Compositions and methods for detecting analytes

Abstract

A composition comprising a polymer matrix or film having dispersed therein a plurality of glass spheres having affinity groups attached thereto, and a method of using the same to detect a target analyte.

Description

[Technical Field]

[0001] background Non-Patent Document 1 discloses a nanoparticle-based immunosensor and immunoassay for aflatoxins. The disclosed sensors include a fluorescent assay, a chemiluminescent assay, and a light scattering assay. Non-Patent Document 2 discloses a rapid immunofluorescence assay for Staphylococcus aureus enterotoxin A using magnetic nanoparticles. The assay uses antibodies immobilized on magnetic nanoparticles. During the assay, the magnetic nanoparticles are removed from the liquid by a magnet, and the fluorescent signal of the supernatant is measured.

[0002] For example, a lateral flow assay is known as a self-administered pregnancy test. This assay does not require special analytical equipment. Quantification of an analyte by ELISA (enzyme-linked immunosorbent assay) is known.

[0003] Polymers for sustained release are discussed in various references such as "Synthetic Polymers for Sustained Release" by Matthews, "Synthetic Polymers for Sustained Release" and ... [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Wang et al. (Analytica Chimca Acta 912 (2016) 10-23) [Non-patent document 2] Becheva et al. (International Journal of Food Science and Technology 2018) [Non-patent document 3] Paolini, MS, Fenton, OS, Bhattacharya, C. et al. Polymers for extended-release administration. Biomed Microdevices 21, 45 (2019) [Non-patent document 4] Polymers Kamaly, N., Yameen, B., Wu, J. et al. Degradable controlled-release polymers and polymeric nanoparticles: mechanisms of controlling drug release. Chem. Rev. 2016, 116, 4, 2602-2663

[0005] Disclosure Overview The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The following description more particularly illustrates exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 shows a method of the present disclosure using a bind-antibody-elution method, which uses a detector compound molecule that is initially complexed with a bound antibody and detects free (i.e., unbound) target analyte molecules by measuring the displaced (i.e., eluted) detector compound molecule. [Figure 2] FIG. 1 illustrates a method of the present disclosure using a bound antibody depletion method, in which free detector compound molecules and bound antibodies are used to detect free target analyte molecules by measuring the (still free) detector compound molecules. [Figure 3] FIG. 1 shows a method of the present disclosure using bound target elution, which uses a labeled antibody (a detection compound molecule comprising a detection group (i.e., label) attached to a complementary group (i.e., antibody)) complexed to a bound target molecule, and detects free target analyte by measuring the labeled antibody displaced (i.e., eluted) by the initially free target analyte molecule. [Figure 4]FIG. 1 shows a method of the present disclosure using bound target depletion to detect free target analyte using an initially free labeled antibody (a detection compound molecule comprising a detection group (i.e., label) attached to a complementary group (i.e., antibody)) and measuring labeled antibody complexed with the initially free target analyte molecule. [Figure 5A] FIG. 11 is a graph showing the size distribution of glass spheres before and after fractionation. [Figure 5B] FIG. 11 is a graph showing the size distribution of glass spheres before and after fractionation. [Figure 6] 1 is a graph of the percent increase in fluorescence (at two different wavelengths) relative to a control sample for solutions containing 10 ppb, 5 ppb, or 1 ppb of either cortisol or fumonisin, or mixtures containing 10 ppb, 5 ppb, or 1 ppb of each. [Figure 7] 1 is a graph of the percent increase in fluorescence (at two different wavelengths) relative to a control sample for solutions containing 10 ppb, 5 ppb, or 1 ppb of either cortisol or fumonisin, or mixtures containing 10 ppb, 5 ppb, or 1 ppb of each. [Figure 8] 1 is a graph of the percent increase in signal relative to a control sample for 50 ppb of either cortisol, fumonisin, aflatoxin B1, or a mixture of all three, at different excitation and emission wavelengths. [Figure 9] 1 is a graph of the percent increase in signal relative to a control sample for 5 ppb of either cortisol, fumonisin, aflatoxin B1, or a mixture of all three, at different excitation and emission wavelengths. [Figure 10] 1 is a graph of fluorescence from solutions containing fumonisins in grain extract or phosphate buffered saline (PBS). [Figure 11] 1 is a graph of fluorescence intensity in the detection of aflatoxin B1 by the depletion method.

[0007] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The term "affinity group" refers to a covalently attached group capable of specifically binding to another molecule. The affinity group may be further conjugated or attached to a detection compound that can be detected, for example, by fluorescence, emission, or absorbance at a particular wavelength. As used herein, affinity groups are distinct from detection compounds or detection groups.

[0008] The term "antibody" refers to a polypeptide molecule having a region capable of binding, and in particular specifically binding, to a target analyte. Thus, the term includes both complete antibodies and, optionally, antibody fragments.

[0009] The terms "specifically bind" and "specific binding" mean that an affinity group complexes with another molecule (i.e., its complementary molecule) with greater affinity than it complexes with other molecules under assay conditions. For example, the binding of an antibody to a complementary target molecule is a specific binding interaction. In certain cases, "specifically bind" can mean that the affinity group complexes with a complementary molecule with at least 106-fold greater affinity, at least 107-fold greater affinity, at least 108-fold greater affinity, or at least 109-fold greater affinity than it complexes with a molecule unrelated to the target molecule. In this context, a molecule is unrelated to the target molecule if it lacks the portion of the target molecule's chemical structure that interacts with the affinity group.

[0010] The term "span" refers to the particle size distribution of a sample of particles and can be represented by the following formula: span = (90P - 10P) / 50P, where 90P is the size below which 90 percent (%) of the particles in the distribution are (referred to as the 90th percentile size), 10P is the size below which only 10% of the particles in the distribution are (referred to as the 10th percentile size), and 50P is the size below which 50 percent of the particles in the distribution are (referred to as the 50th percentile size).

[0011] In this application, terms such as "a," "an," and "the" are not intended to refer exclusively to a singular entity, but include the general class of which a specific example may be described. The terms "a," "an," and "the" are used interchangeably with the phrases "at least one" and "one or more." The phrases "at least one of" and "including at least one of," followed by a list, refer to any one item in the list and any combination of two or more items in the list.

[0012] Terms such as "common," "generally," "often," "frequent," and "frequently" are used to refer to features typically employed in the present invention, but unless otherwise indicated, are not intended to imply that the features so described were known or common prior to the present disclosure.

[0013] Magnetic beads or nanoparticles have been used as solid support components in heterogeneous assays to detect target analytes. A typical prior art assay using magnetic beads or nanoparticles is a competitive assay in which the beads are modified with molecules, such as antibodies, that can bind to the target analyte. A certain amount, usually a known amount, of the target analyte bound to a detection molecule, such as a luminescent dye, is added to an aqueous liquid containing metal beads or nanoparticles. In this case, the assay is a competitive binding assay in which a luminescent analyte bound to a detector and unbound analyte from the sample compete for binding to the antibody bound to the magnetic beads or nanoparticles, and the luminescence of the liquid is measured. A magnet is then applied to the aqueous liquid to remove the magnetic beads or nanoparticles from the liquid, along with anything bound to them. The fluorescence of the supernatant is then measured, and the change in luminescence can be related to the concentration of the target analyte in the sample.

[0014] The use of magnetic beads or nanoparticles presents problems. First, the effect of a magnet on an aqueous liquid depends on the proximity of the liquid to the magnet. With large sample volumes, which may be particularly required when the target analyte is in low concentration, the magnet may not be close enough to the entire sample to ensure that all of the magnetic beads or nanoparticles are removed from the liquid and that the supernatant is free of magnetic beads and nanoparticles.

[0015] Second, detection of small differences in weak fluorescence or luminescence intensity, which may be required to accurately detect or quantify low concentrations of target analytes, is best accomplished with photomultiplier tubes (sometimes known as PMTs), which are more sensitive than other photodetectors. PMTs are sensitive to magnetic fields and should not be operated near magnets for good performance. This makes the use of PMTs in conjunction with magnetic beads or nanoparticles problematic or impossible.

[0016] Third, magnetic nanoparticles may not be compatible with all samples; for example, samples containing ferromagnetic materials may not be usable with magnetic particles.

[0017] Fourth, particles used in two-phase assays with liquid and solid components, whether magnetic or not, can be prone to agglomeration, which can reduce the accuracy of the assay.

[0018] Fifth, samples, especially samples from animals, plants or the environment, may contain chemical compounds that interfere with the detection of the target analyte.

[0019] The fourth and fifth problems may be due in part to the issue of quenching the luminescence of the detection molecule. More specifically, typical assays rely on the luminescence (e.g., fluorescence or phosphorescence) of the detection compound, which, depending on the type of assay, can be turned on or off when the affinity group binds to the target analyte. Therefore, the accuracy of luminescence-based assays is compromised if the luminescence is quenched by processes other than those related to the binding of the target analyte to the affinity group. Aggregation of particles (e.g., those bearing affinity groups) can cause self-quenching of the detection compound. Magnetic beads are often luminescence quenchers. That is, magnetic beads can reduce assay sensitivity even in the absence of aggregation. Similarly, some components of the sample other than the target analyte can quench the luminescence of the detection compound. This quenching can result in a loss of assay accuracy, precision, and sensitivity, limiting the use of fluorescent immunoassays to the detection of relatively high concentrations of compounds in complex matrices.

[0020] Furthermore, with regard to issues involving compounds other than the target analyte in the sample, such compounds may also interfere with the assay by mechanisms other than quenching, such as competitive binding to the target analyte and binding (even without quenching) to the detector compound, among other interference mechanisms.

[0021] Furthermore, when non-magnetic particles are used, the particles must be removed from the assay by one or more procedures such as centrifugation, filtration, or both, which increases the possibility of user error.

[0022] Notably, many of these problems, such as those related to aggregation, quenching, and interference from compounds other than the target analyte, are not unique to magnetic particles and can be problems with any type of particle used in heterogeneous assays. Thus, another problem is reducing or eliminating sample interference, self-quenching, or both in multiplexed assays, i.e., a single assay that can detect more than one analyte.

[0023] Yet another problem is providing a heterogeneous assay that allows for multiplexing, ie, the detection of more than one analyte in the same assay.

[0024] The above problems all relate to the general problem of how to detect target analytes, especially those present in low concentrations, those requiring high sensitivity, or both. More specifically, these problems relate to problems that reduce the sensitivity, accuracy, or precision of heterogeneous assays, or problems that reduce the robustness of such assays. One type of target analyte that is relevant to these problems is mycotoxins, such as aflatoxins. Other types of targets that may be relevant to these problems include whole cells, e.g., bacterial cells, viruses, virions, toxins, proteins, and peptides.

[0025] Solutions to one or more of the above-mentioned problems, and possibly others, can be found in the present disclosure, which provides compositions and methods for detecting analytes using the compositions. The analytes to be detected are commonly referred to as "target analytes." In particular, the compositions and methods allow for multiplexing, which simultaneously measures the levels of two or more analytes at a time.

[0026] The composition includes a first plurality of hollow glass spheres covalently bonded to a first affinity group. The first plurality of glass spheres are embedded in a first polymer matrix material. The first polymer matrix material is a component of a film disposed on a substrate. Optionally, additional affinity groups, such as second, third, and fourth affinity groups, may be used.

[0027] Certain embodiments of the compositions and methods described herein that are configured for multiplexing allow for sequential binding of two or more target analytes in the same assay, rather than simultaneous binding of all target analytes, thereby solving yet another problem, namely, interference between different affinity groups when multiple affinity groups are simultaneously present in a multiplexed assay.

[0028] affinity group Any suitable affinity group can be used as the first affinity group, depending on the analyte to be detected. If additional affinity groups, such as second, third, fourth, etc. affinity groups, are used, they can be independently selected from the same types of affinity groups described herein.

[0029] Typically, an affinity group is selected that binds to an analyte, such as a target analyte. Examples include antibodies, antibody substrates, etc. Examples include antibodies; alternatively, any of the affinity groups can be selected to bind to a quencher or other substance that may interfere with detection of the target analyte. Examples include adsorbents, zeolites, and chelators. It is also possible to select an affinity group that binds to a compound or substance other than the target analyte, for example, to select an affinity group that binds to a compound or substance that may interfere with the assay, in order to reduce or eliminate interference with the assay.

[0030] One type of affinity group that can be used is an antibody. Those skilled in the art can select an appropriate antibody depending on the target analyte or analytes to be detected. Examples include rabbit anti-mouse IgG antibody, anti-cortisol antibody, anti-fumonisin antibody, and anti-aflatoxin B1 antibody. Suitable antibodies can be obtained from various sources, including Jackson ImmunoResearch, West Grove, PA; Abeam, Cambridge, MA; Lifespan Biosciences, Seattle, WA; and Creative Diagnostics, Shirley, NY.

[0031] Another type of affinity group that can be used is an aptamer, i.e., a short polynucleotide. Examples of aptamers include the anti-aflatoxin and anti-ochratoxin aptamers available from Aptagen, Jacobus, PA. The particular aptamer will likewise depend on the target analyte or analytes to be detected.

[0032] In some cases, one or more target analyte molecules, such as heavy metals or small organic molecules, can be used as affinity groups. As used herein, small organic molecules are those having a molecular weight of 5000 grams / mole or less.

[0033] hollow glass sphere The hollow glass spheres used herein require a density that allows them to float in the liquid used in the assay, which is typically an aqueous liquid. Therefore, the hollow glass spheres have a density of less than 0.60 grams / mL, optionally 0.40 grams / mL or less. While there is no required lower limit for the glass spheres, in practice, most glass spheres used have a density of 0.05 grams / mL or greater. Therefore, the particular hollow glass spheres used have a density in the range of 0.05 to 0.40 grams / mL.

[0034] In certain embodiments, the hollow glass spheres have a span of less than 1.0, less than 0.8, or less than 0.7. In certain embodiments, the hollow glass spheres have a span of at least 0.1, at least 0.2, at least 0.3, at least 0.4, or at least 0.5. In preferred embodiments, the hollow glass spheres have a span of 0.1-1.0, 0.1-0.8, 0.1-0.7, or 0.1-0.5. Typically, the narrower the span, the more uniform the hollow glass spheres, the shorter the assay, and the better the assay reproducibility.

[0035] The size of the hollow glass spheres can be selected depending on the needs of the particular assay in which they are used. Considerations include the rate at which the hollow glass spheres need to be released from the polymer matrix in which they are embedded, minimizing clumping, the rate at which the hollow glass spheres float to the top of the liquid, and, in some cases, light scattering effects. Most common hollow glass spheres used herein have an average diameter of at least 30 micrometers, optionally at least 40 micrometers. Typically, hollow glass spheres have an average diameter of 80 micrometers or less. In certain cases, hollow glass spheres have an average diameter of 30 to 80 micrometers, optionally 40 to 80 micrometers. Typically, larger hollow glass spheres float to the top concentrated portion of a liquid more quickly at a given density.

[0036] Suitable hollow glass spheres are commercially available from a variety of sources. These include, for example, those sold under the name XLD3000 silica spheres and 3M™ Glass Bubbles S60HS, available from 3M Company, St. Paul, MN. Typically, commercially available hollow glass spheres are fractionated to obtain a desired size distribution, determined by mean diameter and span. Such fractionated hollow glass spheres contribute to assay reproducibility.

[0037] The advantage of using glass spheres as a solid support compared to other particle-based assays is their inherent buoyancy properties, which allow for easy, tunable, and rapid separation from solution. Therefore, unlike other particles, partial separation of the glass spheres from the liquid components of the assay can be achieved without the application of mechanical forces, such as stirring or centrifugation, or magnetic forces. Instead, the glass spheres can be suspended on the surface of the liquid, and the bottom portion of the liquid, free of glass spheres, can be tested for the presence of analyte. It is also possible, although not always necessary, to easily recover or harvest the glass spheres bound to the target analyte for analysis.

[0038] An additional differentiator of this method is that the fluorescent signal can be detected from assay components in the liquid phase rather than from components on a recovered solid phase, eliminating the need to wash the solid phase, a major advantage in both time to result and elimination of operator error.

[0039] The hollow glass spheres can be covalently attached to affinity groups by any method that functionalizes glass. Many such methods are known, and the appropriate method depends on the nature of the affinity group. In some cases, the hollow glass spheres are activated to facilitate the attachment of affinity groups. Activation can be achieved, for example, by silanization. Conventional silanization reagents (e.g., 3-glycidoxypropyltrimethoxysilane) and techniques are known in the art. Alternatively, commercially available functionalized glass spheres can be used, such as glycidyl-functionalized H20 silica spheres available from 3M Company, St. Paul, MN.

[0040] The affinity group can be further attached, e.g., complexed or non-covalently attached, to a detection compound that can be detected, e.g., by fluorescence. For convenience, the affinity group will be described herein as distinct from the detection compound / group, even if the detection compound or detection group is attached to the affinity group.

[0041] Detection compound molecules and detection groups The detection compound molecule includes a detection group that can be detected, for example, by luminescence, especially fluorescence, and a complementary group. Such a detection compound or group typically has a wavelength of maximum emission (λ) of the detection group. max ) When multiple detection groups or compounds are used, such as in a multiplexed composition or method, a first detection group or compound can be detected at a first wavelength, a second detection group or compound can be detected at a second wavelength, a third detection group or compound can be detected at a third wavelength, and so on.

[0042] A variety of detection compounds and detection groups are known for use with biological assays. In principle, any detection compound or group can be used with the disclosed compositions and methods as long as it is compatible with the assay conditions; incompatibility may include chemical reactions with the target analyte or other components of the assay, instability under the assay conditions, etc. For example, amine-reactive TEXAS RED-X succinimidyl ester can be used to produce a bright red fluorescent detection compound with excitation / emission maxima of approximately 595 / 615 nanometers (nm). Fluorescein isothiocyanate can be used to fluoresce with an emission of 520 nm upon excitation at 488 nm. Many other examples are known to those skilled in the art, who can select appropriate detection groups and compounds depending on the assay requirements, the detection system used, and other factors.

[0043] In any embodiment herein involving detection or detectability at a first wavelength, the first wavelength is particularly in the visible or UV portion of the electromagnetic spectrum, more particularly in the visible portion of the electromagnetic spectrum, and most particularly a wavelength between 600 nm and 850 nm.

[0044] In any embodiment herein involving detection or detectability at a second wavelength, the second wavelength is particularly in the visible or UV portion of the electromagnetic spectrum, more particularly in the visible portion of the electromagnetic spectrum, and most particularly wavelengths between 600 nm and 850 nm.

[0045] In any embodiment herein involving detection or detectability at a third wavelength, the third wavelength is particularly in the visible or UV portion of the electromagnetic spectrum, more particularly in the visible portion of the electromagnetic spectrum, and most particularly a wavelength between 600 nm and 850 nm.

[0046] Detection of the detection compound or group can be achieved using a luminometer, particularly a fluorometer, of which a wide variety are known and which typically include a photomultiplier tube or a charge-coupled device (CCD) for detecting emitted light, such as fluorescence.

[0047] Substrates, Films and Polymer Matrices The composition comprises a film, which itself comprises a first polymer matrix having a first plurality of hollow glass spheres embedded therein, the film resting on a substrate, which is typically a container such as a PCR tube, cuvette, Eppendorf tube, test tube, etc., in which the assay is performed.

[0048] The embedded hollow glass spheres do not need to be in any particular arrangement within the polymer matrix, although certain arrangements may provide more desirable results depending on the assay in which the composition is to be used. For example, the majority of the hollow glass spheres may be located on the surface of the polymer matrix, may be uniformly or nearly uniformly distributed throughout the polymer matrix, or may be distributed in some other manner within the polymer matrix. For example, the hollow glass spheres may be distributed to provide primary release from the polymer matrix, if desired.

[0049] With respect to polymeric matrix materials, the terms "over" and "under" are used to refer to the proximity of a particular matrix material or layer of matrix material to the substrate on which the film is disposed, but do not necessarily indicate an adjacent relationship. For example, if a first polymeric matrix is ​​disposed "over" a second polymeric matrix, the second polymeric matrix is ​​closer to the substrate than the first polymeric matrix, but the first and second polymeric matrices are not necessarily adjacent to each other, and the second polymeric matrix is ​​not necessarily adjacent to the substrate.

[0050] The first polymeric matrix material is designed to release the first plurality of glass spheres from the matrix, e.g., into a liquid composition. To this end, the film and first polymeric matrix material are typically placed on the surface of a container, such as a cuvette, Eppendorf tube, or PCR tube, and the glass spheres can be released therein. The film and first polymeric matrix material can be placed on a surface using various techniques known in the art, such as solvent-casting a solution or suspension of the film and first polymeric matrix material and glass spheres followed by drying, or mixing the glass spheres with a pre-cured adhesive, placing the mixture on the surface, and allowing it to cure (in which case the cured adhesive becomes the polymeric material and film).

[0051] A second polymeric matrix material may be used, for example, disposed over at least a portion of the first polymeric matrix. Third, fourth, and additional polymeric matrix materials may be used, each disposed over at least a portion of the underlying polymeric matrix material. Any of the various polymeric matrix materials may have glass spheres disposed therewith, or may not have glass spheres but rather may be used to control the release of glass spheres from the underlying polymeric matrix material.

[0052] For example, any of the polymer matrix materials can be selected from immediate-release polymers, sustained-release polymers, and enteric polymers. The immediate-release polymer matrix releases all of the glass spheres from the polymer matrix very quickly, for example, after 5 minutes, 2 minutes, 1 minute, or 30 seconds after a liquid, such as an aqueous liquid, contacts the film, and can include one or more of hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, polyvinylpyrrolidone-polyvinyl acetate copolymer, polyvinyl alcohol, copolymers of polyvinyl alcohol and polyvinyl acetate, copolymers of polyvinyl alcohol and polyethylene glycol, water-soluble (meth)acrylic polymers, water-soluble (meth)acrylic copolymers, water-soluble copolymers of (meth)acrylate and polyethylene glycol, and polyethylene glycol.

[0053] The sustained-release polymer matrix releases the glass spheres over an extended period of time, for example, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, or more, and may comprise one or more of poly(glycolic acid), poly(lactic acid), poly(lactic-co-glycolic acid), poly(caprolactone), poly(orthoesters), poly(anhydrides), poly(amides), poly(ester amides), poly(phosphoesters), chitosan, hyaluronic acid, poly(alkyl cyanoacrylates), enteric polymers, or enzymatically degradable polymers. In some cases, the matrix may be chemically modified or have some degree of crosslinking to control the release rate.

[0054] The delayed-release polymer matrix can delay the time between when the liquid contacts the film and when the glass beads are released. For example, the delayed-release matrix can include an immediate-release layer with a coating layer that does not have any glass beads, so that the glass beads are not released until the coating layer dissolves or disintegrates.

[0055] Typically, the various polymer matrix materials are water-soluble or water-degradable, but it is also possible to have matrix materials that are enzymatically degradable, that degrade or dissolve only at a specific pH, temperature, or salt concentration, or that are not soluble or degradable but release the glass beads in other ways, such as having pores through which the glass beads can be released.

[0056] Multilayer films can be used when compounds or substances need to be removed from the sample matrix, for example, when such compounds or substances may interfere with the assay. In one example, a first polymer matrix, such as a controlled- or sustained-release polymer matrix, can be positioned beneath a second polymer matrix. The first polymer matrix can be a sustained- or controlled-release matrix, and can have embedded therein a plurality of glass spheres covalently bonded with affinity groups selected to bind to the target analyte. The second polymer matrix can be an immediate-release polymer, and can have embedded therein a plurality of hollow glass spheres covalently bonded with affinity groups selected to bind to interfering compounds or substances. During use, after a sample-containing liquid, typically an aqueous liquid, contacts the film, the second polymer matrix first releases its hollow glass spheres. These hollow glass spheres float to the surface, bound to the interfering compounds or substances, thereby removing the interfering compounds or substances from the liquid portion of the sample. The first polymer matrix then releases the hollow glass spheres bearing the affinity groups that bind to the target analyte. In this way, interfering compounds or substances can be removed before the portion of the assay that involves binding of the target analyte to the affinity group begins.

[0057] In some embodiments where the composition is suitable for multiplexing, hollow glass spheres are multiplexed. In such cases, there may be two or more affinity groups covalently attached to the hollow glass spheres. This can be accomplished in at least two ways. First, multiple hollow glass spheres may have multiple affinity groups attached to them, such as a first affinity group and a second affinity group (optionally a third, fourth, fifth, sixth, seventh, etc.), so that two or more types of affinity groups may be attached to the same glass sphere.

[0058] Another method for achieving multiplexing is to use at least two plurality of glass beads. A first plurality of glass beads is covalently bound to a first affinity group, and a second plurality of glass beads is covalently bound to a second affinity group that is different from the first affinity group. It is also possible to use third, fourth, fifth, sixth, seventh, etc. affinity groups, each covalently bound to a different plurality of glass beads. A combination of the two approaches is also possible; for example, a first plurality of glass beads can be covalently bound to a first affinity group and a second affinity group, and a second plurality of glass beads can be bound to a third affinity group and a fourth affinity group (additional plurality of glass beads and additional affinity groups may also be used).

[0059] When two plurality of hollow glass spheres are used, they can be present in the same or different polymer matrices. For example, a first plurality of hollow glass spheres can be present in a first polymer matrix, and a second plurality of hollow glass spheres can be present in a second polymer matrix that is located on the first polymer matrix. As a result, the second plurality of hollow glass spheres is released before the first plurality of hollow glass spheres. This can be advantageous in some multiplexed assays, where it may be useful to bind multiple target analytes sequentially rather than simultaneously in the same assay. For example, this configuration and detection method is particularly useful when the second affinity group has some cross-affinity for the first target analyte. This is because binding the first target analyte before the second affinity group is released can prevent the first target analyte from being exposed to the second affinity group.

[0060] A combination of these approaches can also be used. For example, when using two polymer matrices as described above, a third polymer matrix having a third plurality of glass spheres covalently bonded to an adsorbent can be placed on top of the first and second polymer matrices. In this way, the adsorbent can first remove interfering compounds or substances, followed by removal of the first and second plurality of glass spheres.

[0061] In an embodiment in which the multiplexed hollow glass spheres comprise two different covalently bound affinity groups, the composition comprises a plurality of first detection compound molecules that are not covalently bound to the plurality of multiplexed hollow glass spheres, wherein the first detection compound molecules comprise a first detectable group that is detected at a first wavelength, and a plurality of second detection compound molecules that are not covalently bound to the plurality of multiplexed hollow glass spheres, wherein the second detection compound molecules comprise a second detectable group that is detected at a second wavelength that is different from the first wavelength.

[0062] In embodiments in which the multiplexed hollow glass spheres comprise three or more different covalently bound affinity groups, the composition comprises a plurality of first detection compound molecules not covalently bound to the plurality of multiplexed hollow glass spheres, the first detection compound molecules comprising a first detectable group that is detected at a first wavelength, and a plurality of second detection compound molecules not covalently bound to the plurality of multiplexed hollow glass spheres, the second detection compound molecules comprising a second detectable group that is detected at a second wavelength different from the first wavelength. and a plurality of third (optionally fourth, fifth, sixth, seventh, etc.) detection compound molecules that are not covalently bound to the plurality of multiplexed hollow glass spheres, wherein the third (optionally fourth, fifth, sixth, seventh, etc.) detection compound molecules comprise a third (optionally fourth, fifth, sixth, seventh, etc.) detectable group that is detected at a third (optionally fourth, fifth, sixth, seventh, etc.) wavelength that is different from the first or second wavelength (or any other wavelength).

[0063] Sample and Target Analyte The compositions and methods described herein can be applied to assays for any target analyte to which an affinity group can bind. Examples include antibodies, substrates, small molecules, and heavy metals, among others. Many target analytes are food (e.g., animal feed) and water contaminants (i.e., food or water contaminants), as well as those used as biomarkers. However, in principle, any target analyte can be detected as long as at least a portion of the target analyte in the sample can bind to an affinity group.

[0064] Heavy metals are one type of target analyte that can be detected. Certain heavy metals are metals or metalloids with atomic numbers greater than 20. Typically, such heavy metals have atomic numbers of 92 or less. Examples include Pb, As, and Hg.

[0065] The method is particularly effective for small organic molecules. Such small organic molecules have a molecular weight of 5000 grams per mole or less. Examples of such small organic molecules include mycotoxins, such as aflatoxins (e.g., B1, B2, G1, G2, and M1), trichothecenes (e.g., deoxynivalenol, HT-2, and T-2), zearalenone, ochratoxins (e.g., A, B, C, and TA), fumonisins (e.g., B1, B2, B3, and B4), and patulin; shellfish toxins, such as saxitoxin, okadaic acid, domoic acid, and dinophysistoxin derivatives; hormones, such as cortisol, progesterone, and estrogens. radiol, androstenedione, diethylstilbestrol, ractopamine, clenbuterol, stanozolol, triamcinolone, zilpaterol, zeranol, and trenbolone; antibiotics and antivirals, such as β-lactams, tetracyclines, sulfonamides, nitrofurans, ciprofloxacin, chloramphenicol, acyclovir, triclosan, streptomycin, amphenicol, flunixin, florfenicol, fluoroquinolones, gentamicin, lincomycin, neomycin, tylosin, diclazuril, dimetridazole, colistin, and dapsone; biomarkers, such as coprostanol, cholesterol, and creatinine; pesticides, such as atrazine, N,N-diethyl-meta-toluamide (DEET), glyphosate, polychlorinated biphenyls (e.g., DDT and DDE), bromacil, avermectins, organophosphates, and N-methylcarbamates; drugs of abuse, such as opioids These include steroids (e.g., fentanyl and heroin), cocaine, tetrahydrocannabinol (THC), and amphetamines; and other biologically active compounds such as dexamethasone, albuterol, caffeine, warfarin, ibuprofen, codeine, lidocaine, celecoxib, benzatropine, metoprolol, omeprazole, nadolol, statins (e.g., atorvastatin and simvastatin), lisinopril, ketamine, and nandrolone. Such small organic molecules do not include proteins. Small organic molecules of particular interest to be detected include mycotoxins.

[0066] The present disclosure also provides methods for detecting an analyte, such as a contaminant, in a sample, the methods including: a) providing a composition including a liquid including water, a plurality of first hollow glass spheres in the liquid, the first hollow glass spheres having a plurality of first covalently bound affinity groups covalently bound to at least a portion of the plurality of first hollow glass spheres, and a plurality of first detection compound molecules not covalently bound to the plurality of first hollow glass spheres, the first detection compound molecules comprising a first detectable group detected at a first wavelength; b) adding a sample containing first free target analyte molecules to the composition; c) binding the first free target analyte molecules to the first affinity groups or the first detection compound molecules; d) suspending the plurality of first hollow glass spheres in an upper, concentrated portion of the liquid; and e) measuring the amount of the first detection compound molecules in a second portion of the liquid below the upper, concentrated portion without removing the plurality of first hollow glass spheres from the liquid. In this method, the first hollow glass spheres have a density of less than 0.60 grams / mL, a span of less than 1.0, and a plurality of first covalently bound affinity groups. It is understood that although not all spheres may float to the upper concentrated portion, a more efficient separation results in greater sensitivity.

[0067] The methods described herein can be used to detect heavy metals and small organic compounds that are typically found to contaminate food (e.g., animal feed) and water (i.e., food or water contaminants), as well as those used as biomarkers.

[0068] As used herein, a "heavy metal" is a metal or metalloid having an atomic number greater than 20 (calcium). Typically, such heavy metals have an atomic number equal to or less than 92 (uranium). Examples include Pb (lead), As (arsenic), and Hg (mercury).

[0069] The method is particularly effective for small organic molecules. Such small organic molecules have a molecular weight of 5000 grams per mole or less. Examples of such small organic molecules include mycotoxins, such as aflatoxins (e.g., B1, B2, G1, G2, and M1), trichothecenes (e.g., deoxynivalenol, HT-2, and T-2), zearalenone, ochratoxins (e.g., A, B, C, and TA), fumonisins (e.g., B1, B2, B3, and B4), and patulin; shellfish toxins, such as saxitoxin, okadaic acid, domoic acid, and dinophysistoxin derivatives; hormones, such as cortisol, progesterone, and estrogens. radiol, androstenedione, diethylstilbestrol, ractopamine, clenbuterol, stanozolol, triamcinolone, zilpaterol, zeranol, and trenbolone; antibiotics and antivirals, such as β-lactams, tetracyclines, sulfonamides, nitrofurans, ciprofloxacin, chloramphenicol, acyclovir, triclosan, streptomycin, amphenicol, flunixin, florfenicol, fluoroquinolones, gentamicin, lincomycin, neomycin, tylosin, diclazuril, dimetridazole, colistin, and dapsone; biomarkers, such as coprostanol, cholesterol, and creatinine; pesticides, such as atrazine, N,N-diethyl-meta-toluamide (DEET), glyphosate, polychlorinated biphenyls (e.g., DDT and DDE), bromacil, avermectins, organophosphates, and N-methylcarbamates; drugs of abuse, such as opioids These include steroids (e.g., fentanyl and heroin), cocaine, tetrahydrocannabinol (THC), and amphetamines; and other biologically active compounds such as dexamethasone, albuterol, caffeine, warfarin, ibuprofen, codeine, lidocaine, celecoxib, benzatropine, metoprolol, omeprazole, nadolol, statins (e.g., atorvastatin and simvastatin), lisinopril, ketamine, and nandrolone. Such small organic molecules do not include proteins. Small organic molecules of particular interest to be detected include mycotoxins.

[0070] Samples containing such analytes may be solid or liquid, but are in liquid form when measuring fluorescence intensity. The sample is typically a food product (e.g., grains such as oats, corn, wheat, etc.) or a water sample. When analyzing solid samples, it is preferable to extract the target analytes using a water extraction method to avoid handling and disposal of organic solvents. Typical water extraction methods use surfactant-based extraction. For example, TWEEN 20 and TRITON-X non-ionic detergents can be used to extract the target analytes. If interference is observed, it can be overcome by diluting with a buffer (e.g., phosphate-buffered saline).

[0071] Binding antibody composition / method The films described herein, containing one or more polymer matrices and at least a first plurality of glass spheres, can be used in various types of assays. Regardless of the type of assay, the film is contacted with a liquid that causes at least a portion of the hollow glass spheres to be released from the polymer matrix, for example, by dissolving the matrix, degrading the matrix, or eluting the hollow glass spheres from the matrix.

[0072] The liquid may be a sample-containing composition, such as one from a sample that is to be extracted, diluted, or both, as discussed herein. The liquid may also contain free detection compounds, which are detection compounds or groups that are not bound to the hollow glass spheres. The assay method typically involves contacting the sample as a liquid, most typically a solution, with a film comprising a polymer matrix and hollow glass spheres. In some cases, the sample can be added as a solid that dissolves in the liquid, causing the hollow glass spheres to be released from the polymer matrix.

[0073] Most assays also use at least a plurality of first detection compound molecules that are not covalently bound to the plurality of first hollow glass spheres. Some assays, such as multiplexed assays, may also use a plurality of second, third, fourth, or further detection compound molecules that are not covalently bound to the plurality of first hollow glass spheres. Any detection compound molecules that are not covalently bound to the plurality of first hollow glass spheres are typically contacted with the polymer matrix in liquid form, for example, by adding them as a solution or dispersion to a container in which a film containing the polymer matrix is ​​placed. In this case, the detection compound molecules may be added in the same liquid as the sample, or in a different liquid. The detection compound molecules that are not covalently bound to the plurality of first hollow glass spheres may also be placed within the polymer matrix and released along with the glass spheres.

[0074] The antibody binding method of the present disclosure is typically a competitive binding (e.g., competitive conjugation) assay, in which a detection compound competes with a target analyte present in a liquid sample to conjugate with a limited number of affinity groups, typically antibodies, that are available binding sites on hollow glass spheres. After binding, the hollow glass spheres float in the upper, concentrated portion of the liquid, and luminescence, typically fluorescence, emitted from unbound (i.e., free) detection compound molecules in the liquid below the upper, concentrated portion is measured. It is understood that not all glass spheres will be able to float in the upper, concentrated portion of the liquid.

[0075] The compositions described herein can be used in either elution or depletion assays. In elution assays, hollow glass spheres to which affinity groups, typically antibodies, are bound are complexed with free detection compound molecules before exposure to a sample. This can be achieved, for example, by first contacting a liquid containing free detection compound molecules with a polymer film to release the hollow glass spheres, and then adding a sample containing the target analyte. The target analyte in the sample displaces the detection compound molecules from the spheres, i.e., elutes them. In depletion assays, detection compound molecules are added to a liquid sample containing the target analyte, and then, after the glass spheres are released from the polymer matrix, the two molecules compete for binding with the affinity groups, typically antibodies, on the glass spheres.

[0076] In the elution method, as shown in FIG. 1, an antibody is an affinity group covalently bound to a hollow glass sphere. The antibody complexes with a detection compound molecule before contacting the free target analyte molecule, thereby forming a bound antibody composition. The detection compound molecule and the target analyte molecule both contain complementary groups and can compete for binding with the bound antibody. In this method for detecting an analyte, the method includes preparing a bound antibody composition (comprising hollow glass spheres having a first binding antibody complexed with a first detection compound molecule as an affinity group), adding a sample containing a first free target analyte molecule to the composition, allowing the first free target analyte molecule to bind to the first binding antibody (thereby displacing, i.e., eluting, the first detection compound molecule from the first binding antibody covalently bound to the hollow glass sphere), suspending the plurality of first hollow glass spheres in an upper, concentrated portion of the liquid, and measuring the amount of the (displaced) first detection compound molecule in a second portion of the liquid below the upper, concentrated portion without removing the plurality of first hollow glass spheres from the liquid. In FIG. 1, a first detection compound, which can be detectable (ie, a detection group bound to a complementary group), is first complexed with the bound antibody.

[0077] In the depletion method, the antibody is an affinity group covalently attached to a hollow glass sphere, as shown in Figure 2. Competition between the target analyte molecule and the detection compound molecule for binding to the bound antibody is induced. In the composition used in this method, a first detection compound molecule contains a first complementary group, and a first free target analyte molecule contains a first complementary group, and the first detection compound molecule competes with the first free target analyte molecule for complexation with the first bound antibody. In this method for detecting an analyte, the method includes providing a bound antibody composition and free detection compound molecules, adding a sample containing a first free target analyte molecule to the composition, allowing the first free target analyte molecule to bind to the first bound antibody (thereby competing with the first free detection compound molecule), suspending a plurality of first hollow glass spheres in an upper concentrated portion of the liquid, and measuring the amount of the first detection compound molecules (which remain free) in a second portion below the upper concentrated portion of the liquid without removing the plurality of first hollow glass spheres from the liquid.

[0078] In Figure 2, the detection compound is initially in the form of a free molecule. In the bound antibody depletion method, the target analyte molecule is covalently or otherwise bound to the hollow glass sphere and then bound to the glass sphere by complexing with a labeled antibody (not covalently). In this method of detecting an analyte, the method includes preparing a bound target composition (including hollow glass spheres having bound target analyte molecules complexed with a labeled antibody as affinity groups), adding a sample containing a first free target analyte molecule to the composition, complexing the first free target analyte molecule with the first labeled antibody (thereby displacing, i.e., eluting, the first labeled antibody from the first bound target analyte molecule bound to the hollow glass sphere), suspending a plurality of first hollow glass spheres in an upper, concentrated portion of the liquid, and measuring the amount of first detection compound molecules (the displaced first labeled antibody complexed with the initially free first target analyte molecule) in a second portion of the liquid below the upper, concentrated portion without removing the plurality of first hollow glass spheres from the liquid. The complexation step may optionally involve agitation, such as shaking, tumbling, or mixing, to keep the hollow glass spheres dispersed and non-aggregated in the liquid. In Figure 3, the detection compound molecule is a labeled antibody, which is first complexed with the bound target molecule in the bound target elution method.

[0079] In the depletion method, as shown in Figure 4, competition is caused between bound and free target analyte molecules for complexing with a labeled antibody. The composition used in this method includes a first free labeled antibody and hollow glass spheres to which a first bound target analyte molecule is bound. The first bound target analyte molecule competes with the first free target analyte molecule for complexing with the first free labeled antibody. In this method for detecting an analyte, the method includes preparing a bound target composition (comprising hollow glass spheres having a first bound target analyte molecule as an affinity group and a free, labeled antibody as a first detection compound molecule), adding a sample containing the first free target analyte molecule to the composition, complexing the first free target analyte molecule with the first labeled antibody (thereby competing with the first bound target analyte molecule), suspending a plurality of the first hollow glass spheres in an upper, concentrated portion of the liquid, and measuring the amount of the first labeled antibody complexed to the initially free target analyte molecule in a second portion below the upper, concentrated portion of the liquid without removing the plurality of first hollow glass spheres from the liquid. In Figure 4, the detection compound is initially in the form of a free, labeled antibody in the bound target depletion method.

[0080] Typically, in detection, the intensity of the fluorescent signal is directly or inversely proportional to the amount of target analyte in the sample. Thus, in such embodiments, the methods described herein can be used to quantify the amount of target analyte. This can be done by comparing the signal of an unknown sample with the signals of standards (e.g., typically three or more standard samples) of known target analyte concentrations.

[0081] The assays and methods described herein function essentially similarly when multiplexing is used. The primary differences are that the hollow glass spheres are one or more of the types described herein useful for multiplexing, and that two or more free detection compound molecules are used. After suspending the hollow glass spheres on a surface, detection is achieved by measuring emission, such as fluorescence, at multiple wavelengths. The emission measured at each wavelength corresponds, for example, by a direct or inverse proportional relationship, to the concentration of the target analyte associated with the detection compound whose emission is being measured.

[0082] Example The objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed as limiting the invention. These examples are for illustrative purposes only. Unless otherwise specified, all amounts are in weight percent.

[0083] material Polyvinyl alcohol (PVOH, 4-88 grade) was obtained from Kuraray America, Inc., Houston, TX.

[0084] XLD3000 glass (silica) spheres, S60HS glass (silica) spheres, and glycidyl-functionalized H20 glass (silica) spheres were obtained from 3M Company, St. Paul, MN.

[0085] Sodium sulfate and 0.1 N NaOH solution were obtained from Avantor, Radnor, PA.

[0086] Ethanol was obtained from Decon Labs, King of Prussia, PA.

[0087] 3-Glycidoxypropyltrimethoxysilane, dry dimethyl sulfoxide (DMSO), and ethanolamine were obtained from Alfa Aesar (Haverhill, MA).

[0088] Bovine serum albumin (BSA), TWEEN 20 non-ionic detergent, fumonisin B1, aflatoxin B1 (AFB1), and cortisol were obtained from Sigma Aldrich, St. Louis, MO.

[0089] N-hydroxysuccinimide (NHS) and ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) were obtained from Thermo Scientific (Rockford, IL).

[0090] Acetic acid, acetone, chloroform, dimethylformamide, methanol, and triethylamine were obtained from EMD Millipore, Billerica, MA.

[0091] TEXAS RED-X succinimidyl ester, EZ-LINK hydrazide-PEG4-biotin and QDOT streptavidin sampler kit (Q10151MP) were obtained from Thermo Fisher Scientific, Waltham, MA.

[0092] Cy3-labeled rabbit anti-mouse IgG was obtained from Jackson ImmunoResearch, West Grove, PA.

[0093] Anti-cortisol antibody (XM-210) was obtained from Abeam, Cambridge, MA.

[0094] Anti-fumonisin antibody (PY715173) and anti-aflatoxin B1 antibody (DMAB2948) were obtained from Creative Diagnostics, Shirley, NY.

[0095] Deionized (DI) water was purified using a MILLI-Q water purification system (EMD Millipore, Burlington, MA).

[0096] SUPELCLEAN LC-Si SPE cartridges and AMICON Ultra 0.5 mL centrifugal filter units (3000 NMWL) were obtained from Millipore Sigma, St. Louis, MO.

[0097] NANOSEP tubing was obtained from Pall Corporation, Port Washington, NY.

[0098] Size distribution was measured using a Flex dynamic light scattering system from Microtrac Inc. (Montgomery, PA).

[0099] LCMS (liquid chromatography-mass spectrometry) data were recorded on an Agilent 1260 Infinity HPLC system equipped with a 6130 quadrupole LC / MS (Agilent Technologies, Santa Clara, CA).

[0100] Fluorescence was measured on either a Tecan Infinite 200 plate reader (Tecan Group, Ltd., Switzerland) or a Synergy Neo2 plate reader (BioTek Instruments, Winooski, VT).

[0101] FITC-cortisol (fluorescein isothiocyanate-labeled cortisol) was synthesized according to the procedure of M. Pourfarzaneh et al., Clin. Chem. 26(6), 730-733 (1980).

[0102] The anti-AFB1 antibody was labeled with ALEXA FLUOR 680 according to the instructions provided with the ALEXA FLUOR 680 labeling kit (#A20188) from Thermo Fisher Scientific, Waltham, MA.

[0103] Preparation Example 1: Fractionation of Glass Beads 1A - Fractionation of XLD3000 Glass Beads: XLD3000 glass beads (3M ID98-0212-3469-9; 10 grams (g)) were added to 400 milliliters (mL) of deionized (DI) water in a separatory funnel. The suspension was shaken, and the beads were allowed to suspend for 1 minute (min), after which 390 mL of liquid (containing debris and small beads) was drained from the bottom. This process was repeated five times, after which the fractionated beads were recovered by filtration through a Whatman 4 filter under low pressure. The recovered beads were then rinsed with acetone (100 mL) and allowed to dry overnight at room temperature. The size distribution of the fractionated sample (determined by the mean (i.e., averaged) diameter and span) is shown in Figure 5A.

[0104] 1B - Fractionation of H20 Glass Bulbs: Glass sphere H20 (3M ID70-0704-8399-8; 10 grams (g)) was added to 400 milliliters (mL) of DI water in a separatory funnel. The suspension was shaken, and the glass spheres were allowed to suspend for 1 minute (min), after which 390 mL of liquid (containing debris and small glass spheres) was drained from the bottom. This process was repeated five times, after which the fractionated glass spheres were recovered by filtration through a Whatman 4 filter under low pressure. The glass spheres were then rinsed with acetone (100 mL) and allowed to dry overnight at room temperature. The size distribution of the fractionated samples (determined by the mean (i.e., averaged) diameter and span) is shown in Figure 5B.

[0105] Preparation Example 2: Activation and silanization of XLD3000 glass spheres The fractionated glass spheres from Preparative Example 1 (6.4 g; mean = 43.7 micrometers; span = 0.70) were cleaned by gentle rotation (ThermoFisher rotator) in 400 mL of 0.1 normal (N) NaOH at 10 revolutions per minute (rpm) for 3 hours, followed by vacuum filtration through Whatman #54 filter paper. After rinsing with Milli-Q water, the cleaned glass spheres were transferred to a plastic dish and air-dried at room temperature overnight.

[0106] Cleaned fractionated glass spheres (500 milligrams (mg)) were added to a 40 mL solution of 95% ethanol / 5% 50 mM acetate buffer (pH 5.2) and 300 microliters (μL) of 3-glycidoxypropyltrimethoxysilane, dispersed by vortexing, and then rotated vertically at 20 rpm on a rotator for 30 minutes to 2 hours. The silanized spheres were then separated from the solution by vacuum filtration (Whatman #54 filter paper), rinsed with ethanol, transferred to a glass vial, and air-dried overnight at room temperature.

[0107] Preparation Example 3: Antibody functionalization of XLD3000 glass spheres The silanized glass beads (3 mg) from Preparation Example 2 were added to a 0.5 mL centrifuge tube and suspended in 90 microliters of coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) and 10 microliters of a 1.5 mg / mL IgG reconstituted stock solution (rabbit anti-mouse IgG antibody, labeled with cyanine 3 (Cy3) fluorescent dye). After rotating the tube vertically at 20 rpm in the dark for 30 minutes, the glass beads were vacuum filtered (Whatman #1 filter paper) and rinsed with 1 M NaCl solution followed by deionized (DI) water.

[0108] The purpose of this example was to demonstrate efficient binding of antibodies to glass beads. Fluorescence microscopy showed consistent coverage of the beads with antibodies.

[0109] Preparation Example 4: General procedure for antibody immobilization on H20 glass beads precoated with glycidyl groups A NANOSEP tube (0.45 micron) was loaded with 10 mg of glycidyl-functionalized H2O glass beads. 300 microliters of antibody solution (approximately 0.1 mg / mL) in coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) was added, and the suspension was gently rotated at 25 rpm for 1 hour. The liquid was removed by centrifugation, and 0.5 mL of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added. The contents of the tube were mixed by rotating at 25 rpm for 1 hour. The liquid was removed by centrifugation, and the glass beads were washed three times with 0.5 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4).

[0110] Preparation Example 5: Synthesis of TEXAS RED-X fumonisin conjugate (detection compound) A 50-microliter sample of a 2 mg / mL fumonisin B1 toxin solution in DMF was mixed with 50 microliters of 2 mg / mL TEXAS RED-X succinimidyl ester in methanol (Invitrogen) and 10 microliters of triethylamine in a small plastic reaction vial. The reaction mixture was allowed to react overnight (approximately 20 hours) at room temperature, protected from light. A 10-microliter aliquot of the reaction mixture was loaded onto a SUPELCLEAN LC-Si SPE cartridge and eluted with chloroform / methanol / acetic acid (20:5:0.5). Fractions of 0.5 mL each were collected in clean vials and analyzed by LC / MS. The fractions were dried under a gentle stream of nitrogen.

[0111] Preparation Example 6: Immobilization of aflatoxin B1 (target analyte) on H20 glass spheres A NANOSEP tube (0.45 microns) was loaded with 10 mg of glycidyl-functionalized H2O glass beads. A 400 microliter volume of BSA-AFB1 solution (approximately 0.5 mg / mL) in coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) was added, and the suspension was mixed by rotating overnight at 4 °C. The liquid was then removed by centrifugation, and 0.5 mL of quenching buffer (3 M ethanolamine, pH 9) was added and mixed by rotating at 25 rpm for 4 hours. The liquid was then removed by centrifugation, and the glass beads were washed three times with 0.5 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4). The glass beads were finally resuspended in dilution buffer and stored at 4 °C until use.

[0112] Proof-of-concept example 1: Detection of aflatoxins by bound target elution method A tube containing 10 mg of aflatoxin B1-coated glass beads in dilution buffer (prepared according to Example 6) was centrifuged to remove the dilution buffer, washed with dilution buffer (phosphate buffer, pH 7.4, containing 0.01% TWEEN 20 non-ionic detergent), and centrifuged again. A solution of ALEXA FLUOR 680-labeled anti-AFB1 antibody (400 microliters of a 10 microgram / mL solution in phosphate-buffered saline (PBS)) was added and incubated for 1.5 hours with rotation and protected from light. The beads were then washed three times with dilution buffer (phosphate buffer, pH 7.4, containing 0.01% TWEEN 20 non-ionic detergent) to remove excess antibody, resuspended in dilution buffer (300 microliters), and transferred to a 2 mL vial.

[0113] The glass beads with bound target analyte molecules and complexed labeled antibodies were kept in suspension by vortexing, and 25 microliter volumes were carefully aliquoted into separate clean 1.5 mL tubes. Standard solutions of aflatoxin B1 in dilution buffer (50 ppb, 5 ppb, 0.5 ppb, and 0 ppb, 500 microliters each; ppb = parts per billion) were added to each tube, one standard solution per tube. Each tube was rotated for 30 min in the dark. The tubes were tilted to allow the glass beads to float to the surface, and 200 microliters of liquid from each tube was carefully transferred to a NANOSEP tube, centrifuged to filter out any remaining glass beads, and then transferred to a single well of a black half-area 96-well plate. Fluorescence was measured at 670 nm excitation and 715 nm emission.

[0114] Table 1 shows the average fluorescence signal intensities measured from several replicates of solutions containing the indicated amounts of aflatoxin B1 after 30 min of incubation with functionalized glass beads. The antibodies tested gave good results, with increasing signal from solutions containing higher concentrations of target in the sample, and clear discrimination between 0.5 ppb and 0 ppb.

[0115] [Table 1]

[0116] Proof-of-concept example 2: Multiplexed detection of fumonisin and cortisol in the same sample using a bound antibody elution method 2A - Preparation of Cortisol Spheres: XLD3000 glass spheres were first silanized with 3-glycidoxypropyltrimethoxysilane as in Example 2. Then, 10 mg of epoxy-functionalized XLD3000 glass spheres were placed in a NANOSEP tube. A solution of anti-cortisol antibody (XM-210) was prepared by mixing 25 microliters of a 2 mg / mL antibody solution with 375 microliters of coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) and then added to the tube containing the glass spheres. The tube was gently rotated at 25 rpm for 1 hour. The liquid was removed by brief centrifugation. An aliquot (0.5 mL) of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added, and the suspension was rotated at 25 rpm for 1 hour. The liquid was again removed by brief centrifugation, and the glass beads were washed three times with 0.5 mL of dilution buffer (phosphate buffer, pH 7.4, containing 0.1% TWEEN 20 non-ionic detergent). A solution of FITC-cortisol conjugate (500 microliters, 100 nM) was added, and the suspension was rotated for 1.5 hours in the dark. The liquid was removed by centrifugation. The glass beads were washed three times with dilution buffer to remove excess conjugate, and then resuspended in 0.25 mL of dilution buffer.

[0117] 2B - Preparation of Fumonisin Spheres: XLD3000 glass spheres were first silanized with 3-glycidoxypropyltrimethoxysilane as in Example 2. Then, 10 mg of epoxy-functionalized XLD3000 glass spheres were placed in a NANOSEP tube. A solution of anti-fumonisin antibody (Creative Diagnostics PY715173) was prepared by mixing 10 microliters of a 5.3 mg / mL antibody solution with 390 microliters of coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) and then added to the tube containing the glass spheres. The tube was gently rotated at 25 rpm for 1 hour. The liquid was removed by brief centrifugation. An aliquot (0.5 mL) of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added, and the suspension was rotated at 25 rpm for 1 hour. The liquid was again removed by brief centrifugation, and the glass beads were washed three times with 0.5 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4). A solution of TEXAS RED-X fumonisin conjugate (500 microliters, 100 nM, prepared according to Preparation 5) was added, and the suspension was rotated for 1.5 hours in the dark. The liquid was removed by centrifugation. The glass beads were washed three times with dilution buffer to remove excess conjugate, and then resuspended in 0.25 mL of dilution buffer.

[0118] 2C - Multiplexed Competitive Assay by Elution Method: The glass bead suspensions from 2A and 2B were vortexed to maintain suspension, and 25 microliter aliquots were carefully transferred into clean 1.5 mL tubes. Aliquots of nine different target sample and control solutions (cortisol alone: ​​10 ppb, 5 ppb, 1 ppb; fumonisin alone: ​​10 ppb, 5 ppb, 1 ppb; cortisol + fumonisin mixture: 10 ppb of each component, 5 ppb of each component, 1 ppb of each component; and control: 0 ppb; n=2 for each sample) were prepared in dilution buffer and added individually to the separate tubes containing glass beads (single aliquot per tube). Each tube was rotated for 1 hour, protected from light. The tubes were tilted to allow the glass beads to float to the surface, and 180 microliters of liquid from each tube was carefully transferred into one well of a black half-area 96-well plate. The fluorescence intensity was measured under two sets of conditions: excitation 488 nm / emission 520 nm (for cortisol detection) and excitation 590 nm / emission 620 nm (for fumonisin detection).

[0119] The results are presented in Tables 2 and 3 and Figures 6 and 7 as percent increase in signal relative to the control. The data in Table 2 and Figure 6 clearly show that solutions containing both cortisol and fumonisin closely track the response of solutions containing only cortisol when probed at wavelengths appropriate for the FITC-cortisol conjugate. The data in Table 3 and Figure 7 clearly show that solutions containing both cortisol and fumonisin closely track the response of solutions containing only fumonisin when probed at wavelengths appropriate for the TEXAS RED-X conjugate.

[0120] [Table 2]

[0121] [Table 3]

[0122] Proof-of-concept example 3: Multiplexed detection of aflatoxins, fumonisins, and cortisol in the same sample using a bound antibody elution method 3A - Preparation of Cortisol Spheres: A NANOSEP tube was loaded with 12 mg of epoxy-coated H20 glass spheres. A solution of anti-cortisol antibody (XM-210) was prepared by mixing 25 microliters of a 2 mg / mL antibody solution with 375 microliters of coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) and then added to the tube containing the glass spheres. The tube was gently rotated at 25 rpm for 1 hour. The liquid was removed by brief centrifugation. An aliquot (0.5 mL) of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added, and the suspension was rotated at 25 rpm for 1 hour. The liquid was again removed by brief centrifugation, and the glass spheres were washed three times with 0.5 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4). A solution of FITC-cortisol conjugate (400 microliters, 100 nM) was added, and the suspension was rotated for 1.5 hours in the dark. The liquid was removed by centrifugation. The glass beads were washed three times with dilution buffer and then resuspended in 0.3 mL of dilution buffer.

[0123] 3B - Preparation of Fumonisin Spheres: A NANOSEP tube was loaded with 12 mg of epoxy-coated H20 glass spheres. A solution of anti-fumonisin antibody (Creative Diagnostics PY715173) was prepared by mixing 10 microliters of a 5.3 mg / mL antibody solution with 390 microliters of coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) and then added to the tube containing the glass spheres. The tube was gently rotated at 25 rpm for 1 hour. The liquid was removed by brief centrifugation. An aliquot (0.5 mL) of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added, and the suspension was rotated at 25 rpm for 1 hour. The liquid was again removed by brief centrifugation, and the glass spheres were washed three times with 0.5 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4). A solution of TEXAS RED-X fumonisin conjugate (400 microliters, 100 nM, prepared according to Preparation Example 5) was added, and the suspension was rotated for 1.5 hours protected from light. The liquid was removed by centrifugation. The glass beads were washed three times with dilution buffer and then resuspended in 0.3 mL of dilution buffer.

[0124] 3C - Preparation of Aflatoxin B1 Spheres: A NANOSEP tube was loaded with 12 mg of epoxy-coated H20 glass spheres. A BSA-AFB1 solution (0.5 mg / mL in coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5)) was added to the tube containing the spheres, and the tube was gently rotated at 25 rpm for 1 hour. The liquid was removed by brief centrifugation. An aliquot (0.5 mL) of quenching buffer (3 M ethanolamine, pH 9, without excess BSA) was added, and the suspension was rotated at 25 rpm for 1 hour. The liquid was again removed by brief centrifugation, and the spheres were washed three times with 0.5 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4). A solution of Alexa Fluor 680-labeled anti-aflatoxin antibody (400 microliters, 10 micrograms / mL) was added, and the suspension was rotated for 1.5 hours, protected from light. The liquid was removed by centrifugation. The glass beads were washed three times with dilution buffer and then resuspended in 0.3 mL of dilution buffer.

[0125] 3D - Multiplexed Competitive Assay by Elution Method: The glass bead suspensions from 3A, 3B, and 3C were combined, floated, and 0.54 mL of buffer solution was removed from the bottom to achieve a glass bead concentration of 100 mg / mL. The beads were kept suspended by vortexing, and 20 microliter aliquots were carefully dispensed into clean 2 mL tubes. Aliquots of eight different target sample and control solutions (cortisol alone: ​​50 ppb, 5 ppb; fumonisin alone: ​​50 ppb, 5 ppb; aflatoxin alone: ​​50 ppb, 5 ppb; cortisol + fumonisin + aflatoxin B1 mixture: 50 ppb each, 5 ppb each; and control: 0 ppb; n=2 for each sample) were prepared in dilution buffer and added individually to separate tubes containing glass beads (single aliquot per tube). Each tube was rotated for 30 min protected from light. The tubes were tilted to allow the glass beads to float to the surface. Liquid (130 microliters) was removed from each duplicate, combined, and filtered into a NANOSEP tube. The combined filtered sample (170 microliters) was transferred to one well of a black half-area 96-well plate. Fluorescence intensity of the filtered samples was measured under three sets of conditions: excitation 488 nm / emission 520 nm (cortisol); excitation 590 nm / emission 620 nm (fumonisin); and excitation 670 nm / emission 715 nm (aflatoxin B1).

[0126] Tables 4 and 5 and Figures 8 and 9 show the data as percent increase in signal relative to the control. Table 4 shows the percent increase in fluorescence at different wavelengths for solutions containing 50 ppb of cortisol, fumonisin, or aflatoxin B1, or mixtures containing 50 ppb of each component. Table 5 shows the percent increase in fluorescence at different wavelengths for solutions containing 5 ppb of cortisol, fumonisin, or aflatoxin B1, or mixtures containing 5 ppb of each component. The data clearly show that all three targets can be readily detected at 50 ppb, and that fumonisin in particular can be readily detected at 5 ppb, even in the presence of other targets and fluorescent conjugates.

[0127] [Table 4]

[0128] [Table 5]

[0129] Proof-of-concept example 4: Detection of fumonisins in cereal extracts using a bound antibody elution method Samples (2 g) of three different grains (oats, corn, and wheat) were weighed into separate 50 mL centrifuge tubes. Each was treated with 10 mL of a 3% solution of TWEEN 20 nonionic detergent in PBS, shaken by hand for 3 minutes, and allowed to settle for 30 seconds. The supernatant was decanted, filtered through a 0.45-micrometer syringe filter, and then diluted 10-fold with PBS. A 10 ppm (parts per million) standard solution of fumonisins in water was added to either the grain extract or dilution buffer (phosphate buffer, pH 7.4, containing 0.1% TWEEN 20 nonionic detergent) in sufficient volume to produce individual samples containing 100 ppb, 50 ppb, 5 ppb, and 0 ppb of fumonisins.

[0130] XLD3000 glass beads were first silanized with 3-glycidoxypropyltrimethoxysilane as in Preparation 2. Next, 5 mg of epoxy-functionalized XLD3000 glass beads were each placed in a NANOSEP tube, followed by a solution of anti-fumonisin antibody (Creative Diagnostics PY715173) prepared by mixing 5 microliters of a 5.3 mg / mL antibody solution with 395 microliters of coupling buffer (phosphate buffer containing 0.9 M sodium sulfate, pH 7.5). After gently rotating the tube at 25 rpm for 1 hour, the liquid was removed by brief centrifugation, and 0.35 mL of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added. The resulting suspension was rotated at 25 rpm for 1 hour. The liquid was again removed by brief centrifugation, and the glass beads were washed three times with 0.4 mL of dilution buffer (phosphate buffer containing 0.1% TWEEN 20 non-ionic detergent, pH 7.4). A solution of TEXAS RED-X fumonisin conjugate (400 microliters, 100 nM, prepared according to Preparation Example 5) was added, and the suspension was rotated for 1 hour protected from light. The liquid was removed by centrifugation. The glass beads were washed three times with dilution buffer to remove excess conjugate, then resuspended in 0.2 mL of dilution buffer.

[0131] The glass beads were kept suspended by vortexing, and 25-microliter aliquots were carefully dispensed into clean 1.5-mL tubes. Target solutions (100 ppb, 50 ppb, 5 ppb, and 0 ppb; 250 microliters of each grain extract or PBS control) were added individually to the separate tubes containing the glass beads (a single target solution per tube). Each tube was rotated for 1 hour, protected from light. The tubes were tilted to allow the glass beads to float to the surface, and 160 microliters of liquid from each tube was carefully transferred to one well of a black, half-area 96-well plate. Fluorescence intensity was measured at 620 nm emission with 590 nm excitation and is reported in Table 6 and Figure 10. These data demonstrate that fumonisins can be efficiently extracted from grain and analyzed using the methods described herein.

[0132] [Table 6]

[0133] Preparation Example 7: Synthesis of aflatoxin B1-QD 585 conjugate Dry DMSO (0.4 mL) was added to 50 mg of EZ-LINK hydrazide-PEG4-biotin to make a 250 mM stock solution. A 10 microliter aliquot of this solution (2.5 micromoles) was added to 100 microliters of a 10 mg / mL solution of aflatoxin B1 in DMSO in an amber vial. Acetic acid (110 microliters) was added to the vial, which was then heated to 50°C overnight in a water bath. LCMS revealed the consumption of EZ-LINK hydrazide-PEG4-biotin and the aflatoxin conjugate (biotin-PEG4-AFB1, MH). + A 5 microliter aliquot (2 × 10 -8 A 0.02 nM solution was made by diluting 100 nM of QDOT 585 (100 nM) with 975 microliters of phosphate buffer (0.1 N pH 7.6). This solution was added to 20 microliters of a 1 micromolar solution of QDOT 585 streptavidin conjugate (obtained as part of the Q10151MP Sampler Kit from Thermo Fisher Scientific) and gently rotated at room temperature for 2 hours.

[0134] After conjugation, the solution was concentrated using two 0.5 mL AMICON Ultra centrifugal filters by centrifugation at 14,000 G for 15 minutes. The eluate (approximately 400 microliters) was removed, and the concentrated solution remaining on the filter was washed three more times (14,000 G, 15 minutes) with 0.4 mL of pH 7.6 buffer. The two concentrated final solutions were combined, refrigerated, and purified using approximately 10% PEG-1000 ribonucleotides (RIs) functionalized with aflatoxin B1 via a streptavidin-biotin-PEG4 linker. -11 Approximately 110 microliters of a bright orange fluorescent solution containing 100 moles of QDOT 585 was obtained.

[0135] Proof-of-concept example 5: Detection of aflatoxins by depletion method using conjugated DMAB2948 antibody Ten milligrams of glycidyl-functionalized XLD3000 glass beads (Example 2) were individually placed into NANOSEP tubes (0.45 microns). To each tube, 400 microliters of an anti-aflatoxin (DMAB2948) antibody solution (approximately 0.07 mg / mL) in coupling buffer (0.1 M phosphate buffer containing 0.9 M sodium sulfate, pH 7.5) was added, and the suspension was gently rotated at 25 rpm for 1 hour. The liquid was removed by centrifugation, and 0.4 mL of quenching buffer (3 M ethanolamine, pH 9) containing 1% BSA was added. The contents were mixed by rotating at 25 rpm for 1 hour. The liquid was removed by centrifugation, and the glass beads were washed three times with 0.5 mL of dilution buffer (phosphate buffer, pH 7.4 containing 0.1% TWEEN 20 non-ionic detergent).

[0136] The antibody-functionalized glass spheres were then suspended in 200 microliters of dilution buffer (phosphate buffer, pH 7.4, containing 0.1% TWEEN 20 non-ionic detergent), and a 25 microliter aliquot of this suspension was carefully transferred to a clean 1.5 mL tube.

[0137] A stock solution of aflatoxin B1 (20 micrograms / mL in methanol, Sigma-Aldrich, St. Louis, MO) was diluted (PBS, 0.1% Tween-20, pH 7.4) to obtain a 1000 ppb solution. This solution was mixed with 20 microliters of the QD-PEG-AFB1 conjugate solution from Preparation 7 and the appropriate amount of dilution buffer to obtain final aflatoxin B1 concentrations of 100 ppb, 10 ppb, 1 ppb, and 0 ppb. Aliquots (175 microliters) of these solutions were added individually in duplicate to separate tubes containing antibody-functionalized glass beads (single aliquot per tube). Each tube was incubated for 30 min with rotation. The tubes were tilted to allow the glass beads to float to the surface, and 200 microliters of liquid from each tube was carefully transferred to one well of a black half-area 96-well plate. Fluorescence intensity was measured at 585 nm emission with 400 nm excitation and is reported in Table 7 and Figure 11. These data indicate that aflatoxin concentrations of 10 ppb or greater can be effectively measured by the depletion method.

[0138] [Table 7]

[0139] Example 1: PVOH (100 g) was dissolved in deionized water (233 g) by heating at 70° C. for 20 hours. The PVOH solution was cooled to room temperature. S60HS glass spheres (0.3 g, density 0.6 g / mL, average diameter 30 micrometers) were added to a bottle containing 30 g of the PVOH solution, and the contents were mixed using a bottle roller mixer at 50° C. for 1 hour to obtain the coating formulation.

[0140] Film samples containing immobilized glass spheres were prepared by coating the coating formulation onto the release-coated side of polyester release film using a notch bar coater with an 8-mil gap setting. The coated samples were dried at 65°C for 20 minutes. Disks (2.54 cm diameter) were punched out of the samples, and the release film was removed. A single film disk was placed at the bottom of a vial, and 2 mL of room-temperature water was then added to the vial. Within 30 seconds, the film dissolved, and the functionalized glass spheres were observed to float to the top of the water.

[0141] Example 2: The same procedure as described in Example 1 was followed, except that a smaller amount of S60HS glass spheres (0.05 g) was added to a bottle containing 30 g of PVOH solution. The resulting film disc dissolved in water within 30 seconds, and the functionalized glass spheres were observed to float to the top of the water surface.

[0142] Example 3: The same procedure as described in Example 1 was followed, except that 0.3 g of XLD3000 glass spheres (density 0.23 g / mL, average diameter 30 micrometers) were added to a bottle containing 30 g of PVOH solution. The resulting film disc dissolved in water within 30 seconds, and the functionalized glass spheres were observed to float to the top water surface.

Claims

1. A composition comprising a film disposed on a substrate, The film, a first polymer matrix material; a plurality of first hollow glass spheres embedded in the first polymer matrix material; Including, The composition, wherein the first hollow glass spheres have a density less than 0.60 grams / mL, a span less than 1.0, and a plurality of first affinity groups covalently bonded to the first hollow glass spheres.

2. A composition according to claim 1, a plurality of second hollow glass spheres embedded in a first water soluble or water degradable polymer matrix material, the second hollow glass spheres having a density less than 0.60 grams / mL, a span less than 1.0, and a plurality of second affinity groups covalently bonded to the first hollow glass spheres, the second affinity groups being different from the first affinity groups.

3. The composition of claim 1 or 2, wherein at least a portion of the first affinity groups comprises a first binding antibody, and optionally, at least a portion of the second affinity groups comprises a binding antibody.

4. The composition of any one of claims 1 to 3, wherein at least a portion of the first affinity groups comprises a first binding target analyte molecule, and optionally, at least a portion of the second affinity groups comprises a second binding target analyte molecule.

5. The composition of any one of claims 1 to 4, wherein at least a portion of the first affinity groups comprises at least one group selected from the group consisting of adsorbents, zeolites and chelating agents.

6. at least two film layers, a first film layer comprising said first water-soluble or water-degradable polymer matrix material and a second film layer comprising a second water-soluble or water-degradable polymer matrix material; 6. The composition of any one of claims 1 to 5, optionally further comprising a plurality of third hollow glass spheres embedded in the second water-soluble or water-degradable polymer matrix material, the second hollow glass spheres having a density less than 0.60 grams / mL, a span less than 1.0, and a plurality of affinity groups covalently bonded to the first hollow glass spheres, the plurality of affinity groups being selected from a first affinity group, a second affinity group, or a combination thereof.

7. The composition of any one of claims 1 to 6, wherein the second water-soluble or water-degradable polymeric matrix material is disposed over at least a portion of the first water-soluble or water-degradable polymeric matrix material.

8. The composition of any one of claims 1 to 7, wherein the first polymeric matrix material comprises a sustained release polymer.

9. The composition of any one of claims 1 to 8, wherein the second water-soluble or water-degradable polymer matrix material comprises an immediate release polymer.

10. 10. The composition of claim 9, wherein the immediate release polymer comprises hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, polyvinylpyrrolidone-polyvinyl acetate copolymer, polyvinyl alcohol, copolymers of polyvinyl alcohol and polyvinyl acetate, copolymers of polyvinyl alcohol and polyethylene glycol, water soluble (meth)acrylic polymers, water soluble (meth)acrylic copolymers, water soluble copolymers of (meth)acrylates and polyethylene glycol, and polyethylene glycol.

11. 9. The composition of claim 8, wherein the sustained release polymer comprises poly(glycolic acid), poly(lactic acid), poly(lactic-co-glycolic acid), poly(caprolactone), poly(orthoester), poly(anhydride), poly(amide), poly(ester amide), poly(phosphoester), chitosan, hyaluronic acid, poly(alkyl cyanoacrylate), an enteric polymer, or an enzymatically degradable polymer.

12. The composition of any one of claims 1 to 11, further comprising a plurality of first detection compound molecules not covalently bound to said plurality of first hollow glass spheres, said first detection compound molecules comprising a first detectable group detectable at a first wavelength.

13. 13. The composition of claim 12, wherein at least a portion of the first detector compound molecules are unbound.

14. The composition of any one of claims 1 to 13, further comprising a plurality of second detection compound molecules not covalently bound to the plurality of first hollow glass spheres, the second detection compound molecules comprising a second detectable group that is detectable at a second wavelength.

15. 15. The composition of claim 14, wherein at least a portion of the second detector compound molecules are unbound.

16. The composition of any one of claims 1 to 15, wherein the substrate is a container, optionally selected from an Eppendorf tube, a PCR tube, a test tube, and a cuvette.

17. 1. A method for detecting an analyte, comprising: Providing a composition according to any one of claims 1 to 16; contacting a sample containing a first free target analyte molecule with the composition; contacting a liquid, optionally an aqueous liquid, with the composition; binding said first free target analyte molecule to said first affinity group or said first detection compound molecule; suspending the first plurality of hollow glass spheres in an upper concentrated portion of the liquid; measuring the amount of said first detectable compound molecules in a second portion of said liquid below said upper concentrated portion, optionally without removing said plurality of first hollow glass spheres from said liquid; A method comprising:

18. 20. The method of claim 17, wherein measuring the amount of the first detector compound molecule comprises measuring a luminescence signal at a first wavelength or measuring absorbance at a first wavelength.

19. binding a second target analyte molecule to said second affinity group or said second detection compound molecule; suspending the plurality of second hollow glass spheres in an upper concentrated portion of the liquid; measuring the amount of second detectable compound molecules in a second portion of said liquid below said upper concentrated portion, optionally without removing said plurality of second hollow glass spheres from said liquid; 19. The method of claim 17 or 18, further comprising:

20. 20. The method of any one of claims 17 to 19, further comprising releasing the first plurality of hollow glass spheres from the first water soluble or water degradable polymer matrix material, and optionally releasing the second plurality of hollow glass spheres from the second water soluble or water degradable polymer matrix material.

21. releasing a first portion of the hollow glass spheres from the first water soluble or water degradable polymeric matrix material within a first period of time, the first period of time beginning when the liquid contacts the composition and optionally ending when all of the first portion of the hollow glass spheres have been released; releasing a second portion of the hollow glass spheres from the first or second water-soluble or water-degradable polymer matrix material within a second period of time longer than the first period of time, the second period of time beginning when the liquid contacts the composition and optionally ending when all of the second portion of the hollow glass spheres have been released. Further comprising: the first portion of the hollow glass spheres is a portion of the first plurality of hollow glass spheres or all of the first plurality of hollow glass spheres; The second portion of the hollow glass spheres is a portion of the first plurality of hollow glass spheres, a portion of the second plurality of hollow glass spheres, or all of the second plurality of hollow glass spheres. The method according to any one of claims 17 to 20.

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