achieved a linear detection range of 10 pM to 10 nM in 2 times diluted serum (FIG. 3, Panel C, curves b-f). While previous studies have utilized 10 to 100-fold serum dilution to enhance sensor response by diminishing nonspecific signals from the complex matrix (Mirsalari et al., Spectrochim Acta A Mol Biomol Spectrosc (2020) 240:118617), such extensive dilution can decrease the analyte protein concentration in actual samples, potentially compromising the sensor's ability to detect and overall assay performance. Application of the designed colorimetric sensor serum calibration plot (FIG. 3, Panel D) to a type 1 diabetes patient and healthy human serum samples and correlation of the results with an enzyme-linked immunosorbent assay (ELISA)run by an independent laboratory located at The University Health Services (Oklahoma State University) inferred good agreement at a 95% confidence level (FIG. 3, Panel E). The percentage recovery of the ELISA kit was reported as 91%, and the correlation with the present colorimetric sensor infers a similar level of accuracy between the two methods.

[0084] Detection in 20-times diluted artificial saliva. Fabre et al. (Endocr Connect (2012) 1(2):58-61) reported a 92% confidence interval for the correlation between salivary and serum insulin levels, highlighting saliva as a valuable biofluid for non-invasive detection. However, detecting insulin in the saliva is challenging due to its approximately 10-fold lower concentration compared to serum and the complex nature of saliva's composition, which can introduce biological noise and affect the assay's signal response (Josephy et al., Journal of Biological Chemistry (1982) 257(7):3669-3675; and De et al., Nat Chem (2009) l(6):461-465). To identify the optimal dilution that minimizes background noise and maximizes signal response to spiked insulin, a series of tests were conducted using different dilutions of commercially available artificial saliva (Pickering Laboratories), each spiked with 1 nM insulin.

[0085] In FIG. 4, the highest signal response relative to the control was noted for the 20- times diluted artificial saliva matrix. The undiluted saliva's background color was more pronounced at lower dilutions than that of the spiked sample response, indicating significant interference. The absorbance changes in 5-times diluted artificial saliva were minimal. Consequently, a 20-times dilution for the saliva matrix was chosen to reduce the impact of interfering agents effectively.

[0086] In FIG. 5 (Panel A for visual color changes and Panel B for 3D representation), a linear response was observed in concentrations ranging from 50 pM to 1 nM (FIG. 5, Panels C and D, respectively). The intensity for the sample spiked with 10 pM was nearly indistinguishable from the 20-times diluted saliva samples (no spiked insulin). Visually, the intensities at 10 pM and 1 pM appeared lower or comparable to the 20-times diluted saliva samples with no spiked insulin. UV-visible spectral analysis further confirmed that the absorbance at 10 pM closely resembled that of the insulin-unspiked saliva, and the absorbance at 1 pM was equivalent to that of the artificial saliva.

[0087] Detecting insulin in human saliva is complex due to its constituents like water, salts, mucus, enzymes, and proteins. Additionally, the viscosity of saliva complicates sample homogenization, leading to frothing even after diligent mixing. In this Example, 20 times diluted, non-fasting, healthy human saliva samples were spiked with three differentconcentrations of insulin. Saliva samples from fasting diabetic patients and non-fasting healthy individuals were collected in 5 mL glass vials and refrigerated at 2-8°C. Prior to experimentation, samples were taken out of refrigeration, diluted 20-times in PBS, and spiked with insulin. In FIG. 6 (Panel A type-1 diabetic fasting saliva and Panel B for the 20-times diluted human healthy saliva, and Panels C and D for the respective 3D representations), a significant background signal was noted in the human saliva samples (FIG. 6, Panels E and F) compared to the less complex artificial saliva made of salts (FIG. 5, Panel C). Detection was achieved at 1 nM and 10 nM concentrations above the insulin unspiked saliva control for both the real human saliva and artificial salivary matrix (FIG. 6, Panel G).

[0088] Optimizing sensor performance and sensitivity typically involves refining various components, such as the type of material and bio-recognizing elements. However, one of the most critical yet often overlooked factors is the background color of the sample matrix. This can substantially affect the sensor's analytical parameters and its ability to detect the analyte effectively. For instance, in this Example, the comparative detection limits shown in Table 1 indicate better performance for insulin spiked in a buffer. This is likely due to fewer obstructions for insulin binding to the capture antibody in the buffer, which lacks the complex biological components and viscosity present in serum and saliva that can amplify background signals. Additionally, the sensor design of the present disclosure relies on direct modification of magnetic particles, avoiding complex co-materials or hybrid materials that might otherwise increase the background peroxidase-like color from TMB.Table 1. Comparison of analytical parameters for a buffer, two times diluted serum, and 20-times diluted saliva matrices.

[0089] Validation using Surface Plasmon Resonance Imaging (SPRi) Analysis. To validate the colorimetric results, Surface Plasmon Resonance Imaging (SPRi), a label-free, real-time optical biosensing technique, was employed. This method is particularly adept at monitoring molecular interactions on a metallic sensor surface populated with oscillating electrons known as plasmons.

[0090] The experimental setup included a 5-nm thin gold layer on a glass surface, with incident light interacting with surface-bound molecules via an evanescent wave. SPR gold chips were placed with the colorimetric insulin sensor solutions, and reflectivity changes were measured against a buffer background. As the concentration of spiked insulin increased in the immunoassay solution, a corresponding increase in pixel intensity was noted. Data acquisition from the SPRi difference images was conducted using the Digital Optics V++ software, and the 3D representation of the data was generated using ImageJ software (FIG. 7). These results agreed with the findings obtained from UV-vis spectrometry.

[0091] The sensor produced herein is compared with other colorimetric sensors in Table 2. One such sensor uses a G-quadruplex insulin assembly coupled with DNA nanotubes and nanoparticles for picomolar detection of insulin. It's important to note the complexity and time intensity of the G-quadruplex insulin assembly method, which requires around 5 hours to complete, with multiple stages including an 80-minute incubation in a thermocycler, a 60- minute ligation period, and additional steps totaling 2 hours and 20 minutes. Additionally, said assay demands specialized techniques like electrophoresis for nanotube separation and gel extraction columns for isolating MNP-coupled DNA nanotubes. Consequently, the G- quadruplex insulin assembly process is laborious and requires the skills of trained professionals (Rafati et al., R Soc Open Sci (2018) 5(3):171835).Table 2. Comparison of insulin / glucose detection using colorimetry.28.7 plU = 1 ng m L1= 172 pM50l plU = 6 pM

[0092] Colorimetric sensors developed on peroxidase activity of magnetic particles by Martinkova et. Al (Mol Biotechnol (2016) 58(5):373-380) have good selectivity and stability; however, the detection limit is affected by the interference of the plasma matrix. Wang et al. (Taianta (2015) 134:712-717) developed a sensor based on core-shell material and demonstrated the detection of glucose detection with good reproducibility; however, the limitation of this method is that study was conducted in PBS buffer. Tan et al. (Taianta (2019) 204:285-293) developed a more straightforward ratiometric assay capable of differentiating between type 1 and type 2 diabetes mellitus, which is also cost-effective. Their method uses core-shell nanorods based on GNR@Au2S / AuAgS / CuS exhibiting peroxidase-like activity for colorimetric signal generation. This assay involves glucose oxidation by glucose oxidase (GOx), producing hydrogen peroxide as a byproduct, which then oxidizes TMB, leading to a visible color change. However, it should be noted that this method requires a 100-fold dilution of serum samples. Additionally, a simpler assay using graphene oxide / gold nanoparticle nanocomposites modified with Triton X-100 was able to detect insulin levels consistently over five days, with intra-day and inter-day sample variations of 2.7% and 4.8%, respectively. This approach, which utilizes straightforward spectrometric analysis, is notable for its simplicity, although the long-term stability of the sensor was not evaluated (Mirsalari et al., Spectrochim Acta A Mol Biomol Spectrosc (2020) 240:118617). Despite this, such methods represent rapid and sensitive platforms, employing innovative strategies like self-assembling nanocomposites for insulin detection in human serum.

[0093] It is recognized that the inherent peroxidase activity of the magnetic particles presents a potential limitation in the sensor, leading to background interference; however, it has been demonstrated that this activity can be mitigated. By limiting the detection step reaction to a brief 30 seconds, it is ensured that the HRP-d riven oxidation of TMB by hydrogen peroxide occurs more rapidly than the non-enzymatic peroxidase-like activity of the MPs, which originates from the iron centers. Additionally, conducting the detection reaction at 4°C and maintaining a low hydrogen peroxide concentration further minimizes the non-enzymatic catalytic activity of the MPs, thereby reducing the background signal attributable to non- enzymatic TMB oxidation (Gao et al., Nat Nanotechnol (2007) 2(9):577-583).

[0094] Table 3 presents a comparative account of the developed colorimetric sensor with a representative set of commercial ELISA kits. The commercial colorimetric ELISA kits use different labeling methods, such as HRP, Alkaline phosphatase (ALP), and high-affinity HRP- streptavidin-biotin, and cost between $450 to $730 per kit. The Mercodia ELISA kit employs an HRP-labeled sandwich antibody platform, but its detection limit and linear range are not as good. However, it offers high specificity and minimal cross-reactivity with other proteins in the matrix. The Enzo Lifesciences ELISA kit also utilizes a sandwich antibody approach with a biotinylated sandwich antibody, which is conjugated to the streptavidin-HRP label for colorimetric detection. This kit requires a similar sample dilution as utilized in this Example for the serum and plasma samples; however, the assay of this Example well outperforms the Enzo kit in terms of detection limit and assay time. Abcam's kit, though pricier, offers a comparable detection limit and dilution factor for serum samples, but the longer assay time of 5 h may be a drawback. The Novus Biologicals and ALPCO kits do not define the required sample dilution, and it is up to the users to determine the optimal sample dilution. Therefore, the assay of this Example exceeds all commercially available kits in terms of assay time, dilution factor, and linear ranges when considered as a whole. In addition, compared to the commercial methods, the approach disclosed herein offers advantages such as costeffectiveness that can be useful in resource-limited settings.Table 3. Comparison with commercial colorimetric enzyme-linked immunosorbent assay (ELISA) kits for insulin detection.*lto et al. (Biotechniques (2015) 59(6) :359— 367)

[0095] Functional stability of the sensor. The functional stability of the sensor is a critical factor in determining its longevity, reliability, and repeatability over time. Stability refers to the sensor's ability to maintain consistent performance and provide accurate readings over extended periods. It encompasses resistance to physical and chemical changes, such as degradation of sensor components, loss of bioactivity in biological elements, or drift in signal response due to environmental factors. Ensuring functional stability means that the sensor can be reliably used in various settings, maintaining accuracy and precision without frequent recalibration or replacement. This is especially important in clinical settings and remotesensing, where consistent and dependable readings are essential for patient diagnosis and continuous monitoring applications.

[0096] The antibodies are covalently bound to the MPs via stable amide bonds formed with free amine groups on lysine residues. Despite the stability of these bonds, proteins can denature when exposed to water or temperature fluctuations. To evaluate the durability of the sensor, 32 individual vials of the capture antibody-MP conjugates were prepared and stored at 2-8°C. Over a period of 8 days, new vials were taken from storage for each assay to assess their insulin detection response. Although the conjugates settled at the bottom of the Eppendorf tubes, they were readily resuspended with gentle agitation. Each assay was spiked with 1 nM insulin in 10 mM PBS, and it was noted that the control buffer sample (no-insulin) consistently showed a lower response than the spiked samples throughout the 8 days (FIG. 8). The average absorbance response of the spiked samples was consistently higher than that of the buffer samples containing the MP-capture antibody conjugate (no spiked insulin), indicating maintained functional stability over the tested duration in buffer solutions.

[0097] Conclusions. The ability to quickly and easily detect biomolecules using a visual sensor is highly advantageous in resource-limited and remote settings. The colorimetric sensor disclosed herein effectively measured spiked insulin across various matrices, including buffer, serum, and saliva. It demonstrated good functional stability and reproducibility, as evidenced by consistent performance over an at least 8-day storage period and in replicate measurements. The sensor can be applied for insulin measurements in real serum samples and can differentiate between type 1 diabetes and healthy human samples. Independent validation using absorbance spectroscopy and surface plasmon resonance imaging confirmed the reliability of the sensor results. This validation underscores the use of the sensor as a robust and reliable tool for insulin detection and indicates its broader applicability for various molecular targets in real samples.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0098] Illustrative embodiment 1. A sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample, wherein the immunoassay comprises: a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label; and wherein the capture antibody and the detection antibody specifically bind to non-overlapping epitopes ofinsulin so that a sandwich is formed.

[0099] Illustrative embodiment 2. A kit for a sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample, wherein the kit comprises: a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label; and wherein the capture antibody and the detection antibody specifically bind to non-overlapping epitopes of insulin so that a sandwich is formed.

[0100] Illustrative embodiment 2A. An immunoassembly, comprising: insulin; a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label; and wherein the capture antibody and the detection antibody are specifically bound to non-overlapping epitopes of insulin so that a sandwich is formed.

[0101] Illustrative embodiment 3. The immunoassay / kit / immunoassembly of illustrative embodiment 1, 2, or 2A, wherein the functionalized magnetic nanoparticle comprises a citrate-stabilized magnetic nanoparticle.

[0102] Illustrative embodiment 4. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-3, wherein the functionalized magnetic nanoparticle has a size in a range of from about 100 nm to about 2 microns.

[0103] Illustrative embodiment 5. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-4, wherein the functionalized magnetic nanoparticle has a size of about 200 nm.

[0104] Illustrative embodiment 6. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-5, wherein the colorimetric label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta-galactosidase.

[0105] Illustrative embodiment 7. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-6, further comprising a substrate for the enzyme of the colorimetric label.

[0106] Illustrative embodiment 8. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-7, further comprising at least one magnetic device for capture of the sandwich.

[0107] Illustrative embodiment 9. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-8, wherein the immunoassay can detect an insulin concentrationin a range of from about 10 pM to about 100 nM.

[0108] Illustrative embodiment 10. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-9, wherein the immunoassay is detected manually by visual inspection.

[0109] Illustrative embodiment 11. The immunoassay / kit / immunoassembly of any of illustrative embodiments 1-10, further comprising a receptacle in which the immunoassay is performed.

[0110] Illustrative embodiment 12. A sensor for performing a sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample, wherein the sensor comprises: a housing comprising at least one compartment, wherein the at least one compartment comprises: a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label; and wherein the capture antibody and the detection antibody specifically bind to nonoverlapping epitopes of insulin so that a sandwich is formed.

[0111] Illustrative embodiment 13. The sensor of illustrative embodiment 12, wherein the conjugate and the labeled detection antibody are disposed in the same compartment.

[0112] Illustrative embodiment 14. The sensor of illustrative embodiment 12 or 13, wherein the housing comprises at least two compartments, and wherein the conjugate and labeled detection antibody are disposed in different compartments.

[0113] Illustrative embodiment 15. The sensor of any of illustrative embodiments 12-14, further comprising at least one substrate for the colorimetric label, and wherein the at least one substrate is disposed in a different compartment than the labeled detection antibody.

[0114] Illustrative embodiment 16. The sensor of any of illustrative embodiments 12-15, wherein the functionalized magnetic nanoparticle comprises a citrate-stabilized magnetic nanoparticle.

[0115] Illustrative embodiment 17. The sensor of any of illustrative embodiments 12-16, wherein the functionalized magnetic nanoparticle has a size in a range of from about 100 nm to about 2 microns.

[0116] Illustrative embodiment 18. The sensor of any of illustrative embodiments 12-17, wherein the functionalized magnetic nanoparticle has a size of about 200 nm.

[0117] Illustrative embodiment 19. The sensor of any of illustrative embodiments 12-18, wherein the colorimetric label is an enzyme selected from the group consisting of alkalinephosphatase, horseradish peroxidase, and beta-galactosidase.

[0118] Illustrative embodiment 20. The sensor of any of illustrative embodiments 12-19, wherein the housing further comprises at least one read compartment in fluidic communication with the at least one compartment containing the conjugate and labeled detection antibody.

[0119] Illustrative embodiment 21. The sensor of illustrative embodiment 20, wherein the at least one read compartment comprises a magnetic device for capture of the sandwich thereon.

[0120] Illustrative embodiment 22. The sensor of any of illustrative embodiments 12-21, wherein the immunoassay detects an insulin concentration in a range of from about 10 pM to about 100 nM.

[0121] Illustrative embodiment 23. The sensor of any of illustrative embodiments 12-22, wherein the immunoassay is detected manually by visual inspection.

[0122] Illustrative embodiment 24. The sensor of any of illustrative embodiments 12-23, wherein the housing is configured for insertion within an instrument for detection.

[0123] Illustrative embodiment 24A. The immunoassay / kit / immunoassembly / sensor of any of illustrative embodiments 1-24, further defined as a diabetes insulin monitor.

[0124] Illustrative embodiment 24B. The immunoassay / kit / immunoassembly / sensor of illustrative embodiment 24A, further defined as a type 1 diabetes insulin monitor.

[0125] Illustrative embodiment 24C. The immunoassay / kit / immunoassembly / sensor of illustrative embodiment 24A, further defined as a type 2 diabetes insulin monitor.

[0126] Illustrative embodiment 24D. The immunoassay / kit / immunoassembly / sensor of any of illustrative embodiments 1-24, further defined as a sports insulin monitor.

[0127] Illustrative embodiment 25. A method of detecting an insulin concentration in at least one fluidic biological sample, the method comprising the steps of: combining the at least one fluidic biological sample with a conjugate and a labeled detection antibody to form a mixture, wherein the conjugate comprises a capture antibody attached to a functionalized magnetic nanoparticle, the detection antibody is labeled with a colorimetric label, and wherein the capture antibody and the detection antibody specifically bind to non-overlapping epitopes of insulin; incubating the mixture under conditions so that a sandwich is formed when the capture and detection antibodies bind to insulin; exposing the mixture to a magnetic device to capture the sandwich; adding a substrate for the colorimetric label; anddetermining the insulin concentration present in the at least one fluidic biological sample based on a resulting color intensity in the mixture at a location to which the magnetic device is exposed.

[0128] Illustrative embodiment 26. The method of illustrative embodiment 25, wherein the at least one fluidic biological sample is selected from the group consisting of urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0129] Illustrative embodiment 26A. The method of illustrative embodiment 26, wherein the at least one fluidic biological sample comprises at least one of serum or saliva.

[0130] Illustrative embodiment 27. The method of illustrative embodiment 25 or 26, wherein the determining step is performed manually by visual inspection.

[0131] Illustrative embodiment 28. The method of any of illustrative embodiments 25-27, wherein the determining step is performed by an instrument.

[0132] Illustrative embodiment 29. The method of any of illustrative embodiments 25-28, wherein the functionalized magnetic nanoparticle comprises a citrate-stabilized magnetic nanoparticle.

[0133] Illustrative embodiment 30. The method of any of illustrative embodiments 25-29, wherein the functionalized magnetic nanoparticle has a size in a range of from about 100 nm to about 2 microns.

[0134] Illustrative embodiment 31. The method of any of illustrative embodiments 25-30, wherein the functionalized magnetic nanoparticle has a size of about 200 nm.

[0135] Illustrative embodiment 32. The method of any of illustrative embodiments 25-31, wherein the colorimetric label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta-galactosidase.

[0136] Illustrative embodiment 33. The method of any of illustrative embodiments 25-32, wherein the method detects an insulin concentration in a range of from about 10 pM to about 100 nM.

[0137] Illustrative embodiment 34. A method of detecting an insulin concentration in at least one fluidic biological sample, the method using the immunoassay / kit / immunoassembly of any of illustrative embodiments 1-11 or the sensor of any of illustrative embodiments 12- 24, wherein the method comprises one or more of any of the steps of illustrativeembodiments 25-33.

[0138] Illustrative embodiment 35. A system comprising the immunoassay / kit / immunoassembly of any of illustrative embodiments 1-11 or the sensor of any of illustrative embodiments 12-24.

[0139] Thus, in accordance with the present disclosure, there have been provided compositions, reagents, kits, systems, sensors, and devices, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth herein. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, 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 present disclosure.

Claims

CLAIMS1. A kit for a sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample, wherein the kit comprises: a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label; and wherein the capture antibody and the detection antibody specifically bind to nonoverlapping epitopes of insulin so that a sandwich is formed.

2. The kit of claim 1, wherein the functionalized magnetic nanoparticle comprises a citrate-stabilized magnetic nanoparticle.

3. The kit of claim 1, wherein the functionalized magnetic nanoparticle has a size in a range of from about 100 nm to about 2 microns.

4. The kit of claim 3, wherein the functionalized magnetic nanoparticle has a size of about 200 nm.

5. The kit of claim 1, wherein the colorimetric label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta-galactosidase.

6. The kit of claim 1, further comprising a substrate for the enzyme of the colorimetric label.

7. The kit of claim 1, further comprising at least one magnetic device for capture of the sandwich.

8. The kit of claim 1, wherein the immunoassay is detected manually by visual inspection.

9. The kit of claim 1, further comprising a receptacle in which the immunoassay is performed.

10. A sensor for performing a sandwich immunoassay for detecting an insulin concentration in at least one fluidic biological sample, wherein the sensor comprises: a housing comprising at least one compartment, wherein the at least one compartment comprises: a conjugate comprising a capture antibody attached to a functionalized magnetic nanoparticle; and a detection antibody labeled with a colorimetric label; and wherein the capture antibody and the detection antibody specifically bind to non-overlapping epitopes of insulin so that a sandwich is formed.

11. The sensor of claim 10, wherein the conjugate and the labeled detection antibody are disposed in the same compartment.

12. The sensor of claim 10, wherein the housing comprises at least two compartments, and wherein the conjugate and labeled detection antibody are disposed in different compartments.

13. The sensor of claim 12, further comprising at least one substrate for the colorimetric label, and wherein the at least one substrate is disposed in a different compartment than the labeled detection antibody.

14. The sensor of claim 10, wherein at least one of: the functionalized magnetic nanoparticle comprises a citrate-stabilized magnetic nanoparticle; the functionalized magnetic nanoparticle has a size in a range of from about 100 nm to about 2 microns; the functionalized magnetic nanoparticle has a size of about 200 nm; and / or the colorimetric label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta-galactosidase.

15. The sensor of claim 10, wherein the housing further comprises at least one read compartment in fluidic communication with the at least one compartment containing the conjugate and labeled detection antibody, wherein the at least one read compartment comprises a magnetic device for capture of the sandwich thereon.

16. The sensor of claim 10, wherein the immunoassay is detected manually by visual inspection.

17. The sensor of claim 10, wherein the housing is configured for insertion within an instrument for detection.

18. A method of detecting an insulin concentration in at least one fluidic biological sample, the method comprising the steps of: combining the at least one fluidic biological sample with a conjugate and a labeled detection antibody to form a mixture, wherein the conjugate comprises a capture antibody attached to a functionalized magnetic nanoparticle, the detection antibody is labeled with a colorimetric label, and wherein the capture antibody and the detection antibody specifically bind to nonoverlapping epitopes of insulin; incubating the mixture under conditions so that a sandwich is formed when the capture and detection antibodies bind to insulin; exposing the mixture to a magnetic device to capture the sandwich; adding a substrate for the colorimetric label; and determining the insulin concentration present in the at least one fluidic biological sample based on a resulting color intensity in the mixture ata location to which the magnetic device is exposed.

19. The method of claim 18, wherein the at least one fluidic biological sample comprises at least one of serum or saliva.

20. The method of claim 18, wherein the determining step is performed manually by visual inspection.

21. The method of claim 18, wherein the determining step is performed by an instrument.

22. The method of claim 18, wherein at least one of: the functionalized magnetic nanoparticle comprises a citrate-stabilized magnetic nanoparticle; the functionalized magnetic nanoparticle has a size in a range of from about 100 nm to about 2 microns; the functionalized magnetic nanoparticle has a size of about 200 nm; and / or the colorimetric label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta-galactosidase.

23. The method of claim 18, wherein the method detects an insulin concentration in a range of from about 10 pM to about 100 nM.

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