WO2025144637A1

WO2025144637A1 - Donor acceptor cyclodextrin composition and methods of using same

Info

Publication number: WO2025144637A1
Application number: PCT/US2024/060519
Authority: WO
Inventors: Iaroslav KHOMUTNYK; Khaledur S. Rashid; Barry A. Schoenfelner; Guoping Wang
Original assignee: Beckman Coulter Inc
Current assignee: Beckman Coulter Inc
Priority date: 2023-12-29
Filing date: 2024-12-17
Publication date: 2025-07-03
Anticipated expiration: 2026-06-29

Abstract

Chemiluminescent compositions and systems having at least one donor chemiluminescent molecule, at least one energy acceptor molecule, and at least one host molecule accommodating at least two guest molecules such as the donor chemiluminescent and energy acceptor molecules are described. The two guest molecules are structurally spaced for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule. Methods of using the described chemiluminescent compositions and systems are also provided.

Description

DONOR ACCEPTOR CYCLODEXTRIN COMPOSITION AND METHODS OF USING SAME BACKGROUND [0001] Chemiluminescence is the generation of electromagnetic radiation as light by the release of energy from a chemical reaction. Chemiluminescent reagents are used in applications including Western Blotting, Southern Blotting, ELISA, lateral flow assays, detection of hydrogen peroxide, and proximity based homogenous immunoassays. BRIEF SUMMARY [0002] This disclosure describes compositions, systems, and methods of light emission enhancement from intermolecular energy transfer by chemiluminescent compounds. Donor chemiluminescent molecules and energy acceptor molecules can be complexed into a host molecule configured for resonance energy transfer between guest molecules. Supramolecular complexation of both triggerable donor chemiluminescent molecules and energy acceptor molecules may provide for control of the timing of light release during a chemiluminescent reaction; amplification of the chemiluminescent signal; and optimization of the reagents for specific applications. [0003] In at least one aspect, the present technology provides a composition or system that includes at least one donor chemiluminescent molecule, at least one energy acceptor molecule, and at least one host molecule accommodating the at least two guest molecules. A first guest molecule includes, for example, the donor chemiluminescent molecule. A second guest molecule includes, for example, the energy acceptor molecule. When accommodated in the host molecule, the at least two guest molecules are structurally spaced for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule. [0004] In some aspects, the donor chemiluminescent molecule and/or the energy acceptor molecule include(s) at least a portion that has a suitable size and affinity for an enclosing portion of the host molecule. [0005] In other aspects, the host molecule and the at least two guest molecules form a combined inclusion complex. In a first inclusion of the combined inclusion complex, the host molecule 1 spatially encloses at least a portion of the donor chemiluminescent molecule. In a second inclusion of the combined inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule. [0006] In a further aspect, the composition does not include a micelle. Additionally or alternatively, the composition does not require suspension in solution to produce an enhanced signal. [0007] In another aspect, the host molecules include a dimeric cyclodextrin, a multimeric cyclodextrin, a polymeric cyclodextrin molecule, and/or a combination thereof. [0008] In some aspects, the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule is 12 angstroms to 13.5 angstroms; alternatively, from 12 angstroms to 15 angstroms; alternatively, from 12 angstroms to 16.5 angstroms; alternatively, from 12 angstroms to 18 angstroms; alternatively, from 12 angstroms to 19.5 angstroms; alternatively, from 12 angstroms to 21 angstroms; alternatively, from 12 angstroms to 22.5 angstroms; alternatively, from 12 angstroms to 24 angstroms; alternatively, from 12 angstroms to 25.5 angstroms; and/or, alternatively, from 12 angstroms to 50 angstroms. [0009] In a still further aspect, the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule produces a chemiluminescent signal inversely proportional to the sixth power of the distance between the chemiluminescent molecule and the energy acceptor molecule within the host molecule. [0010] In another aspect, the host molecule includes: 2

wherein R comprises: OH, -O-Alk (alkyl-containing), -O-PEG (PEG containing), -O(CH2)x-PR’3 (alkylphosphonium containing), -O(CH2)x-NR’3 (alkylammonium containing), -O(CH2)x- (aryl)-CH2-NR’3 (alkylammonium containing), or -O(CH2)x-(aryl)-CH2-NR’3 (alkylammonium containing), or a combination thereof; and n is an integer of at least 2. Additionally, in a further aspect, including, for example, where the host molecule has the structure provided above, the average molecular weight of an exemplary linear polymeric host molecule includes a range of n (where n is the number of subunits of the cyclodextrin) multiplied by one-thousand one hundred Daltons to n multiplied by one-thousand five hundred Daltons (that is, n*1100 to n*1500 Da). [0011] In an aspect, the donor chemiluminescent molecule includes a triggerable dioxetane. In some aspects, the triggerable dioxetane includes a removable protecting group trigger, a dioxetane moiety, and an adamantyl group. Moreover, in a still further aspect, the triggerable dioxetane includes a triggerable phenoxy-dioxetane wherein a trigger comprises any moiety that forms a phenolate upon change in an environmental condition or the presence of a specific analyte or an enzyme. In some exemplary aspects, the trigger includes -OH, -OP(O)(OR), -OAcyl, -B(OH)₂, aryl-boronate esters, beta (β)-D-galactoside, -OSiMe2tBu, -OSiR3, or –OR, wherein R comprises a group removable by a selectable analyte or by a selectable enzyme. In a still further aspect, the triggerable phenoxy-dioxetane has an electron-withdrawing or electron-accepting substituent at the ortho position of the phenoxy-dioxetane. [0012] In certain aspects, the triggerable phenoxy-dioxetane includes 3-(4'- methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)phenyl dihydrogen phosphate (PPD), 5-(4'- 3 methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD), 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD), or a combination thereof. [0013] In some aspects, the energy acceptor molecule includes a fluorescent molecule, an anchor, and/or optionally a linker. [0014] In a further aspect, the fluorescent molecule includes a fluorescent molecule with an excitation frequency in a range of 300 nm to 750 nm; in a range of 350 nm to 700 nm; in a range of 400 nm to 650 nm; in a range of 450 nm to 600 nm; or in a range of 500 nm to 550 nm. [0015] In another aspect, the anchor comprises a group that has a strong binding affinity with a host molecule, suitable for the formation of an inclusion complex comprising the host molecule and the energy acceptor molecule. Exemplary anchors include long chain n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl, and/or adamantyl group. [0016] In a further aspect, the linker connects the anchor and the fluorescent molecule. Additionally, in some aspects, the linker includes a long chain alkane, a long chain alkyl ether, a long chain alkyl ester, an anhydride, an isocyanate, a carbodiimide, or a glutaraldehyde, or a combination thereof. [0017] In another aspect, the donor chemiluminescent molecule is not covalently bound to the host molecule. In a further aspect, the energy acceptor molecule is not covalently bound to the host molecule. [0018] In some aspects, the composition of the presently described technology is water soluble. [0019] In another aspect, the composition of the presently described technology produces a detectable signal including, for example, when triggered. [0020] For example, the detectable signal may have a wavelength in a range of 300 nm to 850 nm; alternatively, in a range of 350 nm to 700 nm; alternatively, in a range of 400 nm to 650 nm; alternatively, in a range of 450 nm to 600 nm; or, alternatively, in a range of 500 nm to 550 nm. [0021] In a further aspect, the active intermediate of a reaction between the donor chemiluminescent molecule and the energy acceptor molecule has an extended half-life compared to the half-life of the intermediate without complexation with the host molecule. 4 [0022] In other aspects, the composition produces a detectable signal when the temperature of the composition falls within a range of 20 degrees Celsius to 80 degrees Celsius; within a range of 20 degrees Celsius to 70 degrees Celsius; within a range of 20 degrees Celsius to 60 degrees Celsius; within a range of 20 degrees Celsius to 50 degrees Celsius; within a range of 25 degrees Celsius to 40 degrees Celsius; or within a range of 35 degrees Celsius to 40 degrees Celsius. Additionally or alternatively, the composition may produce a detectable signal when the pressure is in a range of 0.8 arm to 1.2 atm; in a range of 0.9 atm to 1.1 atm; or is 1 atm. [0023] In further aspects, the composition produces the detectable signal when the pH of the composition is in a range of 2.0 to 13.0; in a range of 3.0 to 12.0; in a range of 4.0 to 11.0; in a range of 5.0 to 10.0; in a range of 6.0 to 9.0; or in a range of 6.0 to 8.0. [0024] In still further aspects, the composition produces a detectable signal when deposited on solid media and triggered. In further aspects, the composition exhibits no relative attenuation of chemiluminescence when adsorbed on a filter media (versus in solution) and triggered. [0025] This disclosure further provides methods of using the compositions and systems described herein. For example, this disclosure provides a method of using the compositions or systems described herein in an immunoassay, in a lateral flow assay, or in a Western blot assay. [0026] This disclosure further describes systems that use the compositions described herein. [0027] In still further aspects, disclosed herein are methods of enhancing transient chemiluminescence that include providing a host molecule, complexing a donor chemiluminescent molecule to the host molecule, and complexing an energy acceptor molecule to the host molecule. In some aspects, the method includes configuring the distance between the inclusion complexes formed to produce a triggerable, detectable signal. [0028] In a further aspect, the host molecule includes a dimeric, multimeric, or polymeric cyclodextrin molecule. [0029] In a still further aspect, at least a portion of the donor chemiluminescent molecule or at least a portion of the energy acceptor molecule or both have a suitable size and a strong affinity for an enclosing portion of the host molecule. [0030] In a further aspect, the host molecule and the at least two guest molecules form a combined inclusion complex; wherein the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule; and, wherein the host molecule spatially encloses at least a portion 5 of the energy acceptor molecule. In a still further aspect, the method does not include forming a micelle or maintaining micelle kinetic stability. [0031] In another aspect, the method does not include forming a covalent bond between the donor chemiluminescent molecule and a cyclodextrin molecule; nor, forming a covalent bond between the energy acceptor molecule and a cyclodextrin molecule. [0032] In a further aspect, the method also includes extending the half-life of an active intermediate of a reaction between the donor chemiluminescent molecule and the energy acceptor molecule. In a still further aspect, the method also includes configuring a distance between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule so that the reaction between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule produce a chemiluminescent signal that is inversely proportional to the sixth power of the distance between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule. [0033] In another aspect, the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule is 12 angstroms to 13.5 angstroms; alternatively, from 12 angstroms to 15 angstroms; alternatively, from 12 angstroms to 16.5 angstroms; alternatively, from 12 angstroms to 18 angstroms; alternatively, from 12 angstroms to 19.5 angstroms; alternatively, from 12 angstroms to 21 angstroms; alternatively, from 12 angstroms to 22.5 angstroms; alternatively, from 12 angstroms to 24 angstroms; alternatively, from 12 angstroms to 25.5 angstroms; and/or, alternatively, from 12 angstroms to 50 angstroms. [0034] In other aspects and embodiments of the presently technology, there is disclosed herein a diagnostic test system including a test strip comprising one or more test regions and one or more control regions, wherein the one or more test regions include a donor chemiluminescent molecule, an acceptor comprising a fluorescent molecule, and a host molecule configured to form a combined inclusion complex with the donor chemiluminescent molecule and the energy acceptor molecule and configured to be structurally spaced for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule. [0035] In a further aspect, the test strip supports lateral flow of a fluid along a lateral flow direction, and wherein at least one test region includes an area that is exposed for optical inspection and is characterized by a first dimension transverse to the lateral flow direction and a second dimension parallel to the lateral flow direction. 6 [0036] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative 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 DESCRIPTION OF THE DRAWINGS [0037] FIG.1 illustrates a perspective view of an exemplary cartoon schema of an example method of enhancing transient chemiluminescence. [0038] FIG.2 illustrates a perspective view of an exemplary cartoon embodiment of a triggerable donor chemiluminescent molecule. [0039] FIG. 3 illustrates a perspective view of an exemplary cartoon embodiment of an energy acceptor molecule. [0040] FIG.4 shows an annotated chemical formula of an exemplary dimeric β-cyclodextrin. [0041] FIG.5 shows light intensity (in relative light units (RLUs) as a function of time (in minutes) for PPD alone or PPD combined with different concentrations of cyclodextrins, as further described in Example 1. [0042] FIG.6a shows chemiluminescent spectral analysis of PPD combined with fluorescein (Fls + PPD); PPD and fluorescein complexed in a host polymeric cyclodextrin (bCDpol+PPD+Fls); and LUMI-PHOS 530, as further described in Example 3. [0043] FIG.6b shows spectral analysis of PPD chemiluminescence alone, as further described in Example 3. [0044] FIG. 7 shows the light intensity of LUMI-PHOS 530 (blue) or VPPD and polymeric β- cyclodextrin (bCDpol) (red) adsorbed on filtration paper, after activation with Alkaline Phosphatase conjugate (AP-conjugate), as further described in Example 5. The lower panels show an image view of the autoexposure setting of LUMI-PHOS 530 on filtration paper after activation (left), and an image view of autoexposure setting of VPPD and polymeric β-cyclodextrin on filtration paper after activation (right), as further described in Example 5. 7 [0045] FIG. 8 shows the light intensity of LUMI-PHOS 530 (blue) or VPPD and polymeric β- cyclodextrin (bCDpol) (red) adsorbed on nitrocellulose membrane, after activation with Alkaline Phosphatase conjugate (AP-conjugate), as further described in Example 6. The lower panels show an image view of the autoexposure setting of LUMI-PHOS 530 on nitrocellulose membrane after activation (left), and an image view of the autoexposure setting of VPPD and polymeric β- cyclodextrin on nitrocellulose membrane after activation (right), as further described in Example 6. [0046] FIG. 9 shows the light intensity of LUMI-PHOS 530 (blue) or VPPD and polymeric β- cyclodextrin (bCDpol) (red) adsorbed on PVDF membrane, after activation with Alkaline Phosphatase conjugate (AP-conjugate), as further described in Example 7. The lower panels show an image view of the autoexposure setting of LUMI-PHOS 530 on PVDF membrane after activation (left), and an image view of the autoexposure setting of VPPD and polymeric β-cyclodextrin on PVDF membrane after activation (right), as further described in Example 7. [0047] FIG.10 illustrates a perspective view of an example lateral flow assay. [0048] FIG.11 provides an exemplary lateral flow assay incorporating an example immunoassay as further described in Example 9. DETAILED DESCRIPTION [0049] This disclosure relates to compositions, systems, and methods of light emission _{enhancement from intermolecular energy transfer in chemiluminescence compounds. Donor} chemiluminescent molecules and energy acceptor molecules are complexed into a host molecule, creating a composition configured for resonance energy transfer between guest molecules. Described herein are compositions, systems, and methods that provide for or include enhancement of chemiluminescence by supramolecular complexation of both triggerable donor chemiluminescent molecules and energy acceptor molecules with host molecules. [0050] In one embodiment, this disclosure describes a composition including a donor chemiluminescent molecule, an energy acceptor molecule, and a host molecule. The host molecule accommodates at least two guest molecules. A first guest molecule includes the donor chemiluminescent molecule. A second guest molecule includes the energy acceptor molecule. When accommodated in the host molecule, the at least two guest molecules are structurally spaced 8 for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule. [0051] In some aspects, the energy transfer is chemiluminescence resonance energy transfer (CRET), a non-radiative transfer of energy from the donor chemiluminescent molecule to energy acceptor molecule during a chemiluminescence reaction. Similar to Förster Resonance Energy Transfer (FRET), the chemiluminescent donor molecule undergoes a chemical reaction, emitting light. The chemiluminescent donor molecule serves as the source of the initial energy and the energy acceptor molecule absorbs the energy emitted by the chemiluminescent donor molecule. In some aspects, the probability of an energy-transfer event occurring per donor excitation event may depend on physical parameters such as: (1) the distance between the donor molecule and the energy acceptor; (2) the spectral overlap of the donor molecule’s emission spectrum and the energy acceptor’s absorption spectrum; and, (3) the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. (King, C., Barbiellini, B., Moser, D., & Renugopalakrishnan, V. (2012). Exactly soluble model of resonant energy transfer between molecules. Physical Review B, 85(12), 125106.) [0052] Spectral overlap refers to the degree to which the emission spectrum of one molecular entity overlaps with the absorption spectrum of another molecular entity (such as the donor chemiluminescent molecule and energy acceptor molecule). In CRET, spectral overlap influences the efficiency of energy transfer between the two molecules. When there is a significant overlap between the emission spectrum of the donor molecule and the absorption spectrum of the energy acceptor (or high spectral overlap), the probability of energy transfer is higher. In theory, a larger spectral overlap increases the likelihood that the energy acceptor molecule will absorb the emitted energy from the donor chemiluminescent molecule. If there is less spectral overlap, the efficiency of energy transfer decreases. [0053] The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment refer to the direction and magnitude of the polarity associated with the emission of energy and the direction and magnitude of the dipole associated with the absorption of energy. The efficiency of energy transfer between the donor and acceptor depends on how well aligned these two dipole moments are. The more aligned the dipole moments, the greater the probability of an energy transfer and light emission. 9 [0054] In some aspects, the enhancement of light emission also depends on concentrations of the donor chemiluminescent molecules, energy acceptor molecules, and host molecule (e.g., a polymeric beta cyclodextrin); the number and variety of guest molecules complexed within the host molecule (which will, in turn, be influenced by polymeric chain length); the composition and concentration of a buffer in which the molecules are situated, the presence and concentration of a triggering enzyme or analyte; and/or environmental conditions such as temperature, atmospheric pressure, and pH. As discussed further below, the compositions and methods described herein may further include optimizing these additional variables. [0055] FIG. 1 shows an exemplary host molecule complexing with donor chemiluminescent molecules and energy acceptor molecules. After complex formation, the host molecule accommodates the at least two guest molecules, and the guest molecules (the donor chemiluminescent molecule and the energy acceptor molecule) are structurally spaced in the complex for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule. Such resonance energy transfer may be triggered or enhanced by the presence of an enzyme or substrate. [0056] In some embodiments, at least a portion of the donor chemiluminescent molecule has a suitable size and affinity for an enclosing portion of the host molecule. Additionally or alternatively, at least a portion of the energy acceptor molecule has a suitable size and affinity for an enclosing portion of the host molecule. In some embodiments, the host molecule and the at least two guest molecules form a combined inclusion complex. In the combined inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule and at least a portion of the energy acceptor molecule. [0057] The host molecule includes a non-monomeric cyclodextrin, that is, a dimeric cyclodextrin, a multimeric cyclodextrin, or a polymeric cyclodextrin, or a combination thereof. Cyclodextrins are cyclic oligosaccharides that include a macrocyclic ring of glucose subunits joined by α-1,4 glycosidic bonds. Cyclodextrins include five or more linked α-D-glucopyranoside units: α- cyclodextrins include 6 glucose subunits, β-cyclodextrins include 7 glucose subunits, and γ- cyclodextrins include 8 glucose subunits. Typical cyclodextrins may include glucose monomers in a ring arrangement, creating a cone shape. 10 [0058] In some aspects, the cyclodextrin preferably includes a β-cyclodextrin. Without wishing to be bound by theory, it is believed that because the cavity size of the β-cyclodextrin ring is sterically suited to accommodate the adamantyl group of the donor chemiluminescent molecule, the β- cyclodextrin may provide improved results over cyclodextrins with more or fewer glucose molecules (which would provide larger or smaller cyclodextrin rings). In some aspects, a cyclodextrin may include alternating cyclodextrin units or blocks of different cyclodextrin units. (Miyauchi, Masahiko, and Akira Harada. "Construction of supramolecular polymers with alternating α-, β-cyclodextrin units using conformational change induced by competitive guests." Journal of the American Chemical Society 126.37 (2004): 11418-11419). In some aspects, the host molecule may be a linear polymeric cyclodextrin. Additionally or alternatively, the host molecule may include branched, topographical (2D), or stereographical (3D) cyclodextrin architectures. [0059] In some aspects, the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule can be 12 angstroms to 13.5 angstroms; from 12 angstroms to 15 angstroms; from 12 angstroms to 16.5 angstroms; from 12 angstroms to 18 angstroms; from 12 angstroms to 19.5 angstroms; from 12 angstroms to 21 angstroms; from 12 angstroms to 22.5 angstroms; from 12 angstroms to 24 angstroms; from 12 angstroms to 25.5 angstroms; and/or from 12 angstroms to 50 angstroms. An exemplary dimeric β-cyclodextrin is shown in FIG. 4. The cyclodextrins are linked together such that the distance between the midline of the two cyclodextrins is at least 12 angstroms and up to 25 angstroms. In general, the distance between triggerable donor chemiluminescent molecules and energy acceptor molecules is preferably maintained by the host molecule at a distance to optimize donor/acceptor interactions. [0060] In an aspect, the distance between triggerable donor chemiluminescent molecules and energy acceptor molecules when accommodated in the host molecule generally produces a chemiluminescent signal inversely proportional to the sixth power of the distance between the chemiluminescent molecule and the energy acceptor molecule within the host molecule. [0061] In some aspects, the number of covalently linked cyclodextrins may be varied for desired effects. For example, in some aspects, dimeric cyclodextrins may be preferred. In alternate aspects, the cyclodextrin may include at least 3, at least 10, at least 50, at least 100, at least 500, at least 11 1000, or at least 1500 covalently linked cyclodextrins. The host cyclodextrin may accommodate differing amounts of donor chemiluminescent molecules and energy acceptor molecules. The amount and distribution of donor chemiluminescent molecules and energy acceptor molecules affects the amount of light emitted. [0062] In some embodiments, the average molecular weight of the dimeric, multimeric, or polymeric cyclodextrin can be in a range of 2,000 Da to 3,000 Da; alternatively, in a range of 2,000 Da to 4,500 Da; or, alternatively, in a range of 2,000 Da to 6,000 Da. In alternative embodiments, multimeric or polymeric cyclodextrin can be in a range of 3,000 Da to 6,000 Da; alternatively, in a range of 3,000 Da to 12,000 Da; alternatively, in a range of 3,000 Da to 18,000 Da; alternatively, in a range of 3,000 Da to 24,000 Da; alternatively, in a range of 3,000 Da to 30,000 Da; alternatively, in a range of 3,000 Da to 36,000 Da; alternatively, in a range of 3,000 Da to 42,000 Da; alternatively, in a range of 3,000 Da to 48,000 Da; alternatively, in a range of 3,000 Da to 54,000 Da; alternatively, in a range of 3,000 Da to 60,000 Da; alternatively, in a range of 3,000 Da to 72,000 Da; alternatively, in a range of 3,000 Da to 84,000 Da; alternatively, in a range of 3,000 Da to 96,000 Da; alternatively, in a range of 3,000 Da to 108,000 Da; or, alternatively, in a range of 3,000 Da to 120,000 Da. [0063] In some aspects, polymeric cyclodextrin may be cross-linked to form nanostructures or hydrogel networks. In some aspects, if cyclodextrin is cross-linked, the concentration of cyclodextrin may be varied for desired effects rather than a set number. The concentration may depend on the solubility and type of cyclodextrin. For example, in alternate aspects, the concentration of polymeric β-cyclodextrin in water may include between 0.01 mg/mL to 0.333 mg/mL (at 25°C); alternatively between 0.05 mg/mL to 0.333 mg/mL (at 25°C); alternatively between 0.10 mg/mL to 0.333 mg/mL (at 25°C); alternatively between 0.15 mg/mL to 0.333 mg/mL (at 25°C); alternatively between 0.20 mg/mL to 0.333 mg/mL (at 25°C); alternatively between 0.25 mg/mL to 0.333 mg/mL (at 25°C); and, alternatively between 0.3 mg/mL to 0.333 mg/mL (at 25°C). In another example with alternate aspects, a derivative of beta cyclodextrin, hydroxypropyl-β-cyclodextrin, may include concentrations between 2 mg/mL to 11 mg/mL; alternatively between 4 mg/mL to 11 mg/mL; alternatively between 6 mg/mL to 11 mg/mL; alternatively between 8 mg/mL to 11 mg/mL; and, alternatively between 10 mg/mL to 11 mg/mL (at 25°C). 12 [0064] In an exemplary aspect, the host molecule of the composition may have a chemical formula:

[0065] wherein R comprises: OH, -O-Alk (alkyl-containing), -O-PEG (PEG containing), - O(CH2)x-PR’3 (alkylphosphonium containing), -O(CH₂)x-NR’3 (alkylammonium containing), - O(CH2)x-(aryl)-CH2-NR’3 (alkylammonium containing), or -O(CH2)x-(aryl)-CH2-NR’3 (alkylammonium containing), or a combination thereof; and n is an integer of at least 2. Additionally, in a further aspect, including, for example, where the host molecule has the structure provided above, the average molecular weight of an exemplary linear polymeric host molecule includes a range of n (where n is the number of subunits of the cyclodextrin) multiplied by one- thousand one hundred Daltons to n multiplied by one-thousand five hundred Daltons (that is, n*1100 to n*1500 Da). [0066] Typically, the cyclodextrins have a hydrophobic core. The hydrophobic core of cyclodextrins refers to the inner cavity or central region of these cyclic oligosaccharides. The interior of the cyclodextrin molecule is shielded from water molecules in part because the hydrophilic hydroxyl groups of the glucose units on the outer surface of the ring-like structure 13 attracts surrounding water molecules to the exterior. The protected core of cyclodextrin is believed to protect complexed reactants from the water that quenches the chemiluminescent reaction. [0067] At the same time, the environment of the interior can be manipulated by altering the functional groups that face the interior. The inward facing functional groups of cyclodextrins affect the half-life of complexed reactants. The half-life of a reaction refers to the time it takes for half of the initial amount of the reactants to be consumed or transformed in a chemical reaction. The stability of reaction intermediates would affect the time a reaction takes to reach completion or the probability of a reaction taking place. For the chemiluminescent reactions discussed below, in general a more protic environment tends to shorten the half-life of the reactions by destabilizing the reaction intermediates. By a more protic environment in the interior of the cyclodextrin, functional groups would be more able to donate a proton (H+) in a chemical reaction. In an aspect, the internal groups can be replaced to change the characteristics of the reaction between triggerable donor chemiluminescent molecules and energy acceptor molecules to fit desired properties or reaction parameters. [0068] As the size of the host molecule may vary, the number and configuration of donor chemiluminescent molecules and energy acceptor molecules that form inclusion complexes within a particular host molecule will also vary. For a given number and length of linked cyclodextrin, the concentrations of host molecules, triggerable donor chemiluminescent molecules, and energy acceptor molecules can be manipulated to produce an optimal signal. In Example 1, the concentration of polymeric cyclodextrin, the host molecule, is variable and the optimal concentration of polymeric cyclodextrin can be found by choosing the concentration that fits the desired properties or characteristics for a reaction. The example shows different levels of quantum yield over time and the concentration of host molecules can be selected to provide the desired properties or characteristics. Thereby, the host molecule size and concentration can be manipulated for statistically enhancing chemiluminescence in the manner that is desired. [0069] Any suitable donor chemiluminescent molecule may be used. Generally, the donor chemiluminescent molecule is an excited fluorescent molecule capable of transferring energy non- radioactively through intermolecular long-range dipole–dipole coupling to an energy acceptor molecule, producing chemiluminescence. In some aspects, at least a portion of the donor chemiluminescent molecule has a suitable size and affinity for an enclosing portion of the host 14 molecule. In addition, or the alternative, the donor chemiluminescent molecule may be complexed to a molecule that has a suitable size and affinity for an enclosing portion of the host molecule. A suitable size and affinity can refer to a size that is appropriate or fitting for a particular purpose, context, and can vary depending on the specific requirements or expectations of a situation. For example, a suitable affinity could be a portion of donor chemiluminescent molecule with strong affinity for the interior of the host molecule. Additionally or alternatively, a suitable affinity could mean that a portion of donor chemiluminescent molecule has less binding affinity if the desired effect on the chemiluminescent reaction can be realized (such as an extended half-life of a reactant). A suitable size and affinity might be one that meets the functional or spatial requirements for a given task or space. [0070] An exemplary cartoon donor chemiluminescent molecule is shown in FIG.2. As shown in FIG.2, a donor chemiluminescent molecule 200 can include a removable protecting group 210, a an excited fluorescent molecule 220, and a group with a suitable size and affinity for an enclosing portion of the host molecule 230 (for example, an adamantyl group). Exemplary donor chemiluminescent molecules include, but are not limited to, triggerable dioxetanes, triggerable phenoxy-dioxetanes, and/or a combination thereof. [0071] An exemplary donor chemiluminescent molecule includes a triggerable dioxetane. Triggerable dioxetanes are well known in the art. (See, for example, A. P. Schaap, T.-S. Chen, R. S. Handley, R. DeSilva and B. P. Giri, Tetrahedron Lett., 1987, 28, 1155–1158; A. P. Schaap, R. S. Handley and B. P. Giri, Tetrahedron Lett., 1987, 28, 935–938; A. P. Schaap, M. D. Sandison and R. S. Handley, Tetrahedron Lett., 1987, 28, 1159–1162). Typically, a removable protecting group can be selectively removed. A change in environmental conditions or the presence of an enzyme or an analyte of interest ‘triggers’ removal of the protecting group exposing the chemiluminescent molecule, such as dioxetane. Once the removable protecting group is removed, the dioxetane reacts to produce light. [0072] Potential changes in environmental conditions that can trigger a triggerable dioxetane or could affect triggering may include, but are not limited to, varying the pH, the temperature, the amount and content of solvent or reaction medium the concentration of reactants, the presence of catalysts or inhibitors, oxygen concentration (for example, peroxides), the concentration of ions in solution or the reaction mixture, and/or the presence of contaminants. 15 [0073] In some aspects, the addition or presence of an analyte or enzyme may preferably be used to remove a protecting group trigger. In some embodiments, the triggerable dioxetane includes a triggerable group that includes a moiety that forms a phenolate upon change in an environmental condition, or the presence of a specific analyte or an enzyme. For example, chemiluminescence can be triggered by alkaline phosphatase if the removable protecting group trigger is a phosphate group and alkaline phosphatase is added to the reaction solution in sufficient quantities. Upon removal of phosphate group by alkaline phosphatase, phenolate formation initiates chemiexcitation and chemiluminescence following deprotection of a phenoxy group. In another example, β-galactosidase could be added to a solution with the compound including a beta-D- galactoside removable protecting group trigger. The β-galactosidase catalyses the hydrolysis of the β-glycosidic bond releasing the beta-D-galactoside. The hydrolysis exposes the phenoxy group and the formation of the phenolate dioxetane initiates chemiexcitation and light emission. [0074] Exemplary triggers include, but are not limited to, an -OH, -OP(O)(OR), -OAcyl, -B(OH)₂, -aryl-boronate esters, -beta-D-galactoside, -OSiMe2tBu, -OSiR3, or –OR, wherein R comprises a group removable by a selectable analyte or by a selectable enzyme. [0075] In an embodiment, different substituents could be introduced to different positions of the phenol ring to modulate the properties of the dioxetanes (stability, the quantum efficiency of the chemiluminescence, the wavelength of emitted light, water solubility, etc.). In some aspects, the triggerable phenoxy-dioxetane can have an electron-withdrawing or electron accepting substituent at the ortho position of the phenoxy-dioxetane if found to have a chemiluminescent enhancing effect. Various desirable or selectable properties and characteristics have been studied. (Hananya N, Shabat D. Recent Advances and Challenges in Luminescent Imaging: Bright Outlook for Chemiluminescence of Dioxetanes in Water. ACS Cent Sci. 2019 Jun 26;5(6):949-959. doi: 10.1021/acscentsci.9b00372. Epub 2019 May 29. PMID: 31263754; PMCID: PMC6598152.) [0076] In some embodiments, the triggerable phenoxy-dioxetane includes 3-(4'- methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)phenyl dihydrogen phosphate (PPD): 16 (PPD); 5-(4'-methoxyspiro[adamantane , , yl)-2-vinylphenyl dihydrogen phosphate (VPPD): or 5-(4'-methoxyspiro

2-vinylphenyl dihydrogen phosphate (VMPD): (VMPD);

or a combination thereof. As noted above, a donor chemiluminescent molecule includes a group with a suitable size and affinity for an enclosing portion of the host molecule including, for example, an adamantyl group. When included in a donor chemiluminescent molecule, the adamantyl group preferably has a strong binding affinity for cyclodextrin, including an ability to form highly stable complexes with cyclodextrin. In certain embodiments, the presence of the adamantyl group increases the likelihood 17 that a triggerable donor chemiluminescent molecule forms an inclusion complex with a cyclodextrin host molecule in solution. [0077] Any suitable energy acceptor molecule may be used. FIG.3 illustrates a perspective view of an exemplary energy acceptor molecule 300. Generally, the energy acceptor molecule 300 includes an energy transfer accepting fluorescent molecule. In some aspects, as shown in an exemplary embodiment in FIG. 3, the energy acceptor molecule 300 includes a fluorescent molecule 320, an anchor 340, and/or a linker 330. [0078] The energy acceptor molecule may include any suitable fluorescent molecule. In some aspects, the fluorescent molecule includes a fluorescent molecule with an excitation frequency in a range of 300 nm to 750 nm including, for example, a fluorescent molecule with an excitation frequency in a range of 300 nm to 750 nm; in a range of 350 nm to 700 nm; in a range of 400 nm to 650 nm; in a range of 450 nm to 600 nm; or in a range of 500 nm to 550 nm. As mentioned above, the spectral overlap of the donor molecule’s emission spectrum and the energy acceptor’s absorption spectrum influences the efficiency of energy transfer between the two molecules. As further explained in Example 3, FIG.6b shows PPD’s spectral emission wavelength distribution (which overlaps with the energy acceptor’s absorption spectrum). When combined with an appropriate energy acceptor molecule, acceptor emission is observed, as shown in FIG.6a. Some exemplary acceptor fluorescent molecules include, but are not limited to, fluorescent dyes including, for example, fluorescein rhodamine, coumarin, cyanine, luciferins, methylene blue, Congo red, and derivatives thereof. For example, exemplary derivatives of fluorescein include fluorescein isothiocyanate and carboxyfluorescein. It should be understood by a person of skill in the art that any appropriate fluorescent dye capable of accepting energy from decomposition of an unstable oxide intermediate to provide the emission of measurable light may be used. [0079] In an aspect, the energy acceptor molecule can innately form an inclusion complex with the host molecule. Additionally or alternatively, the energy acceptor molecule may be modified to increase its ability to form an inclusion complex with the host molecule. For example, the acceptor fluorescent molecule may be coupled to an anchor, optionally via a linker. When present, the anchor includes a group that has a strong binding affinity with a host molecule, suitable for the formation of an inclusion complex. That is, the anchor may help a guest molecule (that is, the energy acceptor molecule) “insert itself” into a host molecule. For example, when cyclodextrin is the host molecule, the anchor may include a group that forms strong complexes with cyclodextrin. 18 Exemplary groups that are known to form strong complexes with cyclodextrins, include but are not limited to, n-alkyl groups, cyclohexyl groups, biphenyl groups, naphthyl groups, tert-butyl groups, and adamantyl groups. In an aspect, the n-alkyl groups including chains of between 5 to 20 linked carbons, or alternatively 6 to 15 linked carbons. [0080] When the energy acceptor molecule includes an anchor, the energy acceptor molecule may further include a linker that connects the anchor and the fluorescent molecule. When present, as shown in an exemplary embodiment in FIG. 3, the linker 330 may include any suitable group that connects the anchor 340 and the acceptor fluorescent molecule 320. In addition, the linker may have a strong binding affinity with a host molecule. Exemplary linkers include but are not limited to a long chain alkyl ether, a long chain alkyl ester, an anhydride, an isocyanate, a carbodiimide, or a glutaraldehyde, or a combination thereof., A linker could further include any suitable bioconjugate that can connect an energy acceptor molecule to an anchor. In some aspects, long chain alkanes, long chain alkyl esters, long chain alkyl ethers can include chains of between 5 to 20 linked carbons and/or oxygens in the case of ethers or esters; or, alternatively, 6 to 15 linked carbons and/or oxygens in the case of ethers or esters. [0081] Some exemplary energy acceptor molecules and their formulas include, but are not limited to:

a the host molecule to form an inclusion complex and at least a portion of the energy acceptor molecule inserts into the host molecule to form an inclusion complex, forming a complexed host molecule. 19 An inserted portion of the energy acceptor molecule may be the anchor, the linker, and/or the acceptor fluorescent molecule. Additionally or alternatively, the whole energy acceptor molecule may be inserted into the host molecule. An inserted portion of the donor chemiluminescent molecules may be the triggerable donor chemiluminescent molecule and/or an adamantyl group. Additionally or alternatively, the whole donor chemiluminescent molecule may be inserted into the host molecule. The number and placement of triggerable donor chemiluminescent molecules and energy acceptor molecules that form complexes with a host molecule can vary. [0083] In some aspects, the donor chemiluminescent molecule in the composition is not covalently bound to the host molecule. Additionally or alternatively, the energy acceptor molecule in the composition is not covalently bound to the host molecule. Previous work described compositions that included energy acceptor molecules covalently bonded to trimethylated-β-cyclodextrins which were further combined with adamantyl-1,2-dioxetane probes in sufficient concentrations to drive formation of an at least a 1:1 host–guest complex. (Gnaim et al. "Light emission enhancement by supramolecular complexation of chemiluminescence probes designed for bioimaging." Chemical Science (2019) 10(10):2945-2955.) Although, the reduced distance between the tethered fluorophore and complexed probes was reported to enhance chemiluminescent reporting, making this composition requires synthesis of a covalently-attached fluorogenic dye trimethylated-b- cyclodextrin, a process that is too expensive to be commercially viable for most applications. [0084] The compositions described herein include the donor chemiluminescent molecule and the energy acceptor molecule accommodated in the host molecule. Upon selective removal of the protecting group from the donor chemiluminescent molecule by a change in environmental conditions and/or the addition of an enzyme or analyte of interest, the donor chemiluminescent molecule is triggered, initiating chemiluminescence. Energy is transferred from the donor chemiluminescent molecule to the energy acceptor molecule. The host molecule accommodates sufficient numbers of the donor chemiluminescent molecules and the energy acceptor molecules at a distance to produce a signal. [0085] The signal is preferably detectable including, for example, by a human eye or by a sensor, detector, or other device. Detecting chemiluminescent light may include the use of equipment. Such equipment may include, but are not limited to, photomultiplier tubes, photodiodes, Charge- Coupled Device (CCD) cameras, luminometers, scintillation counters, microplate readers, 20 spectrometers, and generally imaging equipment that is capable of detecting light. Detection may even be aided by the use of a darkroom, a light excluding space (for example, a light-excluding box within an immunoassay instrument), or photo-sensitive paper. [0086] In an aspect, the detectable signal produced by the composition may have a peak wavelength in a range of 300 nm to 850 nm; alternatively, in a range of 350 nm to 700 nm; alternatively, in a range of 400 nm to 650 nm; alternatively, in a range of 450 nm to 600 nm; or, alternatively, in a range of 500 nm to 550 nm. In some aspects, the wavelength of the peak signal produced by the composition is a combination of the peak wavelengths at which the donor chemiluminescent molecule and the energy acceptor molecule fluoresce. Different donor chemiluminescent molecules and different energy acceptor molecules will affect the peak wavelength produced by a composition. [0087] In some embodiments, the composition produces a detectable signal when the temperature of the composition falls within a range of 20 to 80 degrees Celsius; alternatively within a range of 20 to 70 degrees Celsius; alternatively within a range of 20 to 60 degrees Celsius; alternatively within a range of 20 to 50 degrees Celsius; alternatively within a range of 25 to 40 degrees Celsius. In some embodiments, the composition produces a detectable signal when pressure is in a range of 0.8 atm to 1.2 atm; in a range of 0.9 atm to 1.1 atm; or is 1 atm. In further aspects, the composition produces the detectable signal when the pH of the composition is in a range of 2.0 to 13.0; in a range of 3.0 to 12.0; in a range of 4.0 to 11.0; in a range of 5.0 to 10.0; in a range of 6.0 to 9.0; or in a range of 6.0 to 8.0. [0088] The signal produced by the composition when triggered is expected to have greater intensity and have a higher quantum yield than a signal produced by the triggering of the donor molecule alone. Quantum yield is generally the ratio of the number of photons emitted to the number of photons absorbed. Expressed as a percentage, chemiluminescent molecules can have a quantum yield greater than 100%. [0089] In an aspect, an active intermediate of the reaction between the donor chemiluminescent molecule and the energy acceptor molecule has an extended half-life as compared to the half-life of the intermediate without complexation with the host molecule as seen in Example 2. It should be appreciated by those skilled in the art that the half-life of the reaction is the time required for the chemiluminescent molecules to decrease by half. It is also appreciated that the extent of the 21 extended half-life depends at least in part on the type of host molecule and/or its concentration in the composition. [0090] In some embodiments, the composition is water soluble. In such embodiment, the signal produced by the composition is preferably detectable in aqueous solution. In some compositions existing at the time of the invention, the chemiluminescence emission of certain chemiluminescence resonance energy transfer systems was extremely weak in aqueous conditions. An aqueous environment can result in water induced quenching of energy transfer. Water-induced quenching can be the result of water molecules colliding with the excited-state fluorescent molecules and deactivating them without fluorescence emission; water molecules accepting energy from the excited-state fluorescent molecules through a non-radiative energy transfer process; or water influencing the fluorescence properties of a fluorescent molecule, such as its polarity. For many chemiluminescence resonance energy transfer systems in use at the time of the invention, surfactants, such as cetyltrimethylammonium bromide (CTAB), were added to reduce water-induced quenching by providing a hydrophobic environment and bringing fluorescent molecules together in the form of micelles, thereby enhancing light emission efficiency considerably. In some chemiluminescence resonance energy transfer systems in use at the time of the invention, a hydrophobic portion was added to fluorescent molecules to reduce solubility and allow self-assembly into micelles. [0091] In some embodiments, the composition does not require suspension in solution to produce an enhanced signal and, indeed, can be deposited on a solid media and produce a triggerable, detectable signal. The ability to produce a signal when deposited on a solid media allows for implementation of the composition, system, or method in lateral flow assays or Western blots. In contrast, formulations such as LUMI-PHOS 530 (Lumigen, Inc., Southfield, MI) that use micelles to bring a donor chemiluminescent molecule and an energy acceptor molecule into close proxomity are not ideal for implementation on solid media because deposition on solid media destroys the micellular structure, significantly attenuating the light emission or signal. See Examples 5-7. Without wishing to be bound by any theory, it is believed the attenuation of signal observed with LUMI-PHOS 530 (Lumigen, Inc., Southfield, MI) in Examples 5-7 is a result of a disruption of micelle formation or destruction of micelles. There are several reasons that deposition on solid media could disrupt micelles. For example, the lack of fluidity and limited molecular mobility may prevent the dynamic rearrangement of surfactant molecules required for micelle formation. 22 Additionally, surfactant molecules may interact strongly with the solid surface through adsorption, and this interaction can disrupt the organization needed for micelle formation. Moreover, the solid surface may also compete with the hydrophobic interactions that drive the assembly of micelles. Thus, it is generally understood to a person of ordinary skill in the art that a liquid media is required for the formation and stability of micelles. See FIGS.7-9 and Example 5-7. [0092] In an embodiment, the composition exhibits no relative attenuation of chemiluminescence when adsorbed on a filter media and triggered versus in solution and triggered. That is, the composition emits light when triggered in solution and when triggered while adsorbed on a filter media, and the light emission is not diminished when triggered while adsorbed on a filter media relative to light emission when triggered in a solution. In some aspects, the composition exhibits no attenuation of chemiluminescence when the filter media comprises 100% cotton fiber, nitrocellulose membrane, or polyvinylidene difluoride (PVDF) membrane. In another aspect, the composition exhibits no attenuation of chemiluminescence when the solution comprises: 3 ng Alkaline Phosphatase (AP)-conjugate solution (ALP/mL in TRIS buffer, pH 9.0) and a VPPD (0.3mM)+Fls(0.3mM)+bCDPol (0.8mM) solution in AMP buffer (0.75 M 2-amino-2-methyl-1- propanol, hydrochloric acid, 1 mM MgCl₂, at pH 9.0). In yet another aspect, the composition exhibits no attenuation of chemiluminescence when the composition when the light intensity is measured at 20°C; 25°C; 30°C; 35°C; 37°C; 40°C; 45°C; or 50°C. In yet another aspect, the composition exhibits no attenuation of chemiluminescence when the composition when the light intensity is measured after 2 minutes; 5 minutes; 8 minutes; 10 minutes; 12 minutes or 15 minutes. In an exemplary aspect, the composition exhibits no attenuation of chemiluminescence (versus chemiluminescence as measured when triggered in solution) when a volume of the composition is triggered when adsorbed on a filter media comprising 100% cotton fiber, using 3 μL of AP- Conjugate solution (3 ng ALP/mL in TRIS buffer, pH 9.0) and measured after 10 minutes at room temperature; and the composition in solution is triggered by adding 10 μL of AP-Conjugate solution to 2x the volume of the composition added to the filter media and measured after 10 minutes at 37°C. [0093] In some aspects, the composition (including the donor chemiluminescent molecule, the energy acceptor molecule, and the host molecule) does not include a micelle. Chemiluminescent substrates such as LUMI-PHOS 530 (that include a dioxetane further include surfactants that organize the donor chemiluminescent molecule and the energy acceptor molecule into micelles) 23 can be used in methods of triggering chemiluminescence in the presence of alkaline phosphatase. See, for example, U.S. Patent No. 5,004,565. While the micellular structures in such alternative compositions bring a donor chemiluminescent compound and an acceptor fluorescent compound together, providing the conditions for enhancement of a chemiluminescent effect, in the compositions described herein, the donor chemiluminescent molecule sand the energy acceptor molecules are brought together by a host molecule in a non-micellular structure. As further described below including, for example, in Examples 5-7, the compositions described herein do not need to form micelles to bring the donor chemiluminescent compounds and the energy acceptor molecule into sufficient proximity to observe enhancement of the energy transfer. Indeed, as described in Example 5-7, when the compositions described herein are adsorbed on a filter media (which would destroy a micellar structure), chemiluminescence resonance energy transfer and the production of a detectable signal is maintained. [0094] Without wishing to be bound by theory, the compositions described herein may have certain advantages over chemiluminescent reagent formulations that rely on micellar structures to bring donor and acceptor molecules into proximity. These advantages may include, for example, the ability to control the spatial relationship between donor chemiluminescent molecules and energy acceptor molecules, potentially producing more robust enhancement in a greater diversity of working environments or allowing for enhanced control of the period of enhanced chemiluminescent effect. [0095] This disclosure also provides methods of using the compositions described herein. For examples, in one aspect, a method of enhancing transient chemiluminescence can include providing a host molecule, complexing a donor chemiluminescent molecule to the host molecule, and complexing an energy acceptor molecule to the host molecule. In some embodiments, the method includes configuring the distance between the formed inclusion complexes to produce a triggerable, detectable signal. In some embodiments, the method may include configuring the distance between the formed inclusion complexes to optimize the signal for a particular use, for example, to provide a calibrated maximum signal, to extend the half-life of an active intermediate of a reaction between the donor chemiluminescent molecule and the energy acceptor molecule, to maximize the signal at a specific time point, or to calibrate the signal to be produced at a specific rate. 24 [0096] In some embodiments, the method does not include forming a covalent bond between the donor chemiluminescent molecule and a cyclodextrin molecule; nor, forming a covalent bond between the energy acceptor molecule and a cyclodextrin molecule. In some embodiments, the method does not include forming a micelle or maintaining micelle kinetic stability. As further discussed above, the compositions described herein, the donor chemiluminescent compounds and the energy acceptor molecule are brought together by a host molecule in a non-micellular structure and do not need to form micelles to bring the donor chemiluminescent compounds and the energy acceptor molecule into sufficient proximity to observe enhancement of the energy transfer. [0098] The compositions described herein can be used in biological assays or diagnostic test systems, including but not limited to, for example, an immunoassay, an enzymatic assay, a protein array analysis, a Western blot assay, a Northern blot assay, a Southern blot assay, an immunoassay test strip, or a lateral flow assay. For example, the compositions described herein can be used as a substrate in any suitable ALP-based immunoassay including, for example, a sandwich immunoassay (including a one-step or two-step immunoassay), a competitive immunoassay, an immunoassay on a test strip, an Enzyme-Linked Immunosorbent Assay (ELISA), an enzyme _{multiplied immunoassay technique (EMIT), etc. Example 8 provides an exemplary use of a} composition described herein in an immunoassay. Example 9 provides an exemplary use of a composition described herein in a lateral flow assay. [0099] In one aspect, the compositions described herein may be used in a diagnostic test system including a test strip. An exemplary test strip is shown in the context of a lateral flow test strip in FIG.10. A lateral flow assay includes, in general, application of a sample at one end of the strip, on an adsorbent sample pad. The sample pad may include components that make the sample suitable for interaction with the detection system. After leaving the sample pad, the sample migrates through a conjugate release pad. The conjugate release pad may include antibodies that are specific to the target analyte and are conjugated to colored or fluorescent particles. The sample, together with the conjugated antibody bound to the target analyte, migrates along the strip into the detection zone. Recognition of the sample analyte results in an appropriate response on the test line, while a response on the control line indicates the proper liquid flow through the strip. The read-out can be assessed by eye or using a dedicated reader. Finally, it should be appreciated by those skilled in the art that the liquid flows “laterally” across the device because of the capillary 25 force of the strip material and, to maintain this movement, an absorbent pad is typically attached at the end of the strip opposite the sample pad. [0100] It should be appreciated by those skilled in the art that the present technology can be adapted for or incorporated within an immunoassay test strip or lateral flow assay. The presently described technology allows for the adaptation of a lateral flow assay with a donor-acceptor-host system that maintains or improves the enhanced signal detection with such assays. Moreover, the adaptation of such lateral flow assays with the present technology may allow for a reduction in costs in producing and using such assays as well. An embodiment of an exemplary lateral flow assay that uses the compositions described herein is described in Example 9. [0101] In addition, the compositions described herein can be used in other types of r immunoassays in addition to lateral flow assays. Immunoassays refer to biochemical tests that rely on the interaction between antibodies and antigens for the detection or quantification of a specific molecule or analyte in a sample. A label helps for detection or quantification of the analyte, which the present technology is well-suited. [0102] There are many different types of immunoassays. For example, there are heterogeneous and homogeneous immunoassays. Heterogeneous immunoassays require a physical separation step where unbound antibodies and/or unbound analyte must be washed away, while homogeneous immunoassays do not require separation before analysis (all reagents can be freely suspended in bulk solution). These immunoassays may be divided further into two groups: competitive and noncompetitive. [0103] In competitive (also known as “limited reagent”) immunoassays, analytes can be labeled. The analyte and the labeled analyte (sometimes called a tracer) are mixed with a limited amount of capture antibody. After incubation for a certain period, the bound or the free fraction of the labeled analyte is measured and related to the concentration of the analyte in the sample. In noncompetitive (also known as “reagent excess”) immunoassays, antibodies can be labeled (sometimes called a secondary antibody). The labeled antibodies are mixed with a sample and bind to the analyte in the sample. Then, the labeled antibodies bound to analyte can be detected and analyzed. [0104] A sandwich style immunoassay presents an example of a heterogeneous, noncompetitive immunoassay. A capture antibody sits immobilized on a solid surface, and coats a solid phase (such as a microplate or membrane). Then, a sample containing the analyte is added to the coated solid 26 phase and allowed to incubate. During this time, if the analyte is present, it binds to the immobilized antibody. After incubation comes a washing step, the unbound substances are washed away to remove any non-specific binding, leaving only the captured analyte on the solid phase. If secondary labeling antibodies are used, a secondary labeling antibody binds to the captured analytes or primary (capture) antibodies, [0105] For detection and visualization in each of these assays, chemiluminescence can be used. For example, an antibody that binds to a target analyte and is conjugated to a triggering enzyme or substrate could be mixed with a sample suspected to contain the analyte; after washing (for example, to remove any antibody not bound to the target analyte), a composition described herein could be added to the mixture and a sensor used detect the chemiluminescence to determine the presence and quantity of the target analyte. Thus, in some aspects, this disclosure describes an immunoassay composition that includes an antibody that binds to a target analyte and is conjugated to a triggering enzyme or substrate and a composition described herein. [0106] The compositions described herein can be adapted for use in many types of immunoassay or bioassay in which chemiluminescent reporters are used. Example 8 presents an exemplary use of the composition in an immunoassay. Further, as noted above, an embodiment of the composition may be used in a lateral flow assay, as shown in an exemplary embodiment in Example 9. [0107] The compositions described herein can also be used in a Western blot. A Western blot or immunoblot are common assays used to study of different aspects of protein biomolecules. Western blot assays can identify and quantify a specific protein in a complex mixture extracted from cells or tissue lysate. For instance, Western blot assays are capable of detecting different isoforms of proteins, discerning protein-protein interactions or protein DNA-interactions, detecting post-translational modifications, localizing subcellular protein function, and development of antibodies and diagnosis of diseases. [0108] In a typical Western blot assay, the process begins with obtaining the native or denatured proteins that are the target of study. They are separated by gel electrophoresis by size and charge. Then, the proteins are transferred to a protein binding membrane, such as nitrocellulose or polyvinylidene difluoride (PVDF). This transfer is the origin the namesake “blotting.” After the transfer, there is usually a blocking step. The membrane with the transferred protein are incubated with a blocking solution. The blocking solution is used to prevent nonspecific binding of antibodies (blocking can fill holes in the membrane where antibodies could find non-specifically or simply 27 get stuck). This step reduces background signals. After blocking, the membrane is incubated with a primary antibody specific to the target protein for protein detection. The anti-body binds to the target protein on the membrane. Then, the membrane is washed and any excess primary antibody is removed. [0109] Visualization is the last step. Sometimes, the primary antibody has a conjugated detectable label, but more often a secondary antibody is used. A secondary antibody is incubated with the membrane, which binds to the primary antibody. The secondary antibody is conjugated with a detectable label or an enzyme. In this example, the secondary antibody could be conjugated with an enzyme used to trigger the removable triggerable dioxetane. The enzyme catalyzes a reaction with the triggerable dioxetane producing light. Alternatively, the secondary antibody could be conjugated with a donor-acceptor-host system and a triggering enzyme could be added to the membrane to trigger the chemiluminescence. Specialized light detecting equipment could capture and record the emitted light for visualization. [0110] After signal detection, the Western blot is analyzed to determine the presence and quantity of the target protein. This involves assessing the intensity and size of the bands corresponding to the target protein. The compositions described herein may provide improvements to existing systems and/or may reduce costs. [0111] Conjugating the compositions described herein to nucleotides, or DNA or RNA strands would allow visualization and detection for assays such as the Southern blot or the Northern blot. A Southern blot is a technique used to detect specific DNA sequences in a complex mixture. Southern blotting involves several steps, including DNA digestion, gel electrophoresis, transfer to a membrane, hybridization with a labeled probe, and detection. Southern blotting can be used for analyzing DNA fragments based on size and sequence. It has been widely used in molecular biology for tasks such as mapping genes, identifying DNA polymorphisms, and confirming the presence or absence of specific DNA sequences in genomic DNA. [0112] Analogous to the Southern blot, a Northern blot is used to study gene expression by detecting and analyzing RNA molecules. Northern blotting involves several analogous steps, including RNA electrophoresis, transfer to a membrane, hybridization with a labeled probe, and detection. Detection using a donor-acceptor-host system could be possible and may improve existing systems. 28 [0111] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order; and, as appropriate, any combination of two or more steps may be conducted simultaneously. [0112] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. [0113] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention. [0114] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. [0115] By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. [0116] Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one. These articles refer to one or to more than one (i.e., to “at least one”). The term “and/or” means any one or more of the items in the list joined by “and/or”. As an example, “x and/or y” means any element of the three-element set {(x), (y), (x, y)}. In other words, “x and/or y” means “one or both of x and y”. As another example, “x, y, and/or z” means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, “x, y and/or z” means “one or more of x, y and z”. 29 [0117] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range. [0118] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects. [0119] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, unless otherwise indicated, the interval of accuracy is +/- 10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 30 [0120] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. [0121] The term “exemplary” means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms “e.g.,” and “for example” set off lists of one or more non-limiting aspects, examples, instances, or illustrations. [0122] The terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting of the disclosure. As used herein, the singular forms are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “includes,” “comprising,” “including,” “has,” “have,” “having,” and the like when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. [0123] It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, for example, a first element, a first component or a first section discussed below could be termed a second element, a second component or a second section without departing from the teachings of the present disclosure. Similarly, various spatial terms, such as “upper,” “lower,” “side,” and the like, may be used in distinguishing one element from another element in a relative manner. It should be understood, however, that components may be oriented in different manners, for example a semiconductor device may be turned sideways so that its “top” surface is facing horizontally and its “side” surface is facing vertically, without departing from the teachings of the present disclosure. [0124] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, 31 "substantially" may refer to being within at least 20%, alternatively at least 10%, alternatively at least 5% of a characteristic or property of interest. [0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. [0126] The invention is defined in the claims. However, below is a non-exhaustive listing of non- limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein. [0127] The features are more fully shown by the following examples which are provided for purposes of illustration, and are not to be construed as limiting the invention in any way. EXAMPLES All reagents, starting materials, and solvents used in the following examples were purchased from commercial suppliers (such as Sigma Aldrich, St. Louis, MO) and were used without further purification unless otherwise indicated. Example 1. Transient chemiluminescence of a donor-acceptor cyclodextrin composition [0128] Light emission from different formulations were measured using a Turner TD-20/20 single tube luminometer (Turner Designs, San Jose, CA). PPD and fluorescein surfactant (N-(3',6'- dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)tetradecanamide) (Fls) were combined with polymeric beta-cyclodextrin (bCDpol) at different concentrations, as shown in Table 1, in an AMP buffer (0.75 M 2-amino-2-methyl-1-propanol, hydrochloric acid, 1 mM MgCl2, at pH 9.0). Then, 10 μL alkaline phosphatase (ALP) (0.04 U/μL in diethanolamine buffer) was added, and light emission was measured at 37°C for a window of time that typically corresponds to a maximum of emission (~10-15 minutes). [0129] Light emission was measured for a control, baseline condition (PPD alone). For a positive control, the light emission of LUMI-PHOS 530 (Lumigen, Inc., Southfield, MI), a chemiluminescent reagent formulation including PPD, Fls and cetyltrimethylammonium bromide (CTAB) incorporated in a micellar structure, was also measured. Table 1 shows the results for the different 32 conditions, as reported in relative light units (RLU), and the amount of enhancement relative to PPD alone. Table 1. Formulation RLU Enhancement PPD 20 x1 PPD+Fls 60 x3 PPD+Fls+bCDpol 1.6mM 600 x30 PPD+Fls+bCDpol 0.4mM 1100 x55 PPD+Fls+bCDpol 0.8mM 1250 x61 Lumi-Phos-530 3900 x190 Example 2. Half-life data during quantum yield measurement conditions [0130] A quantum yield experiment was set up with a 100uL buffer solution of (AMP, described in the previous example), containing PPD (0.3mM), mixed in different combinations and concentrations of β-cyclodextrin (bCD), polymeric β-cyclodextrin (bCDpol), and or trimethylated- β-cyclodextrin (TMbCD), as shown in Table 2. Alkaline Phosphatase (0.04 U/μL in diethanolamine buffer) was added, and the sample was mixed on the vortex and immediately transferred to a single-tube luminometer (Turner TD-20/20, Turner Designs, San Jose, CA). The light output was constantly measured at 37°C for a period of 60 minutes. FIG. 5 shows light intensity (in relative light units (RLUs) as a function of time (in minutes) for the various donor/acceptor reactions and illustrates host guest-complexes formation that affects the half-life of the active chemiluminescent intermediate. Table 2. F_ormulation ^t _1/2 ^{, min} PPD 2.2 PPD+0.9mM bCD 4.4 PPD+1.6mM bCD 8.1 PPD+0.9mM bCDpol 6.2 PPD+0.9mM TMbCD 4.2 PPD+9.0mM TMbCD 7.2 33 Example 3. Chemiluminescence spectral study [0131] Light emission from LUMI-PHOS 530 (PPD, Fls, and CTAB) [LP530]; PPD combined with Fls [Fls+PPD]; or PPD combined with a polymeric β-cyclodextrin (bCDpol) and Fls [bCDpol+PPD+Fls] were analyzed with a spectrofluorometer (FP-8650, Jasco, Easton, MD). After mixing on vortex, the sample was transferred to the luminometer immediately and spectra of chemiluminescence emission from these reactions are shown in FIG.6a; the peak wavelength for each formulation was 525 nm. As a control, the light emission of PPD alone was measured, and results are shown in FIG.6b; the peak wavelength was 455 nm. Example 4. Chemiluminescent measurements [0132] Light emission was measured with a single tube luinometer (Turner TD-20/20) for various formulations including PPD, VMPPD, and VPPD (0.3mM) in combination with Fls (0.03mM) and polymeric β-cyclodextrin (0.8 mM, based on cyclodextrin unit). Emission of LUMI-PHOS 530 was used as a positive control, and a solution of PPD (0.3mM) and Fls (0.03 mM) was used as a baseline control. Table 3 shows the results for the different conditions, as reported in relative light units (RLU), and the amount of enhancement relative to PPD+Fls. [0133] Table 3. Formulation RLU

PPD+Fls 30 x1 PPD+Fls+bCDpol 640 x20 VMPD+Fls+bCDpol 1240 x41 VPPD+Fls+bCDpol 2260 x75 Lumi-Phos-530 5410 x180 Example 5. Adsorption on filtration paper [0134] To determine if the chemiluminescent enhancement achieved with polymeric cyclodextrins was due to the formation of micelles, the light intensity of formulations in solution was compared with the light intensity of the same formulation adsorbed on filtration paper. 34 To measure light intensity in solution, 10 μL Alkaline Phosphatase (AP)-conjugate solution (3 ng ALP/mL in TRIS buffer, pH 9.0) was added to 100 μL of LP530 or 100 μL of a VPPD (0.3mM)+Fls(0.3mM)+bCDPol (0.8mM) solution in AMP buffer at 37°C. Light intensity was measured on a single tube luminometer (Turner TD-20/20, Turner Designs, San Jose, CA) after 10 minutes. Results are shown in Table 4 (“Solution” entry). Table 4. M_{edia LP530 (RLU) VPPD+Fls+bCDPol (RLU) Ratio} _{Solution 8788 3686 2.38:1} [0135] To measure light intensity on filtration paper, 5 μL of LP530 or 5 μL of the VPPD+Fls+bCDPol solution described above at 37°C were adsorbed on room temperature filtration paper (MINI TRANS-BLOT Cell filtration paper, BIO-RAD, Hercules, CA). Then 3 μL of AP-Conjugate solution (3 ng ALP/mL in TRIS buffer, pH 9.0) was added in the center of a 75x100 mm piece of filtration paper. After 10 minutes at room temperature the light intensity was measured using an Amersham Imager 600 (GE Life Sciences, Chicago, IL) with the auto exposure setting (which calculates optimal exposure for given light intensity). [0136] FIG. 7 shows the results processed using Image Quant TL Software v8.1 (GE). Light intensity data presented in Table 5 (“Filtration paper” entry) Table 5. M_{edia LP530 (RLU) VPPD+Fls+bCDPol (RLU) Ratio} _{Filtration Paper 3029 12980 1:4.29} [0137] The results of this Example show that when VPPD is combined with Fls and a polymeric cyclodextrin on a solid media (e.g., filtration paper), chemiluminescence is enhanced compared to when VPPD is combined with Fls and a polymeric cyclodextrin in solution. In contrast, LUMI- PHOS 530, a formulation known to form a micellar structure exhibited decreased chemiluminescence on solid media. The observed enhancement of chemiluminescence in a formulation including a polymeric cyclodextrin on solid media – which would destroy the majority of micellular structures – suggests that the enhanced chemiluminescence does not require the formation of micelles. In contrast, the relatively diminished light intensity of LUMI-PHOS 530 on filtration paper is believed to be the result of the substantial reduction (or destruction) of micellular structure. 35 Example 6. Adsorption on nitrocellulose membrane [0138] To determine if the chemiluminescent enhancement achieved with polymeric cyclodextrins could also be achieved on a nitrocellulose membrane (such as those used in lateral flow immunoassays), the light intensity of formulations in solution was compared with the light intensity of the same formulation adsorbed on a nitrocellulose membrane. LP530 light intensity data in solution for comparison was taken from Example 5. [0139] To measure light intensity on nitrocellulose membrane, 5 μL of LP530 or 5 μL of the VPPD +Fls+bCDPol solution described above were adsorbed on nitrocellulose membrane (AMERSHAM Protran Premium 0.45 μm blotting membrane, Cytiva, Marlborough, MA). Then, 3 μL of AP-Conjugate solution (3 ng ALP/mL in TRIS buffer, pH 9.0) was added in the center of a 75x100mm piece of nitrocellulose membrane. Light intensity was measured after 10 minutes at room temperature using an Amersham Imager 600 (GE Life Sciences, Chicago, IL) with auto exposure setting.. FIG.8 shows the results of processed using Image Quant TL Software v8.1 (GE). Light intensity data presented in Table 6. (“Nitrocellulose membrane” entry). Table 6 M_{edia LP530 VPPD+Fls+bCDPol RaƟo} _{Nitrocellulose membrane 1173 11691 1:9.97} [0140] The results of this Example show that VPPD+Fls+bCDPol can be used to detect an analyte of interest on nitrocellulose membrane. Example 7. Adsorption on polyvinylidene difluoride (PVDF) membrane [0141] To determine if the chemiluminescent enhancement achieved with polymeric cyclodextrins could also be achieved on a PVDF membrane, the light intensity of formulations in solution was compared with the light intensity of the same formulation adsorbed on a PVDF membrane. LP530 light intensity data in solution for comparison was taken from Example 5. 36 [0142] To measure light intensity on PVDF membrane, 5 μL of LP530 or 5 μL of the VPPD +Fls+bCDPol solution described above were adsorbed on PVDF membrane (AMERSHAM HYBOND PVDF 0.2 um blotting membrane, Cytiva, Marlborough, MA). Then, 3 μL of AP- Conjugate solution (3 ng ALP/mL in TRIS buffer, pH 9.0) was added in the center of a 75x100mm piece of PVDF membrane. Light intensity was measured after 10 minutes at room temperature using an Amersham Imager 600 (GE Life Sciences, Chicago, IL) with auto exposure setting. [0143] FIG.9 shows the results of processed using Image Quant TL Software v8.1 (GE). Light intensity data presented in Table 7. (“PVDF membrane” entry). Table 7 M_{edia LP530 VPPD+Fls+bCDPol Ratio} _{PVDF membrane 1702 9328 1:5.48} [0144] These results show VPPD+Fls+bCDPol can be used to detect an analyte of interest on PVDF membrane. Example 8. Immunoassay [0145] An immunoassay was performed to detect a goat antigen (Normal Goat IgG Control, R&D Systems, Minneapolis, MN). Magnetic particles (DYNABEADS MyOne Epoxy. Thermo Fisher Scientific, Waltham, MA) were conjugated to capturing antibody (IgG Fraction Monoclonal Mouse Anti-Goat IgG, light chain specific, Jackson ImmunoResearch, West Grove, PA) and suspended in a TRIS-based particle diluent buffer. [0146] 125 μL of particle diluent buffer and 25 μL of 1 mg/mL particles conjugated to capturing antibody were added to a 0.6 mL centrifuge tube. Then either 50 μL of goat antigen (in particle diluent buffer) (S1), or 50 μL of particle diluent buffer (to determine background, S0) was added to the tube. Finally, 50 μL of a 300 ng/mL Mouse Anti-Goat Alkaline Phosphatase conjugate in particle diluent buffer was added (Alkaline Phosphatase IgG Fraction Monoclonal Mouse Anti- Goat IgG, light chain specific, Jackson ImmunoResearch, West Grove, PA). [0147] The mixture was incubated at 37°C for 20 minutes. The supernatant was removed while retaining the magnetic particles with a magnet and after washing 3 times with 0.5 mL wash buffer (ACCESS Wash Buffer II (Beckman Coulter, Brea, CA), 100 μL of a chemiluminescent substrate 37 was added. The chemiluminescent substrate was either LUMI-PHOS 530, (Lumigen, Inc., Southfield, MI) or Formulation 1, which included 0.8mM PPD, Fls, and polymeric cyclodextrin (bCDpol). [0148] Light output was measured after 15 minutes on a Turner TD-20/20 single-tube luminometer (Turner Designs, San Jose, CA) at 37°C. Results are shown in Table 8, and provide proof of concept that a polymeric cyclodextrin-based formulation may be used to detect an antigen in the context of an immunoassay. Table 8 _{Formulation 1,} Formulation,1 LP530 LP530 Signal (S1), Background(S0), Signal (S1), Background(S0), R^LU ^RLU ^RLU ^RLU _{2923.4 1222.9 4830.5 1738.5} _{S1/S0 2.39 2.77} Example 9. Lateral Flow Assay [0149] A two-step lateral flow assay will be performed to detect an antigen (for example, Normal Goat IgG Control, R&D Systems, Minneapolis, MN). The lateral flow assay will be performed on a lateral flow strip including a nitrocellulose membrane, a conjugation pad, and an absorption pad. The conjugation pad will include a Mouse Anti-Goat Alkaline Phosphatase-conjugate (for example, Alkaline Phosphatase IgG Fraction Monoclonal Mouse Anti-Goat IgG, light chain specific, Jackson ImmunoResearch, West Grove, PA). The nitrocellulose membrane will include a test line including an antibody that binds the antigen (for example, IgG Fraction Monoclonal Mouse Anti- Goat IgG, light chain specific, Jackson ImmunoResearch, West Grove, PA) and a control line including the antigen to be detected. The assay will be run by adding a buffer including the antigen to the sample pad, then allowing the buffer containing antigen to flow towards the absorption pad. After a suitable period of time, the lateral flow strip will be exposed to a developing solution including a chemiluminescent substrate. The lateral flow strip may be immersed in the developing solution for a suitable period of time, or the developing solution may be added to the sample pad and then the developing solution allowed to flow toward the absorption pad. The developing solutions will include (1) a formulation which includes a polymeric cyclodextrin, VPPD, and Fls (2) LUMI-PHOS 530 (Lumigen, Inc., Southfield, MI), and a suitable positive control (e.g., APS5, Lumigen, Inc., Southfield, MI). 38 [0150] It is expected that when read on a detector after being exposed to the developing solution, both the control line and the test line will be positive when the positive control developing solution is added. More light is expected to be produced by (1) a formulation which includes a polymeric cyclodextrin, VPPD, and Fls than (2) LUMI-PHOS 530, based on the results of Example 6 which shows that significantly more light can be produced by (1) than (2) on a cellulose membrane. [0151] While examples have been used to describe embodiments of the present invention, it will be understood by those skilled in the art that various changes can be made and equivalents can be substituted for elements in the examples and the examples are not intended to limit the scope of the invention, and it is intended that the invention not be limited to the particular embodiment disclosed as an example. 39

Claims

CLAIMS 1. A composition comprising: a donor chemiluminescent molecule; an energy acceptor molecule; and, a host molecule accommodating at least two guest molecules, a first guest molecule comprising a donor chemiluminescent molecule, a second guest molecule comprising an energy acceptor molecule, w_{herein the at least two guest molecules are structurally spaced for resonance energy} transfer between the donor chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule. 2. The composition of claim 1 wherein at least a portion of the donor chemiluminescent molecule or at least a portion of the energy acceptor molecule or both have a suitable size and affinity for an enclosing portion of the host molecule. 3. The composition of any preceding claim, wherein the host molecule and the at least two guest molecules form a combined inclusion complex; wherein, in the combined inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule; and, wherein, in the combined inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule. 4. The composition of any preceding claim wherein the composition does not comprise a micelle. 5. The composition of any preceding claim wherein the composition does not require suspension in solution to produce an enhanced signal. 6. The composition of any preceding claim wherein the host molecule comprises a dimeric cyclodextrin, a multimeric cyclodextrin, a polymeric cyclodextrin molecule, and/or a combination thereof. 40

7. The composition of any preceding claim, wherein, the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule is 12 angstroms to 13.5 angstroms; from 12 angstroms to 15 angstroms; from 12 angstroms to 16.5 angstroms; from 12 angstroms to 18 angstroms; from 12 angstroms to 19.5 angstroms; alternatively from 12 angstroms to 21 angstroms; alternatively from 12 angstroms to 22.5 angstroms; from 12 angstroms to 24 angstroms; from 12 angstroms to 25.5 angstroms; and/or from 12 angstroms to 50 angstroms. 8. The composition of any preceding claim, wherein a distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule produces a chemiluminescent signal inversely proportional to the sixth power of the distance between the chemiluminescent molecule and the energy acceptor molecule within the host molecule. 9. The composition of any preceding claim, wherein the host molecule comprises:

wherein R comprises: OH, -O-Alk (alkyl-containing), -O-PEG (PEG containing), -O(CH2)x-PR’3 (alkylphosphonium containing), -O(CH2)x-NR’3 (alkylammonium containing), -O(CH2)x- (aryl)-CH2-NR’3 (alkylammonium containing), or -O(CH2)x-(aryl)-CH2-NR’3 (alkylammonium containing), or a combination thereof; and, n is an integer of at least 2. 41

10. The composition of claim 8, wherein the average molecular weight of the host molecule comprises a range of n multiplied by one-thousand one-hundred Daltons to n multiplied by one thousand five hundred Daltons (n*1100 to n*1500 Da). 11. The composition of any preceding claim, wherein the donor chemiluminescent molecule comprises a triggerable dioxetane. _{12. The composition of claim 11, wherein the triggerable dioxetane comprises a removable} protecting group trigger, a dioxetane moiety, and an adamantyl group. 13. The composition of claim 11 or 12, wherein the triggerable dioxetane comprises a triggerable group comprising any moiety that forms a phenolate upon change in an environmental condition, or the presence of a specific analyte or an enzyme. 14. The composition of any of the claims 11-13, wherein the trigger comprises -OH, -OP(O)(OR), -OAcyl, -B(OH)₂, -aryl-boronate esters, -beta-D-galactoside, -OSiMe₂tBu, -OSiR₃, or –OR, wherein R comprises a group removable by a selectable analyte or by a selectable enzyme. 15. The composition of any of claims 11-14, wherein the triggerable dioxetane comprises a triggerable phenoxy-dioxetane that has an electron-withdrawing or electron-accepting substituent at the ortho-position of the phenoxy-dioxetane. 16. The composition of any of claims 11-15, wherein the triggerable dioxetane comprises 3-(4'- methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)phenyl dihydrogen phosphate (PPD), 5-(4'- methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD), or 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD), or combination thereof. 17. The composition of any preceding claim, wherein the energy acceptor molecule comprises a fluorescent molecule, an anchor, and optionally a linker. 42

18. The composition of claim 17, wherein the fluorescent molecule comprises a fluorescent molecule with an excitation frequency in a range of 300 nm to 750 nm; in a range of 350 nm to 700 nm; in a range of 400 nm to 650 nm; in a range of 450 nm to 600 nm; or in a range of 500 nm to 550 nm. 19. The composition of either of claims 17 or 18, wherein the anchor comprises a group that has a strong binding affinity with a host molecule, suitable for the formation of an inclusion complex comprising the host molecule and the energy acceptor molecule. 20. The composition of claim 19, wherein the anchor comprises a long chain n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl, and/or adamantyl group. 21. The composition of any of claims 17-20, wherein the energy acceptor molecule comprises a linker, and wherein the linker connects the anchor and the fluorescent molecule. 22. The composition of claim 21, wherein the linker comprises a long chain alkane, a long chain alkyl ether, a long chain alkyl ester, an anhydride, an isocyanate, a carbodiimide, or a glutaraldehyde, or a combination thereof. 23. The composition of any preceding claim wherein the donor chemiluminescent molecule is not covalently bound to the host molecule. 24. The composition of any preceding claim wherein the energy acceptor molecule is not covalently bound to the host molecule. 25. The composition of any preceding claim, wherein the composition is water soluble. 26. The composition of any preceding claim, wherein the composition produces a detectable signal when triggered. 43

27. The composition of claim 26, wherein the detectable signal has a peak within a wavelength range of 300 nm to 850 nm; in a range of 350 nm to 700 nm; in a range of 400 nm to 650 nm; in a range of 450 nm to 600 nm; or in a range of 500 nm to 550 nm. 28. The composition of any preceding claim, wherein an active intermediate of a reaction between the donor chemiluminescent molecule and the energy acceptor molecule has an extended half-life compared to the half-life of the intermediate without complexation of the donor chemiluminescent molecule and the energy acceptor molecule with the host molecule. 29. The composition of any preceding claim, wherein the composition produces a detectable signal when the temperature of the composition falls within a range of 20 degrees Celsius to 80 degrees Celsius; within a range of 20 degrees Celsius to 70 degrees Celsius; within a range of 20 degrees Celsius to 60 degrees Celsius; within a range of 20 degrees Celsius to 50 degrees Celsius; within a range of 25 degrees Celsius to 40 degrees Celsius; or within a range of 35 to 40 degrees Celsius. 30. The composition of any preceding claim, wherein the composition produces a detectable signal when pressure is in a range of 0.8 atm to 1.2 atm; in a range of 0.9 atm to 1.1 atm; or is 1 atm. 31. The composition of any preceding claim, wherein the composition produces the detectable signal when the pH of the composition is in a range of 2.0 to 13.0; in a range of 3.0 to 12.0; in a range of 4.0 to 11.0; in a range of 5.0 to 10.0; in a range of 6.0 to 9.0; or in a range of 6.0 to 8.0. 32. The composition of any preceding claim, wherein the composition produces a detectable signal when deposited on solid media and triggered. 33. The composition of any preceding claim wherein the composition exhibits no relative attenuation of chemiluminescence when adsorbed on a filter media and triggered versus in solution and triggered. 34. A method of using the composition of any of claims 1-33. 44

35. A method of using the composition of any of claims 1-33 in an immunoassay. 36. A method of using the composition of any of claims 1-33 in a lateral flow assay. 37. A method of using the composition of any of claims 1-33 in a Western blot assay. 38. A transient chemiluminescence enhancement system comprising: a donor chemiluminescent molecule; an energy acceptor molecule; and, a host molecule, wherein the host molecule accommodating at least two guest molecules, a first guest molecule comprising a donor chemiluminescent molecule, a second guest molecule comprising an energy acceptor molecule, wherein the at least two guest molecules are structurally spaced for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule. 39. The system of claim 38, wherein at least a portion of the donor chemiluminescent molecule or at least a portion of the energy acceptor molecule or both have a suitable size and affinity for an enclosing portion of the host molecule. 40. The system of any of claims 38-39, wherein the host molecule and the at least two guest molecules form a combined inclusion complex, wherein, in the combined inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule; and wherein, in the combined inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule. 41. The system of any of claims 38-40, wherein the composition does not comprise a micelle. 45

42. The system of any of claims 38-41, wherein the composition does not require suspension in solution to produce an enhanced signal. 43. The system of any of claims 38-42, wherein the host molecule comprises a dimeric cyclodextrin, a multimeric cyclodextrin, and/or a polymeric cyclodextrin molecule. 44. The system of any of claims 38-43, wherein the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule is 12 angstroms to 13.5 angstroms; from 12 angstroms to 15 angstroms; from 12 angstroms to 16.5 angstroms; from 12 angstroms to 18 angstroms; from 12 angstroms to 19.5 angstroms; from 12 angstroms to 21 angstroms; from 12 angstroms to 22.5 angstroms; from 12 angstroms to 24 angstroms; from 12 angstroms to 25.5 angstroms; or from 12 angstroms to 50 angstroms 45. The system of any of claims 38-44, wherein a distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule produces a chemiluminescent signal inversely proportional to the sixth power of the distance between the chemiluminescent molecule and the energy acceptor molecule within the host molecule. 46. The system of any of claims 38-45, wherein the host molecule comprises: 46

wherein R comprises: OH, -O-Alk (alkyl-containing), -O-PEG (PEG containing), -O(CH2)x-PR’3 (alkylphosphonium containing), -O(CH2)x-NR’3 (alkylammonium containing), -O(CH2)x-(aryl)- CH2-NR’3 (alkylammonium containing), or -O(CH2)x-(aryl)-CH2-NR’3 (alkylammonium containing), or a combination thereof; and, n is an integer of at least 2. 47. The system of claim 46, wherein the average molecular weight of the host molecule comprises a range of n multiplied by one-thousand one-hundred Daltons to n multiplied by one thousand five hundred Daltons (n*1100 to n*1500 Da). 48. The system of any of claims 38-47, wherein the donor chemiluminescent molecule comprises a triggerable dioxetane. 49. The system of claim 48, wherein the triggerable dioxetane comprises a removable protecting group trigger, a dioxetane moiety, and an adamantyl group. 47

50. The system of either claim 48 or 49, wherein the triggerable dioxetane comprises a triggerable group, wherein the trigger comprises any moiety that forms a phenolate upon change in an environmental condition or the presence of a specific analyte or an enzyme. 51. The system of any of claims 48-50, wherein the trigger comprises -OH, -OP(O)(OR), -OAcyl, -B(OH)₂, aryl-boronate esters, beta-D-galactoside, -OSiMe₂tBu, -OSiR₃, or –OR, wherein R comprises a group removable by a selectable analyte or by a selectable enzyme. 52. The system of any of claims 48-51, wherein the triggerable dioxetane comprises a triggerable phenoxy-dioxetane that has an electron-withdrawing or electron-accepting substituent at the ortho- position of the phenoxy-dioxetane. 53. The system of any of claims 48-52, wherein the triggerable dioxetane comprises 3-(4'- methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)phenyl dihydrogen phosphate (PPD), 5-(4'- methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD), or 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxetan]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD), or combination thereof. 54. The system of any of claims 48-53, wherein the energy acceptor molecule comprises a fluorescent molecule, an anchor, and optionally a linker. 55. The system of claim 54, wherein the fluorescent molecule comprises a fluorescent molecule with an excitation frequency in a range of 310 nm to 750 nm; in a range of 350 nm to 700 nm; in a range of 400 nm to 650 nm; in a range of 450 nm to 600 nm; or in a range of 500 nm to 550 nm. 56. The system of either of claims 54 or 55, wherein the anchor comprises a group that has a strong binding affinity with a host molecule, suitable for the formation of an inclusion complex comprising the host molecule and the energy acceptor molecule. 57. The system of any of claims 54-56, wherein the anchor comprises n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl, and/or adamantyl group. 48

58. The system of any of claim 54-57, wherein the energy acceptor molecule comprises a linker, and wherein the linker connects the anchor and the fluorescent molecule. 59. The system of claim 58, wherein the linker comprises a long chain alkane a long chain alkyl ether, a long chain alkyl ester, an anhydride, an isocyanate, a carbodiimide, or a glutaraldehyde, or a combination thereof. 60. The system of any of claims 38-59, wherein the donor chemiluminescent molecule is not covalently bound to the host molecule. 61. The system of any of claims 38-59, wherein the energy acceptor molecule is not covalently bound to the host molecule. 62. The system of any of claims 38-61, wherein the composition is water soluble. 63. The system of any of claims 38-62, wherein the composition produces a detectable signal when triggered. 64. The system of any of claims 38-63, wherein the detectable signal has a peak within a wavelength range of 300 nm to 850 nm; in a range of 350 nm to 700 nm; in a range of 400 nm to 650 nm; in a range of 450 nm to 600 nm; or in a range of 500 nm to 550 nm. 65. The system of any of claims 38-64, wherein an active intermediate of a reaction between the donor chemiluminescent molecule and the energy acceptor molecule has an extended half-life compared to the half-life of the intermediate without complexation of the donor chemiluminescent molecule and the energy acceptor molecule with the host molecule. 66. The system of any of claims 38-65, wherein the composition produces a detectable signal when the temperature of the composition falls in a range of 20 degrees Celsius to 80 degrees Celsius; in a range of 20 degrees Celsius to 70 degrees Celsius; in a range of 20 degrees Celsius to 60 degrees 49 Celsius; in a range of 20 degrees Celsius to 50 degrees Celsius; in a range of 25 degrees Celsius to 40 degrees Celsius; or in a range of 35 to 40 degrees Celsius. 67. The system of any of claims 38-66, wherein the composition produces a detectable signal when the pressure is in a range of 0.8 atm to 1.2 atm; in a range of 0.9 atm to 1.1 atm; or is 1 atm. 68. The system of any of claims 38-67, wherein the composition produces the triggerable, chemiluminescent signal when the pH of the composition is in a range of 2.0 to 13.0; in a range of 3.0 to 12.0; in a range of 4.0 to 11.0; in a range of 5.0 to 10.0; in a range of 6.0 to 9.0; or in a range of 6.0 to 8.0. 69. The system of any of claims 38-68, wherein the composition produces a detectable signal when deposited on solid media and triggered. 70. The system of any of claims 38-69, wherein the composition exhibits no relative attenuation of chemiluminescence when adsorbed on a filter media and triggered versus in solution and triggered. 71. A method of using the system of any of claims 38-70. 72. A method of using the system of any of claims 38-70 in an immunoassay. 73. A method of using the system of any of claims 38-70 in a lateral flow assay. 74. A method of using the system of any of claims 38-70 in a Western blot assay. 75. A method of enhancing transient chemiluminescence comprising: providing a host molecule, complexing a donor chemiluminescent molecule to the host molecule, complexing an energy acceptor molecule to the host molecule. 50

76. The method of claim 75 further comprising: configuring the distance between the inclusion complexes formed to produce a triggerable, detectable signal. 77. The method of either claim 75 or 76, wherein the host molecule comprises a dimeric, multimeric, or polymeric cyclodextrin molecule. 78. The method of any of claims 75-77, wherein at least a portion of the donor chemiluminescent molecule or at least a portion of the energy acceptor molecule or both have a suitable size and a strong affinity for an enclosing portion of the host molecule. 79. The method of any of claims 75-78, wherein the host molecule and the at least two guest molecules form at least a first inclusion complex and at least a second inclusion complex wherein, in the first inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule, and wherein, in the second inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule. 80. The method of any of claims 75-79, wherein the method does not comprise: forming a micelle or maintaining micelle kinetic stability. 81. The method of any of claims 75-80, wherein the method does not comprise: forming a covalent bond between the donor chemiluminescent molecule and a cyclodextrin molecule; nor, forming a covalent bond between the energy acceptor molecule and a cyclodextrin molecule. 82. The method of any of claims 75-81, further comprising: extending the half-life of an active intermediate of a reaction between the donor chemiluminescent molecule and the energy acceptor molecule. 51

83. The method of any of claims 75-82, further comprising: configuring a distance between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule so that the reaction between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule produce a chemiluminescent signal that is approximately inversely proportional to the sixth power of the distance between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule. 84. The method of any of claims 75-83, wherein the distance between the chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule is from 12 angstroms to 13.5 angstroms; from 12 angstroms to 15 angstroms; from 12 angstroms to 16.5 angstroms; from 12 angstroms to 18 angstroms; from 12 angstroms to 19.5 angstroms; from 12 angstroms to 21 angstroms; from 12 angstroms to 22.5 angstroms; from 12 angstroms to 24 angstroms; from 12 angstroms to 25.5 angstroms; or from 12 angstroms to 50 angstroms. 85. A diagnostic test system, comprising: a test strip comprising one or more test regions and one or more control regions, wherein the one or more test regions comprise: a donor chemiluminescent molecule; an energy acceptor molecule; and, a host molecule accommodating at least two guest molecules, a first guest molecule comprising a donor chemiluminescent molecule, a second guest molecule comprising an energy acceptor molecule, wherein the at least two guest molecules are structurally spaced for resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule when accommodated in the host molecule. 86. The diagnostic test system of claim 85, wherein the test strip supports lateral flow of a fluid along a lateral flow direction, and wherein at least one test region includes an area that is exposed for optical inspection and is characterized by a first dimension transverse to the lateral flow direction and a second dimension parallel to the lateral flow direction. 52

87. An immunoassay composition comprising: an antibody that binds to a target analyte and is conjugated to a triggering enzyme or substrate; and a composition of any one of claims 1 to 33. 88. The immunoassay composition of claim 86 further comprising the target analyte. 53