Donor-acceptor cyclodextrin compositions and methods of use thereof

Abstract

Chemiluminescent compositions and systems having at least one donor chemiluminescent molecule, at least one energy acceptor molecule, and at least one host molecule that houses at least two guest molecules, such as a donor chemiluminescent molecule and an energy acceptor molecule, are described. When housed in the host molecule, the two guest molecules are structurally spaced apart to enable resonance energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule. Methods of using the described chemiluminescent compositions and systems are also provided.

Description

Background Technology

[0001] Chemiluminescence generates electromagnetic radiation as light by releasing energy from a chemical reaction. Chemiluminescent reagents are used in applications including Western blotting, Southern blotting, ELISA, lateral flow assays, hydrogen peroxide detection, and proximity-based homogeneous immunoassays. Summary of the Invention

[0002] This disclosure describes compositions, systems, and methods for enhancing light emission via intermolecular energy transfer from chemiluminescent compounds. Donor chemiluminescent molecules and energy acceptor molecules can be complexed into a host molecule configured for resonant energy transfer between guest molecules. Triggering supramolecular complexation of both the donor chemiluminescent molecule and the energy acceptor molecule can provide timing control of light release during a chemiluminescent reaction; amplification of the chemiluminescent signal; and optimization of reagents for specific applications.

[0003] In at least one aspect, the present invention provides a composition or system comprising at least one donor chemiluminescent molecule, at least one energy acceptor molecule, and at least one host molecule housing the at least two guest molecules. The first guest molecule includes, for example, a donor chemiluminescent molecule. The second guest molecule includes, for example, an energy acceptor molecule. When housed in the host molecule, the at least two guest molecules are structurally spaced to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule.

[0004] In some respects, the donor chemiluminescent molecule and / or energy acceptor molecule includes at least a portion of the encapsulation portion of the host molecule that has a suitable size and affinity.

[0005] In other respects, the host molecule and the at least two guest molecules form a combined inclusion complex. In the first inclusion of the combined inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule. In the second inclusion of the combined inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule.

[0006] In another aspect, the composition does not contain micelles. Alternatively or additionally, the composition does not need to be suspended in solution to generate an enhanced signal.

[0007] On the other hand, host molecules include dimer cyclodextrins, polymeric cyclodextrins, polymeric cyclodextrin molecules and / or combinations thereof.

[0008] In some respects, when housed within a host molecule, the distance between the chemiluminescent molecule and the energy acceptor molecule is 12 Å to 13.5 Å; alternatively 12 Å to 15 Å; alternatively 12 Å to 16.5 Å; alternatively 12 Å to 18 Å; alternatively 12 Å to 19.5 Å; alternatively 12 Å to 21 Å; alternatively 12 Å to 22.5 Å; alternatively 12 Å to 24 Å; alternatively 12 Å to 25.5 Å; and / or alternatively 12 Å to 50 Å.

[0009] In another respect, when contained within a host molecule, the chemiluminescent signal generated by the distance between the chemiluminescent molecule and the energy acceptor molecule is inversely proportional to the sixth power of the distance between the chemiluminescent molecule and the energy acceptor molecule within the host molecule.

[0010] On the other hand, host molecules include:

[0011]

[0012] Wherein R comprises: OH, -O-Alk (containing alkyl), -O-PEG (containing PEG), -O(CH2)x-PR'3 (containing alkyl phosphocation), -O(CH2)x-NR'3 (containing alkylammonium), -O(CH2)x-(aryl)-CH2-NR'3 (containing alkylammonium), or -O(CH2)x-(aryl)-CH2-NR'3 (containing alkylammonium) or combinations thereof; and n is an integer of at least 2. Additionally, in another aspect, including, for example, when the host molecule has the structure provided above, the average molecular weight of the exemplary linear polymer host molecule includes the range from n (where n is the number of subunits of the cyclodextrin) multiplied by 1100 Daltons to n multiplied by 1500 Daltons (i.e., n*1100 to n*1500 Da).

[0013] On one hand, the donor chemiluminescent molecule includes triggerable dioxetane. In some aspects, triggerable dioxetane includes a removable protecting group trigger, a dioxetane moiety, and an adamantyl group. Furthermore, in still other aspects, triggerable dioxetane includes triggerable phenoxy-dioxetane, wherein the trigger comprises any moiety that forms a phenolic ester upon changes in environmental conditions or in the presence of a specific analyte or enzyme. In some exemplary aspects, the trigger includes -OH, -OP(O)(OR), -O acyl, -B(OH)2, aryl-boronate, β(β)-D-galactoside, -OSiMe2tBu, -OSiR3, or -OR, wherein R contains a group removable by a selectable analyte or a selectable enzyme. In still other aspects, the triggerable phenoxy-dioxetane has an electron-withdrawing or electron-accepting substituent at the ortho position of the phenoxy-dioxetane.

[0014] In some respects, triggerable phenoxy-dioxanes include 3-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)phenyl dihydrogen phosphate (PPD), 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD), 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD), or combinations thereof.

[0015] In some respects, energy acceptor molecules include fluorescent molecules, anchors, and / or optionally connectors.

[0016] In another aspect, fluorescent molecules include those with excitation frequencies in the range of 300 nm to 750 nm; 350 nm to 700 nm; 400 nm to 650 nm; 450 nm to 600 nm; or 500 nm to 550 nm.

[0017] On the other hand, the anchor contains groups with strong binding affinity to the host molecule, suitable for forming inclusion complexes containing both the host molecule and the energy acceptor molecule. Exemplary anchors include long-chain n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl, and / or adamantyl.

[0018] In another aspect, the connector links the anchor and the fluorescent molecule. Additionally, in some aspects, the connector includes long-chain alkanes, long-chain alkyl ethers, long-chain alkyl esters, acid anhydrides, isocyanates, carbodiimides, or glutaraldehyde, or combinations thereof.

[0019] On the other hand, the donor chemiluminescent molecule does not covalently bind to the host molecule. Conversely, the energy acceptor molecule does not covalently bind to the host molecule.

[0020] In some respects, the compositions of the currently described technology are water-soluble.

[0021] On the other hand, the compositions of the currently described technology generate a detectable signal, for example, when triggered.

[0022] For example, the wavelength of the detectable signal can be in the range of 300 nm to 850 nm; alternatively, in the range of 350 nm to 700 nm; alternatively, in the range of 400 nm to 650 nm; alternatively, in the range of 450 nm to 600 nm; or alternatively, in the range of 500 nm to 550 nm.

[0023] In another respect, the active intermediates of the reaction between the donor chemiluminescent molecule and the energy acceptor molecule have a prolonged half-life compared to the half-life of the intermediate when it does not recombine with the host molecule.

[0024] In other respects, the composition generates a detectable signal when the temperature of the composition is in the following ranges: 20°C to 80°C; 20°C to 70°C; 20°C to 60°C; 20°C to 50°C; 25°C to 40°C; or 35°C to 40°C. Alternatively or alternatively, the composition generates a detectable signal when the pressure is in the range of 0.8 atm to 1.2 atm; 0.9 atm to 1.1 atm; or 1 atm.

[0025] In another aspect, the composition produces a detectable signal when the pH of the composition is in the range of 2.0 to 13.0; in the range of 3.0 to 12.0; in the range of 4.0 to 11.0; in the range of 5.0 to 10.0; in the range of 6.0 to 9.0; or in the range of 6.0 to 8.0.

[0026] In another aspect, the composition generates a detectable signal when deposited on a solid medium and triggered. In yet another aspect, the composition does not exhibit a relative attenuation of chemiluminescence when adsorbed on a filter medium (as opposed to in solution) and triggered.

[0027] This disclosure further provides methods and systems for using the compositions described herein. For example, this disclosure provides a method for using the compositions or systems described herein in immunoassays, lateral flow assays, or Western blot assays.

[0028] This disclosure further describes systems using the compositions described herein.

[0029] In another aspect, this document discloses a method for enhancing transient chemiluminescence, comprising providing a host molecule, recombinating a donor chemiluminescent molecule with the host molecule, and recombinating an energy acceptor molecule with the host molecule. In some aspects, the method includes configuring the distance between the formed inclusion complexes to generate a triggerable, detectable signal.

[0030] In another respect, host molecules include dimers, polymers, or polymeric cyclodextrin molecules.

[0031] In another respect, 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 strong affinity for the encapsulated portion of the host molecule.

[0032] In another 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 of the energy acceptor molecule. In yet another aspect, the method does not include forming micelles or maintaining micelle kinetic stability.

[0033] On the other hand, the method does not include forming a covalent bond between the donor chemiluminescent molecule and the cyclodextrin molecule; nor does it include forming a covalent bond between the energy acceptor molecule and the cyclodextrin molecule.

[0034] In another aspect, the method further includes extending the half-life of the active intermediate of the reaction between the donor chemiluminescent molecule and the energy acceptor molecule. In yet another aspect, the method further includes configuring the distance between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule such that the chemiluminescent signal generated by the reaction between the donor chemiluminescent molecule and the energy acceptor molecule complexed with the host molecule 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.

[0035] On the other hand, when housed within a host molecule, the distance between the chemiluminescent molecule and the energy acceptor molecule is 12 Å to 13.5 Å; alternatively 12 Å to 15 Å; alternatively 12 Å to 16.5 Å; alternatively 12 Å to 18 Å; alternatively 12 Å to 19.5 Å; alternatively 12 Å to 21 Å; alternatively 12 Å to 22.5 Å; alternatively 12 Å to 24 Å; alternatively 12 Å to 25.5 Å; and / or alternatively 12 Å to 50 Å.

[0036] In other aspects and embodiments of the prior art, this document discloses a diagnostic testing system comprising: a test strip including one or more test regions and one or more control regions, wherein the one or more test regions include a donor chemiluminescent molecule; a receptor including a fluorescent molecule; and a host molecule configured to form a composite inclusion complex with the donor chemiluminescent molecule and the energy acceptor molecule, and configured to be structurally spaced apart to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule.

[0037] In another aspect, the test strip supports the lateral flow of fluid along the lateral flow direction, and at least one of the test areas includes an area 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.

[0038] The above description of the invention is not intended to describe every disclosed embodiment or every implementation of the invention. The following description illustrates exemplary embodiments in more detail. Throughout this application, guidance is provided by a list of examples, which may be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be construed as an exclusive list. Attached Figure Description

[0039] Figure 1 A perspective view of an exemplary cartoon schematic diagram illustrating a method for enhancing transient chemiluminescence.

[0040] Figure 2 A perspective view showing an exemplary cartoon embodiment that can trigger donor chemiluminescent molecules.

[0041] Figure 3 A perspective view showing an exemplary cartoon embodiment of an energy acceptor molecule.

[0042] Figure 4 Annotated chemical formulas for an exemplary dimer β-cyclodextrin are shown.

[0043] Figure 5 The light intensity (relative light units (RLU)) of PPD alone or in combination with different concentrations of cyclodextrin is shown as a function of time (in minutes), as further described in Example 1.

[0044] Figure 6a The combination of PPD and fluorescein (Fls + PPD); PPD and fluorescein complexed in the host polymer cyclodextrin (bCDpol + PPD + Fls); and chemiluminescence spectroscopy analysis using Lumi-Phos 530 are shown, as further described in Example 3.

[0045] Figure 6b Spectroscopic analysis of PPD chemiluminescence alone is shown, as further described in Example 3.

[0046] Figure 7 The light intensities of Lumi-Phos530 (blue) or VPPD and polymeric β-cyclodextrin (bCDpol) (red) adsorbed on filter paper after activation with alkaline phosphatase conjugate (AP conjugate) are shown, as further described in Example 5. The lower insets show image views (left) of the automatic exposure setup of activated Lumi-Phos 530 on filter paper, and image views (right) of the automatic exposure setup of activated VPPD and polymeric β-cyclodextrin on filter paper, as further described in Example 5.

[0047] Figure 8The light intensities of Lumi-Phos 530 (blue) or VPPD and polymeric β-cyclodextrin (bCDpol) (red) adsorbed on a nitrocellulose membrane after activation with an alkaline phosphatase conjugate (AP conjugate) are shown, as further described in Example 6. The lower insets show image views (left) of the automated exposure setup of Lumi-Phos 530 on a nitrocellulose membrane after activation, and image views (right) of the automated exposure setup of VPPD and polymeric β-cyclodextrin on a nitrocellulose membrane after activation, as further described in Example 6.

[0048] Figure 9 The light intensities of Lumi-Phos 530 (blue) or VPPD and polymeric β-cyclodextrin (bCDpol) (red) adsorbed on a PVDF membrane after activation with an alkaline phosphatase conjugate (AP conjugate) are shown, as further described in Example 7. The lower insets show image views (left) of the automatic exposure setup of activated Lumi-Phos 530 on a PVDF membrane, and image views (right) of the automatic exposure setup of activated VPPD and polymeric β-cyclodextrin on a PVDF membrane, as further described in Example 7.

[0049] Figure 10 A perspective view of an example lateral flow measurement is shown.

[0050] Figure 11 An exemplary lateral flow assay is provided that incorporates an example immunoassay as further described in Example 9. Detailed Implementation

[0051] This disclosure relates to compositions, systems, and methods for enhancing light emission from intermolecular energy transfer in chemiluminescent compounds. A donor chemiluminescent molecule and an energy acceptor molecule are complexed into a host molecule to create a composition configured for resonant energy transfer between guest molecules. This document describes compositions, systems, and methods for providing or including enhanced chemiluminescence by triggering supramolecular complexation of both the donor chemiluminescent molecule and the energy acceptor molecule with the host molecule.

[0052] In one embodiment, this disclosure describes a composition comprising 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 comprises the donor chemiluminescent molecule. A second guest molecule comprises the energy acceptor molecule. When accommodated in the host molecule, the at least two guest molecules are structurally spaced apart to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule.

[0053] In some respects, energy transfer is chemiluminescent resonance energy transfer (CRET), which is a nonradiative transfer of energy from a donor chemiluminescent molecule to an energy acceptor molecule during a chemiluminescent reaction. Similar to Förster resonance energy transfer (FRET), the chemiluminescent donor molecule undergoes a chemical reaction to emit light. The chemiluminescent donor molecule acts as the initial energy source, and the energy acceptor molecule absorbs the energy emitted by the chemiluminescent donor molecule. In some respects, the probability of an energy transfer event occurring for each donor excitation event can depend on physical parameters such as: (1) the distance between the donor molecule and the energy acceptor; (2) the spectral overlap between the emission spectrum of the donor molecule and the absorption spectrum of the energy acceptor; and (3) the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. (King, C., Barbiellini, B., Moser, D. and Renugopalakrishnan, V. (2012). Exactly soluble model of resonant energy transfer between molecules. Physical Review B, 85(12), 125106.)

[0054] 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 a donor chemiluminescent molecule and an energy acceptor molecule). In CRET, spectral overlap affects the efficiency of energy transfer between the two molecules. A higher probability of energy transfer occurs when there is significant overlap (or high spectral overlap) between the emission spectrum of the donor molecule and the absorption spectrum of the energy acceptor. Theoretically, greater spectral overlap increases the likelihood that the energy acceptor molecule will absorb emission energy from the donor chemiluminescent molecule. Less spectral overlap reduces the efficiency of energy transfer.

[0055] The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment refers to the direction and magnitude of the polarity associated with energy emission and the direction and magnitude of the dipole associated with energy absorption. The energy transfer efficiency between the donor and acceptor depends on the degree of alignment of these two dipole moments. The more aligned the dipole moments are, the greater the probability of energy transfer and light emission.

[0056] In some respects, the enhancement of light emission also depends on the concentrations of the donor chemiluminescent molecule, the energy acceptor molecule, and the host molecule (e.g., the polymer β-cyclodextrin); the number and type of guest molecules complexed within the host molecule (which in turn will be affected by the polymer chain length); the composition and concentration of the buffer in which the molecule is contained, the presence and concentration of the 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 optimization of these additional variables.

[0057] Figure 1 An exemplary host molecule complexed with a donor chemiluminescent molecule and an energy acceptor molecule is shown. Upon complex formation, the host molecule accommodates the at least two guest molecules, and the guest molecules (donor chemiluminescent molecule and energy acceptor molecule) are structurally spaced within the complex to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule. This resonant energy transfer can be triggered or enhanced by the presence of an enzyme or substrate.

[0058] In some embodiments, at least a portion of the donor chemiluminescent molecule has a suitable size and affinity for the encapsulation portion of the host molecule. Alternatively or additionally, at least a portion of the energy acceptor molecule has a suitable size and affinity for the encapsulation portion of the host molecule. In some embodiments, the host molecule and the at least two guest molecules form a composite inclusion complex. In the composite inclusion complex, the host molecule spatially encapsulates at least a portion of the donor chemiluminescent molecule and at least a portion of the energy acceptor molecule.

[0059] The host molecule includes non-monomeric cyclodextrins, namely dimer cyclodextrins, polymeric cyclodextrins, or polymeric cyclodextrins, or combinations thereof. Cyclodextrins are cyclic oligosaccharides comprising a macrocycle of glucose subunits linked by α-1,4 glycosidic bonds. Cyclodextrins comprise five or more linked α-D-glucopyranoside units: α-cyclodextrin comprises 6 glucose subunits, β-cyclodextrin comprises 7 glucose subunits, and γ-cyclodextrin comprises 8 glucose subunits. Typical cyclodextrins may comprise glucose monomers arranged in a ring, thus forming a cone shape.

[0060] In some respects, cyclodextrins preferably comprise β-cyclodextrins. Without being bound by theory, it is believed that β-cyclodextrins can provide improved results than cyclodextrins with more or fewer glucose molecules (which would result in larger or smaller cyclodextrin rings) because the cavity size of the β-cyclodextrin ring is spatially suited to accommodate the adamantyl group of the donor chemiluminescent molecule. In some respects, cyclodextrins can comprise 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 respects, the host molecule can be a linear polymeric cyclodextrin. Alternatively or additionally, the host molecule can include a branched, topological (2D), or stereographic (3D) cyclodextrin architecture.

[0061] In some respects, when housed within a host molecule, the distance between the chemiluminescent molecule and the energy acceptor molecule can be 12 Å to 13.5 Å; 12 Å to 15 Å; 12 Å to 16.5 Å; 12 Å to 18 Å; 12 Å to 19.5 Å; 12 Å to 21 Å; 12 Å to 22.5 Å; 12 Å to 24 Å; 12 Å to 25.5 Å; and / or 12 Å to 50 Å. An exemplary dimer β-cyclodextrin is shown in Figure 4 In the middle, the cyclodextrins are linked together such that the distance between the midlines of the two cyclodextrins is at least 12 angstroms and at most 25 angstroms. Generally, the distance between the triggerable donor chemiluminescent molecule and the energy acceptor molecule is preferably maintained by the host molecule at a certain distance to optimize the donor / acceptor interaction.

[0062] On the one hand, when contained within a host molecule, the chemiluminescent signal generated by the distance between the donor chemiluminescent molecule and the energy acceptor molecule is inversely proportional to the sixth power of the distance between the chemiluminescent molecule and the energy acceptor molecule within the host molecule.

[0063] In some respects, the number of covalently linked cyclodextrins can vary for the desired effect. For example, in some respects, dimer cyclodextrins may be preferred. Alternatively, the cyclodextrin may comprise at least 3, at least 10, at least 50, at least 100, at least 500, at least 1000, or at least 1500 covalently linked cyclodextrins. The host cyclodextrin can accommodate varying amounts of donor chemiluminescent molecules and energy acceptor molecules. The amount and distribution of donor chemiluminescent molecules and energy acceptor molecules affect the amount of light emitted.

[0064] In some embodiments, the average molecular weight of the dimer, polymer, or polymeric cyclodextrin may be in the range of 2,000 Da to 3,000 Da; alternatively, in the range of 2,000 Da to 4,500 Da; or alternatively, in the range of 2,000 Da to 6,000 Da. In alternative embodiments, the polymeric or polymeric cyclodextrin may be in the range of 3,000 Da to 6,000 Da; alternatively, in the range of 3,000 Da to 12,000 Da; alternatively, in the range of 3,000 Da to 18,000 Da; alternatively, in the range of 3,000 Da to 24,000 Da; alternatively, in the range of 3,000 Da to 30,000 Da; alternatively, in the range of 3,000 Da to 36,000 Da; alternatively, in the range of 3,000 Da to 42,000 Da; alternatively, in the range of 3,000 Da to 48,000 Da; alternatively, in the range of 3,000 Da to 54,000 Da; alternatively, in the range of 3,000 Da to 60,000 Da; alternatively, in the range of 3,000 Da to 72,000 Da. The range is: Da; alternatively, the range is: 3,000 Da to 84,000 Da; alternatively, the range is: 3,000 Da to 96,000 Da; alternatively, the range is: 3,000 Da to 108,000 Da; or alternatively, the range is: 3,000 Da to 120,000 Da.

[0065] In some respects, polymeric cyclodextrins can be cross-linked to form nanostructures or hydrogel networks. In other respects, if the cyclodextrin is cross-linked, its concentration can be varied for the desired effect rather than a predetermined amount. The concentration can depend on the solubility and type of the cyclodextrin. For example, in terms of alternatives, the concentration of polymeric β-cyclodextrin in water may include between 0.01 mg / mL and 0.333 mg / mL (at 25°C); alternatively between 0.05 mg / mL and 0.333 mg / mL (at 25°C); alternatively between 0.10 mg / mL and 0.333 mg / mL (at 25°C); alternatively between 0.15 mg / mL and 0.333 mg / mL (at 25°C); alternatively between 0.20 mg / mL and 0.333 mg / mL (at 25°C); alternatively between 0.25 mg / mL and 0.333 mg / mL (at 25°C); and alternatively between 0.3 mg / mL and 0.333 mg / mL (at 25°C). In another example with alternatives, the concentration of the β-cyclodextrin derivative, namely hydroxypropyl-β-cyclodextrin, can be between 2 mg / mL and 11 mg / mL; alternatively between 4 mg / mL and 11 mg / mL; alternatively between 6 mg / mL and 11 mg / mL; alternatively between 8 mg / mL and 11 mg / mL; and alternatively between 10 mg / mL and 11 mg / mL (at 25°C).

[0066] In an exemplary aspect, the host molecule of the composition may have the following chemical formula:

[0067]

[0068] Wherein R comprises: OH, -O-Alk (containing alkyl), -O-PEG (containing PEG), -O(CH2)x-PR'3 (containing alkyl phosphocation), -O(CH2)x-NR'3 (containing alkylammonium), -O(CH2)x-(aryl)-CH2-NR'3 (containing alkylammonium), or -O(CH2)x-(aryl)-CH2-NR'3 (containing alkylammonium) or combinations thereof; and n is an integer of at least 2. Additionally, in another aspect, including, for example, when the host molecule has the structure provided above, the average molecular weight of the exemplary linear polymer host molecule includes the range from n (where n is the number of subunits of the cyclodextrin) multiplied by 1100 Daltons to n multiplied by 1500 Daltons (i.e., n*1100 to n*1500 Da).

[0069] Typically, cyclodextrins possess a hydrophobic core. The hydrophobic core of a cyclodextrin refers to the lumen or central region of these cyclic oligosaccharides. The interior of the cyclodextrin molecule is partially isolated from water molecules because the hydrophilic hydroxyl groups of the glucose units on the outer surface of the cyclic structure attract surrounding water molecules to the outside. It is believed that the protected core of cyclodextrins prevents the complexed reactants from being affected by water that quenches the chemiluminescent reaction.

[0070] Simultaneously, the internal environment can be manipulated by altering the inward-facing functional groups. The inward-facing functional groups of cyclodextrins influence the half-life of the complexed reactants. The half-life of a reaction refers to the time taken to consume or transform half of the initial amount of reactant in a chemical reaction. The stability of reaction intermediates will affect the time required for the reaction to complete or the probability of the reaction occurring. For the chemiluminescent reactions discussed below, generally, a more protonated environment tends to shorten the half-life of the reaction by destabilizing the reaction intermediates. Through the more protonated environment inside the cyclodextrin, the functional groups will be more capable of donating protons (H+) in the chemical reaction. On the one hand, the internal groups can be substituted to modify the characteristics of the reaction that can trigger the chemiluminescent donor molecule and the energy acceptor molecule, to suit the desired properties or reaction parameters.

[0071] Because the size of the host molecule can vary, the number and configuration of the donor chemiluminescent molecules and energy acceptor molecules that form inclusion complexes within a specific host molecule will also vary. For a given number and length of linked cyclodextrins, the concentrations of the host molecule, the triggerable donor chemiluminescent molecules, and the energy acceptor molecules can be manipulated to produce an optimal signal. In Example 1, the concentrations of the polymeric cyclodextrin and the host molecule are variable, and the optimal concentration of the polymeric cyclodextrin can be found by selecting a concentration suitable for the desired properties or characteristics of the reaction. The examples illustrate different levels of quantum yield over time, and the host molecule concentration can be selected to provide the desired properties or characteristics. Thus, the size and concentration of the host molecule can be manipulated in a desired manner for statistically enhanced chemiluminescence.

[0072] Any suitable donor chemiluminescent molecule can be used. Typically, the donor chemiluminescent molecule is an excited fluorescent molecule capable of non-radiatively transferring energy to an energy acceptor molecule via long-range intermolecular dipole-dipole coupling, thereby producing chemiluminescence. In some aspects, at least a portion of the donor chemiluminescent molecule has a suitable size and affinity for the encapsulating portion of the host molecule. Alternatively or additionally, the donor chemiluminescent molecule can be complexed with a molecule having a suitable size and affinity for the encapsulating portion of the host molecule. Suitable size and affinity can refer to a size appropriate for or suitable for a particular purpose or context, and can vary depending on the specific requirements or expectations of the situation. For example, suitable affinity could mean that a portion of the donor chemiluminescent molecule has a strong affinity for the interior of the host molecule. Alternatively or additionally, suitable affinity could mean that a portion of the donor chemiluminescent molecule has a smaller binding affinity if the desired effect on the chemiluminescent reaction (e.g., an extended half-life of the reactants) can be achieved. Suitable size and affinity can also be a size and affinity that meets the functional or spatial requirements of a given task or space.

[0073] An exemplary cartoon donor chemiluminescent molecule is shown in Figure 2 In the middle. For example Figure 2 As shown, the donor chemiluminescent molecule 200 may include a removable protecting group 210, an excited fluorescent molecule 220, and a group having a suitable size and affinity for the encapsulation portion (e.g., adamantyl) of the host molecule 230. Exemplary donor chemiluminescent molecules include, but are not limited to, triggerable dioxane, triggerable phenoxy-dioxane, and / or combinations thereof.

[0074] Exemplary donor chemiluminescent molecules include triggerable dioxanes. Triggerable dioxanes are well known in the art. (See, for example, AP Schaap, T.-S. Chen, RS Handley, R. DeSilva and BP Giri, Tetrahedron Letters, 1987, 28, 1155–1158; AP Schaap, RS Handley and BP Giri, Tetrahedron Letters, 1987, 28, 935–938; AP Schaap, MD Sandison and RS Handley, Tetrahedron Letters, 1987, 28, 1159–1162). Typically, removable protecting groups can be selectively removed. Changes in environmental conditions or the presence of the enzyme or analyte of interest "trigger" the removal of the protecting group from the exposed chemiluminescent molecule, such as dioxane. Once the removable protecting group is removed, dioxane reacts to produce light.

[0075] Potential changes in environmental conditions that can trigger the activation of dioxane or affect the activation may include, but are not limited to, alterations in pH, temperature, amount and content of solvent or reaction medium, concentration of reactants, presence of catalysts or inhibitors, oxygen concentration (e.g., peroxides), concentration of ions in the solution or reaction mixture, and / or presence of contaminants.

[0076] In some aspects, the addition or presence of an analyte or enzyme can preferably be used to remove a protecting group trigger. In some embodiments, the triggerable dioxane includes a triggerable group, which includes any portion that forms a phenolic ester upon changes in environmental conditions or in the presence of a specific analyte or enzyme. For example, if the removable protecting group trigger is a phosphate ester group and alkaline phosphatase is added to the reaction solution in sufficient quantities, chemiluminescence can be triggered by alkaline phosphatase. Upon removal of the phosphatase group by alkaline phosphatase, phenolic ester formation triggers chemiluminescence and excitation following phenoxy group deprotection. In another example, β-galactosidase can be added to solution along with a compound comprising a β-D-galactoside removable protecting group trigger. β-galactosidase catalyzes the hydrolysis of the β-glycosidic bond, thereby releasing β-D-galactoside. Hydrolysis exposes the phenoxy group, and the formation of the phenolic ester dioxane triggers chemiluminescence and excitation.

[0077] Exemplary triggers include, but are not limited to, -OH, -OP(O)(OR), -O acyl, -B(OH)2, -aryl-boronic acid ester, -β-D-galactoside, -OSiMe2tBu, -OSiR3, or -OR, wherein R contains a group that can be removed by a selectable analyte or a selectable enzyme.

[0078] In one embodiment, different substituents can be introduced into different positions on the phenol ring to modulate the properties of dioxetane (stability, quantum efficiency of chemiluminescence, wavelength of emitted light, water solubility, etc.). In some aspects, if a chemiluminescence-enhancing effect is found, it can be triggered that phenoxy-dioxetane has electron-withdrawing or electron-accepting substituents at the ortho position of the phenoxy-dioxetane. Various desired or optional properties and characteristics have been investigated. (Hananya N, Shabat D. Recent Advances and Challenges in Luminescent Imaging: Bright Outlook for Chemiluminescence of Dioxetanes in Water. ACS Cent Sci., 26 June 2019; 5(6):949-959. doi: 10.1021 / acscentsci.9b00372. Electronic version, 29 May 2019. PMID: 31263754; PMCID: PMC6598152.)

[0079] In some embodiments, the triggerable phenoxy-dioxane includes 3-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)phenyl dihydrophosphate (PPD):

[0080] (PPD);

[0081] 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD):

[0082] (VPPD); or

[0083] 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD):

[0084] (VMPD);

[0085] Or a combination thereof.

[0086] As described above, the donor chemiluminescent molecule includes a group of suitable size and affinity for the encapsulating portion of the host molecule, including, for example, an adamantyl group. When included in the donor chemiluminescent molecule, the adamantyl group preferably has a strong binding affinity for cyclodextrin, including the ability to form highly stable complexes with cyclodextrin. In some embodiments, the presence of the adamantyl group increases the likelihood of triggering the formation of inclusion complexes between the donor chemiluminescent molecule and the cyclodextrin host molecule in solution.

[0087] Any suitable energy acceptor molecule can be used. Figure 3 A perspective view of an exemplary energy acceptor molecule 300 is shown. Typically, the energy acceptor molecule 300 includes the ability to accept energy transfer from fluorescent molecules. In some aspects, such as Figure 3 As shown in the exemplary embodiment, the energy acceptor molecule 300 includes a fluorescent molecule 320, an anchor 340, and / or a connector 330.

[0088] Energy acceptor molecules can include any suitable fluorescent molecules. In some aspects, fluorescent molecules include those with excitation frequencies in the range of 300 nm to 750 nm, including, for example, fluorescent molecules with excitation frequencies in the range of 300 nm to 750 nm; in the range of 350 nm to 700 nm; in the range of 400 nm to 650 nm; in the range of 450 nm to 600 nm; or in the range of 500 nm to 550 nm. As mentioned above, the spectral overlap of the emission spectrum of the donor molecule and the absorption spectrum of the energy acceptor affects the efficiency of energy transfer between the two molecules. As further explained in Example 3, Figure 6b The spectral emission wavelength distribution of PPD is shown (which overlaps with the absorption spectrum of the energy acceptor). When combined with a suitable energy acceptor molecule, acceptor emission is observed, such as... Figure 6a As shown. Some exemplary acceptor fluorescent molecules include, but are not limited to, fluorescent dyes, such as fluorescein, rhodamine, coumarin, anthocyanins, fluorescein, methylene blue, Congo red, and derivatives thereof. For example, exemplary derivatives of fluorescein include fluorescein isothiocyanate and carboxyfluorescein. Those skilled in the art will understand that any suitable fluorescent dye capable of accepting energy from the decomposition of unstable oxide intermediates to provide measurable light emission can be used.

[0089] On the one hand, energy acceptor molecules can innately form inclusion complexes with host molecules. Alternatively or additionally, energy acceptor molecules can be modified to increase their ability to form inclusion complexes with host molecules. For example, acceptor fluorescent molecules can optionally be coupled to anchors via linkers. When present, anchors include groups with strong binding affinity to the host molecule, suitable for forming inclusion complexes. That is, anchors can help guest molecules (i.e., energy acceptor molecules) "insert themselves" into the host molecule. For example, when cyclodextrin is the host molecule, anchors can include groups that form strong complexes with cyclodextrin. Exemplary groups known to form strong complexes with cyclodextrin include, but are not limited to, n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl, and adamantyl. On the one hand, n-alkyl comprises chains between 5 and 20 linked carbons, or alternatively, between 6 and 15 linked carbons.

[0090] When the energy acceptor molecule includes an anchor, it may further include a connector linking the anchor and the fluorescent molecule. When present, such as... Figure 3 As shown in the exemplary embodiments, the connector 330 may include any suitable group connecting the anchor 340 and the acceptor fluorescent molecule 320. Additionally, the connector may have a strong binding affinity to the host molecule. Exemplary connectors include, but are not limited to, long-chain alkyl ethers, long-chain alkyl esters, acid anhydrides, isocyanates, carbodiimides, or glutaraldehyde, or combinations thereof. The connector may further include any suitable bioconjugate that can connect the energy acceptor molecule to the anchor. In some aspects, in the case of ethers or esters, long-chain alkanes, long-chain alkyl esters, and long-chain alkyl ethers may include chains between 5 and 20 linked carbons and / or oxygen atoms; or alternatively, in the case of ethers or esters, chains between 6 and 15 linked carbons and / or oxygen atoms.

[0091] Some exemplary energy acceptor molecules and their formulas include, but are not limited to:

[0092]

[0093] Typically, at least a portion of the donor chemiluminescent molecule is inserted into the host molecule to form an inclusion complex, and at least a portion of the energy acceptor molecule is inserted into the host molecule to form an inclusion complex, thereby forming a complex host molecule. The inserted portion of the energy acceptor molecule can be an anchor, a connector, and / or a receptor fluorescent molecule. Alternatively, the entire energy acceptor molecule can be inserted into the host molecule. The inserted portion of the donor chemiluminescent molecule can be a triggerable donor chemiluminescent molecule and / or an adamantyl group. Alternatively, the entire donor chemiluminescent molecule can be inserted into the host molecule. The number and placement of the triggerable donor chemiluminescent molecule and the energy acceptor molecule forming the complex with the host molecule can vary.

[0094] In some respects, the donor chemiluminescent molecule in the composition is not covalently bound to the host molecule. Alternatively or additionally, the energy acceptor molecule in the composition is not covalently bound to the host molecule. Previous work has described compositions comprising an energy acceptor molecule covalently bound to trimethyl-β-cyclodextrin, said trimethyl-β-cyclodextrin being further combined with an adamantyl-1,2-dioxane probe at a concentration sufficient to drive the formation of at least a 1:1 host-guest complex. (Gnaim et al., “Lightemission enhancement by supramolecular complexation of chemiluminescence probes designed for bioimaging”. Chemical Science (2019) 10(10):2945-2955.) Although it has been reported that reducing the distance between the tethered fluorophore and the composite probe enhances chemiluminescence reporting, the preparation of this composition requires the synthesis of covalently linked fluorescent dyes trimethyl-β-cyclodextrin, a process that is too expensive and commercially infeasible for most applications.

[0095] The compositions described herein comprise donor chemiluminescent molecules and energy acceptor molecules housed within a host molecule. The donor chemiluminescent molecule is triggered by the selective removal of a protecting group from the donor chemiluminescent molecule through changes in environmental conditions and / or the addition of an enzyme or analyte of interest, thereby initiating chemiluminescence. Energy is transferred from the donor chemiluminescent molecule to the energy acceptor molecule. The host molecule houses a sufficient number of donor and energy acceptor molecules at a distance to generate a signal.

[0096] The signal is preferably detectable, including, for example, by the human eye or by a sensor, detector, or other device. Detection of chemiluminescent light may include the use of equipment. Such equipment may include, but is not limited to, photomultiplier tubes, photodiodes, charge-coupled device (CCD) cameras, photometers, scintillation counters, microplate readers, spectrometers, and bulk imaging devices capable of detecting light. Detection may even be aided by the use of a darkroom, a light-shielded space (e.g., a light-shielding box within an immunoassay instrument), or photosensitive paper.

[0097] On the one hand, the peak wavelength of the detectable signal generated by the composition can be in the range of 300 nm to 850 nm; alternatively, in the range of 350 nm to 700 nm; alternatively, in the range of 400 nm to 650 nm; alternatively, in the range of 450 nm to 600 nm; or alternatively, in the range of 500 nm to 550 nm. In some aspects, the wavelength of the peak signal generated by the composition is a combination of the peak wavelengths of fluorescence emitted by the donor chemiluminescent molecule and the energy acceptor molecule. Different donor chemiluminescent molecules and different energy acceptor molecules will affect the peak wavelength generated by the composition.

[0098] In some embodiments, the composition generates a detectable signal when the temperature of the composition is in the following ranges: falling within the range of 20°C to 80°C; alternatively falling within the range of 20°C to 70°C; alternatively falling within the range of 20°C to 60°C; alternatively falling within the range of 20°C to 50°C; or alternatively falling within the range of 25°C to 40°C. In some embodiments, the composition generates a detectable signal when the pressure is in the range of 0.8 atm to 1.2 atm; in the range of 0.9 atm to 1.1 atm; or at 1 atm. In another aspect, the composition generates a detectable signal when the pH of the composition is in the range of 2.0 to 13.0; in the range of 3.0 to 12.0; in the range of 4.0 to 11.0; in the range of 5.0 to 10.0; in the range of 6.0 to 9.0; or in the range of 6.0 to 8.0.

[0099] The signal generated by the composition upon triggering is expected to have greater intensity and higher quantum yield than the signal generated by triggering a single donor molecule. Quantum yield is typically the ratio of the number of emitted photons to the number of absorbed photons. Expressed as a percentage, the quantum yield of chemiluminescent molecules can be greater than 100%.

[0100] On the one hand, the reactive intermediate of the reaction between the donor chemiluminescent molecule and the energy acceptor molecule has a prolonged half-life compared to the half-life of the intermediate when it does not recombine with the host molecule, as seen in Example 2. Those skilled in the art will understand that the half-life of the reaction is the time required for the chemiluminescent molecule to decrease by half. It should also be understood that the extent of the prolonged half-life depends at least in part on the type of host molecule and / or its concentration in the composition.

[0101] In some embodiments, the composition is water-soluble. In such embodiments, the signal generated by the composition is preferably detectable in an aqueous solution. In some compositions present in this invention, the chemiluminescence emission of certain chemiluminescent resonance energy transfer systems is extremely weak under aqueous conditions. An aqueous environment can lead to water-induced energy transfer quenching. Water-induced quenching can result from water molecules colliding with excited-state fluorescent molecules and inactivating them in the absence of fluorescence emission; water molecules accepting energy from excited-state fluorescent molecules through a non-radiative energy transfer process; or water affecting the fluorescence properties of the fluorescent molecules, such as their polarity. For many chemiluminescent resonance energy transfer systems used in this invention, the addition of surfactants, such as hexadecyltrimethylammonium bromide (CTAB), reduces water-induced quenching by providing a hydrophobic environment and aggregating fluorescent molecules in micelle form, thereby significantly improving light emission efficiency. In some chemiluminescent resonance energy transfer systems used in this invention, a hydrophobic portion is added to the fluorescent molecules to reduce solubility and allow self-assembly into micelles.

[0102] In some embodiments, the composition does not need to be suspended in solution to generate an enhanced signal and can actually be deposited on a solid medium to generate a triggerable, detectable signal. The ability to generate a signal when deposited on a solid medium allows the composition, system, or method to be implemented in lateral flow assays or protein blotting. In contrast, formulations that use micelles to bring donor chemiluminescent molecules and energy acceptor molecules into close proximity, such as Lumi-Phos 530 (Lumigen, Inc., Southfield, MI), are undesirable for implementation on solid media because deposition on a solid medium disrupts the micelle structure, resulting in significant attenuation of light emission or signal. See Examples 5-7. Without wishing to be bound by any theory, it is believed that the signal attenuation observed in Examples 5-7 with Lumi-Phos 530 (Lumigen, Inc., Southfield, MI) is a result of disrupted micelle formation or destruction of the micelles. Deposition on a solid medium can disrupt micelles for several reasons. For example, a lack of fluidity and limited molecular mobility may prevent the dynamic rearrangement of surfactant molecules required for micelle formation. Furthermore, surfactant molecules can interact strongly with solid surfaces through adsorption, and this interaction can disrupt the organization required for micelle formation. Additionally, the solid surface can compete with the hydrophobic interactions that drive micelle assembly. Therefore, it is generally understood by those skilled in the art that micelle formation and stability require a liquid medium. See also Figure 7-9 And Examples 5-7.

[0103] In one embodiment, the composition did not exhibit a relative attenuation of chemiluminescence when triggered while adsorbed onto a filter medium, compared to when triggered in solution. That is, the composition emits light both when triggered in solution and when triggered while adsorbed onto a filter medium, and the light emission is not reduced when triggered while adsorbed onto a filter medium, compared to the light emission when triggered in solution. In some aspects, the composition did not exhibit attenuation of chemiluminescence when the filter medium contained 100% cotton fiber, nitrocellulose membrane, or polyvinylidene fluoride (PVDF) membrane. On the other hand, the composition did not exhibit attenuation of chemiluminescence when the solution contained: 3 ng alkaline phosphatase (AP) conjugate solution (ALP / mL in TRIS buffer, pH 9.0) and VPPD (0.3 mM) + Fls (0.3 mM) + bCDPol (0.8 mM) in AMP buffer (0.75 M 2-amino-2-methyl-1-propanol, hydrochloric acid, 1 mM MgCl2, pH 9.0). On another aspect, when the light intensity of the composition was measured at 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, or 50°C, the composition did not exhibit any attenuation of chemiluminescence. On yet another aspect, when the light intensity of the composition was measured after 2 minutes, 5 minutes, 8 minutes, 10 minutes, 12 minutes, or 15 minutes, the composition did not exhibit any attenuation of chemiluminescence (relative to chemiluminescence measured when triggered as in solution) when a certain volume of the composition was adsorbed onto a filter medium containing 100% cotton fibers and triggered with 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 when an aqueous solution of the composition was triggered by adding 10 µL of AP conjugate solution to twice the volume of the composition added to the filter medium, and measured after 10 minutes at 37°C.

[0104] In some aspects, the composition (comprising a donor chemiluminescent molecule, an energy acceptor molecule, and a host molecule) does not include micelles. Chemiluminescent substrates, such as Lumi-Phos 530 (which comprises dioxane and further comprises a surfactant that organizes the donor chemiluminescent molecule and the energy acceptor molecule into micelles), can be used in methods that trigger chemiluminescence in the presence of alkaline phosphatase. See, for example, U.S. Patent No. 5,004,565. While the micellar structure in such alternative compositions aggregates the donor chemiluminescent compound and the acceptor fluorescent compound together, thereby providing conditions for enhanced chemiluminescence, in the compositions described herein, the donor chemiluminescent molecule and the energy acceptor molecule are aggregated together by the host molecule in a non-micelle structure. As further described below, including, for example, in Examples 5-7, the compositions described herein do not require the formation of micelles to bring the donor chemiluminescent compound and the energy acceptor molecule close enough to observe an enhancement in energy transfer. Indeed, as described in Examples 5-7, when the compositions described herein are adsorbed onto a filter medium (which would disrupt the micellar structure), chemiluminescent resonant energy transfer and the generation of a detectable signal are maintained.

[0105] Without being bound by theory, the compositions described herein can possess certain advantages over chemiluminescent reagent formulations that rely on micellar structures to bring donor and acceptor molecules into close proximity. These advantages may include, for example, the ability to control the spatial relationship between donor chemiluminescent molecules and energy acceptor molecules, thereby potentially resulting in more robust enhancements in a wider variety of working environments or allowing for control over the timing of the enhancement of the chemiluminescent effect.

[0106] This disclosure also provides methods of using the compositions described herein. For example, in one aspect, a method for enhancing transient chemiluminescence may include providing a host molecule, recombinating a donor chemiluminescent molecule with the host molecule, and recombinating an energy acceptor molecule with the host molecule. In some embodiments, the method includes configuring the distance between the formed inclusion complexes to generate 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 purpose, such as to provide a calibrated maximum signal, to prolong the half-life of an active intermediate of the 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 generated at a specific rate.

[0107] In some embodiments, the method does not include forming a covalent bond between the donor chemiluminescent molecule and the cyclodextrin molecule; nor does it include forming a covalent bond between the energy acceptor molecule and the cyclodextrin molecule.

[0108] In some embodiments, the method does not include forming micelles or maintaining micellar kinetic stability. As further discussed above, for the compositions described herein, the donor chemiluminescent compound is aggregated with the energy acceptor molecule by the host molecule in a non-micelle structure, and it is not necessary to form micelles to bring the donor chemiluminescent compound and the energy acceptor molecule close enough to observe an enhancement in energy transfer.

[0109] The compositions described herein can be used in bioassay or diagnostic assay systems, including but not limited to, immunoassays, enzyme assays, protein array analysis, Western blot assays, Northern blot assays, Southern blot assays, immunoassay strips, or lateral flow assays. For example, the compositions described herein can be used as substrates in any suitable ALP-based immunoassay, including, for example, sandwich immunoassays (including one-step or two-step immunoassays), competitive immunoassays, on-strip immunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme amplification immunoassay (EMIT), etc. Example 8 provides an exemplary use of the compositions described herein in an immunoassay. Example 9 provides an exemplary use of the compositions described herein in a lateral flow assay.

[0110] On the one hand, the compositions described herein can be used in diagnostic testing systems that include test strips. Exemplary test strips are shown in... Figure 10 In the context of lateral flow test strips, lateral flow assays typically involve applying a sample to an absorbent sample pad at one end of the strip. The sample pad may include components that adapt the sample 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 an antibody specific for the target analyte and conjugated to colored or fluorescent particles. The sample, along with the conjugated antibody bound to the target analyte, migrates along the strip into the detection zone. Recognition of the sample analyte produces an appropriate response on the test line, while a response on the control line indicates appropriate liquid flow through the strip. Readouts can be assessed visually or using a dedicated reader. Finally, those skilled in the art will understand that the liquid flows "laterally" through the device due to capillary forces of the strip material, and to maintain this movement, the absorbent pad is typically attached to the end of the strip opposite the sample pad.

[0111] Those skilled in the art will understand that the techniques of this invention can be adapted to or incorporated into immunoassay test strips or lateral flow assays. The techniques described herein allow for the adaptation of lateral flow assays to donor-recipient-host systems that maintain or improve enhanced signal detection with such assays. Furthermore, adapting such lateral flow assays to the techniques of this invention can allow for a reduction in the cost of producing and using such assays. Exemplary examples of lateral flow assays using the compositions described herein are described in Example 9.

[0112] In addition to lateral flow assays, the compositions described herein can also be used in other types of immunoassays. Immunoassays are biochemical tests that rely on the interaction between antibodies and antigens for the detection or quantification of specific molecules or analytes in a sample. Labeling facilitates the detection or quantification of analytes, which is ideally suited to the techniques of this invention.

[0113] There are many different types of immunoassays. For example, there are heterogeneous and homogeneous immunoassays. Heterogeneous immunoassays require a physical separation step in which unbound antibodies and / or unbound analytes must be washed away, while homogeneous immunoassays do not require separation before analysis (all reagents can be freely suspended in the bulk solution). These immunoassays can be further divided into two groups: competitive and non-competitive.

[0114] In competitive (also known as "reagent-limited") immunoassays, the analyte 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 binding or free fraction of the labeled analyte is measured and correlated with the concentration of the analyte in the sample. In non-competitive (also known as "reagent-excess") immunoassays, antibodies (sometimes called secondary antibodies) can be labeled. The labeled antibody is mixed with the sample and binds to the analyte in the sample. The labeled antibody bound to the analyte can then be detected and analyzed.

[0115] Sandwich immunoassays present an example of heterogeneous, non-competitive immunoassays. A capture antibody is immobilized on a solid surface and coated with a solid phase (such as a microplate or membrane). A sample containing the analyte is then added to the coated solid phase and incubated. During this time, if the analyte is present, it binds to the immobilized antibody. A washing step following incubation removes unbound material and any non-specific binding, leaving only the captured analyte on the solid phase. If a secondary labeled antibody is used, it binds to either the captured analyte or the primary (capture) antibody.

[0116] For detection and visualization in each of these assays, chemiluminescence can be used. For example, an antibody bound to a target analyte and conjugated to a trigger enzyme or substrate can be mixed with a sample suspected of containing the analyte; after washing (e.g., to remove any antibodies not bound to the target analyte), the composition described herein can be added to the mixture, and the sensor used detects the chemiluminescence to determine the presence and amount of the target analyte. Thus, in some aspects, this disclosure describes an immunoassay composition comprising an antibody and the composition described herein, said antibody being bound to a target analyte and conjugated to a trigger enzyme or substrate.

[0117] The compositions described herein are suitable for use in many types of immunoassays or bioassays in which a chemiluminescent reporter gene is used. Example 8 illustrates an exemplary use of the composition in an immunoassay. Further, as described above, examples of the compositions can be used in lateral flow assays, as shown in the exemplary embodiments in Example 9.

[0118] The compositions described herein can also be used in Western blotting. Western blotting, or immunoblotting, is a common assay used to study different aspects of protein biomolecules. Western blotting assays can identify and quantify specific proteins in complex mixtures extracted from cell or tissue lysates. For example, Western blotting assays can detect different isoforms of proteins, distinguish protein-protein interactions or protein-DNA interactions, detect post-translational modifications, locate subcellular protein functions, and aid in antibody development and disease diagnosis.

[0119] In a typical Western blot assay, the process begins with obtaining native or denatured proteins as the research target. These are separated by size and charge using gel electrophoresis. The proteins are then transferred to a protein-binding membrane, such as nitrocellulose or polyvinylidene fluoride (PVDF). This transfer is the source of the eponymous "blot." Following the transfer, a blocking step is typically performed. The membrane containing the transferred proteins is incubated with a blocking solution. The blocking solution is used to prevent non-specific binding of antibodies (blocking can fill pores in the membrane where antibodies can be non-specifically found or simply trapped). This step reduces background signal. After blocking, the membrane is incubated with a primary antibody specific to the target protein for protein detection. The antibody binds to the target protein on the membrane. The membrane is then washed to remove any excess primary antibody.

[0120] Visualization is the final step. Sometimes, the primary antibody has a conjugated detectable label, but more often a secondary antibody is used. The secondary antibody and the membrane bound to the primary antibody are incubated together. The secondary antibody is then conjugated to a detectable label or an enzyme. In this example, the secondary antibody may be conjugated to an enzyme used to trigger the removal of triggerable dioxane. The enzyme catalyzes the reaction with the triggerable dioxane, thereby generating light. Alternatively, the secondary antibody may be conjugated to a donor-receptor-host system, and a triggering enzyme may be added to the membrane to trigger chemiluminescence. Dedicated photodetector equipment can capture and record the emitted light for visualization.

[0121] Following signal detection, the protein blot is analyzed to determine the presence and quantity of the target protein. This involves assessing the intensity and size of the band corresponding to the target protein. The compositions described herein can provide improvements to existing systems and / or reduce costs.

[0122] Conjugating the compositions described herein with nucleotides, DNA strands, or RNA strands allows for visualization and detection in assays such as Southern blotting or Northern blotting. Southern blotting is a technique for detecting specific DNA sequences in complex mixtures. It involves several steps, including DNA digestion, gel electrophoresis, transfer to a membrane, hybridization with a labeled probe, and detection. Southern blotting can be used to analyze DNA fragments based on size and sequence. It has been widely used in tasks in molecular biology, such as mapping genes, identifying DNA polymorphisms, and confirming the presence or absence of specific DNA sequences in genomic DNA.

[0123] Similar to Southern blotting, Northern blotting is used to study gene expression by detecting and analyzing RNA molecules. Northern blotting involves several similar steps, including RNA electrophoresis, transfer to a membrane, hybridization with labeled probes, and detection. Detection using a donor-recipient-host system may be possible, and existing systems could be improved.

[0124] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order; and, if necessary, any combination of two or more steps may be performed simultaneously.

[0125] All headings are for the reader's convenience and should not limit the meaning of the text following them unless otherwise specified.

[0126] The terms "preferred" and "ideally" refer to embodiments of the invention that offer certain advantages under specific circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are useless, nor is it intended to exclude other embodiments from the scope of the invention.

[0127] The term "comprising" and its variations are not intended to be limiting in their use in the specification and claims. Such terms are to be understood as implying the inclusion of the stated steps or elements or groups of steps or elements, but not excluding any other steps or elements or groups of steps or elements.

[0128] The phrase "consisting of..." means including and limited to anything that follows the phrase "consisting of...". Therefore, the phrase "consisting of..." indicates that the listed element is necessary or mandatory, and no other elements may be present. The phrase "substantially consisting of..." means including any element listed after this phrase, and is limited to other elements that do not interfere with or contribute to the activities or actions specified in this disclosure of the listed element. Therefore, the phrase "substantially consisting of..." indicates that the listed element is necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activities or actions of the listed element.

[0129] Unless otherwise stated, “a / an,” “the,” and “at least one” are used interchangeably and mean one or more. These articles indicate one or more (i.e., at least one). The term “and / or” means any one or more items in the list connected by “and / or.” As an example, “x and / or y” means any element in 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 in 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.”

[0130] Where a range is given, the endpoints include all numbers contained within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise specified or clearly apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges in different embodiments of this disclosure may take any particular value or subrange within the stated range, accurate to one-tenth of the lower limit unit of the range, unless the context explicitly specifies otherwise. In this document, a “maximum” number (e.g., at most 50) includes that number (e.g., 50). The terms “in the range” or “within the range” (and similar statements) include the endpoints of the stated range.

[0131] Throughout this specification, references to "an aspect," "aspect," "certain aspects," or "some aspects," etc., mean that a particular feature, configuration, composition, or material or property described in connection with said aspect is included in at least one aspect of this disclosure. Therefore, the appearance of such phrases throughout this specification does not necessarily refer to the same embodiment of this disclosure. Furthermore, specific features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0132] Unless otherwise specified, all figures representing amounts of components, molecular weights, etc., used in the specification and claims should be understood to be modified by the term "about" in all cases. As used herein, with respect to a measured quantity, the term "about" refers to the variation in the measured quantity expected by a person skilled in the art when performing the measurement and taking appropriate precautions commensurate with the purpose of the measurement and the accuracy of the measuring equipment used. The term "about," used throughout the specification and claims in conjunction with numerical values, indicates an accuracy range familiar and acceptable to a person skilled in the art. Generally, unless otherwise stated, the accuracy interval is + / - 10%. Therefore, unless otherwise specified, the numerical parameters shown in the specification and claims are approximations that can vary to the desired properties sought to be obtained according to the invention. At least, without attempting to limit the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0133] While the wide range of numerical ranges and parameters described in this invention are approximations, the values ​​shown in the specific examples are reported as precisely as possible. However, all values ​​inherently include a range that is necessarily generated by the standard deviation present in their respective test measurements.

[0134] The term “exemplary” means as a non-limiting example, instance, or illustration. As used herein, the terms “e.g.” and “for example” list one or more non-limiting aspects, examples, instances, or illustrations.

[0135] The terminology used herein is for the purpose of describing particular instances only and is not intended to limit this disclosure. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. It should be further understood that when the terms “comprises,” “includes,” “comprising,” “including,” “has,” “have,” and “having” are used in this specification, they specify the presence of the stated feature, integer, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0136] It will be understood that while 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 used only to distinguish one element from another. Thus, for example, without departing from the teachings of this disclosure, the first element, first component, or first segment discussed below may be referred to as the second element, second component, or second segment. Similarly, various spatial terms, such as “upper,” “lower,” “side,” etc., may be used to distinguish one element from another in a relative manner. However, it should be understood that components may be oriented in different ways without departing from the teachings of this disclosure; for example, a semiconductor device may be rotated laterally such that its “top” surface faces horizontally and its “side” surface faces vertically.

[0137] As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or extent of the characteristic or property of interest. Biological and chemical phenomena rarely (if at all) proceed to completion and / or progress to fullness or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the inherent lack of completeness in many biological and chemical phenomena. For example, "substantially" may mean at least about 20%, alternatively at least about 10%, or alternatively at least about 5% of the characteristic or property of interest.

[0138] 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 pertain.

[0139] The invention is defined in the claims. However, the following is a non-exhaustive list of non-limiting exemplary aspects. Any one or more features of these aspects may be combined with any one or more features of another instance, embodiment, or aspect described herein.

[0140] These features are more fully illustrated by the following examples provided for illustrative purposes and should not be construed as limiting the invention in any way.

[0141] Example

[0142] All reagents, starting materials, and solvents used in the following examples were purchased from commercial suppliers such as Sigma Aldrich, St. Louis, MO, and are ready for use without further purification unless otherwise instructed.

[0143] Example 1. Transient chemiluminescence of donor-acceptor cyclodextrin compositions

[0144] The light emission of different formulations was measured using a Turner TD-20 / 20 single-tube photometer (Turner Designs, San Jose, CA). PPD and the luciferin surfactant (N-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthon]-5-yl)tetradecanoamide) (Fls) were combined with different concentrations of polymeric β-cyclodextrin (bCDpol) (as shown in Table 1) in AMP buffer (0.75 M 2-amino-2-methyl-1-propanol, hydrochloric acid, 1 mM MgCl2, pH 9.0). Then, 10 µL of alkaline phosphatase (ALP) (0.04 U / µL in diethanolamine buffer) was added, and light emission was measured at 37°C for a time window typically corresponding to the emission peak (approximately 10–15 minutes).

[0145] Light emission was measured against baseline conditions (PPD alone). For the positive control, light emission was also measured with Lumi-Phos530 (Lumigen, Southfield, Michigan), a chemiluminescent reagent formulation comprising PPD, Fls, and hexadecyltrimethylammonium bromide (CTAB) incorporated into the micelle structure. Table 1 shows the results for the different conditions, reported as in relative light units (RLU), and the amount of enhancement relative to PPD alone.

[0146]

[0147] Example 2. Half-life data during quantum yield measurement conditions

[0148] The quantum yield experimental setup included 100 μL of buffer solution (AMP, described in a previous example) containing PPD (0.3 mM), mixed with 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 samples were vortexed and immediately transferred to a single-tube spectrophotometer (Turner TD-20 / 20, Turner Corporation, San Jose, CA). Light output was measured over 60-minute intervals at 37 °C. Figure 5 The changes in light intensity (relative light units (RLU)) of various donor / acceptor responses over time (in minutes) are shown, and host-guest complex formation that influences the half-life of active chemiluminescent intermediates is demonstrated.

[0149]

[0150] Example 3. Chemiluminescence Spectroscopy Study

[0151] The light emission from Lumi-Phos 530 (PPD, Fls, and CTAB) [LP530]; combinations of PPD and Fls [Fls+PPD]; or combinations of PPD with polymer β-cyclodextrin (bCDpol) and Fls [bCDpol+PPD+Fls] was analyzed using a spectrophotometer (FP-8650, Jasco, Easton, MD). After vortex mixing, the samples were immediately transferred to the photometer, and the spectra of the chemiluminescent emission from these reactions are shown in […]. Figure 6a In the middle; the peak wavelength of each formulation is 525 nm. As a control, the light emission of individual PPDs was measured, and the results are shown in Figure 6b The peak wavelength is 455 nm.

[0152] Example 4. Chemiluminescence Measurement

[0153] The light emission of various formulations, including combinations of PPD, VMPPD, and VPPD (0.3 mM) with Fls (0.03 mM) and polymeric β-cyclodextrin (0.8 mM, based on cyclodextrin units), was measured using a single-tube luminometer (Turner TD-20 / 20). Emissions from Lumi-Phos 530 were used as a positive control, and solutions of PPD (0.3 mM) and Fls (0.03 mM) were used as baseline controls. Table 3 shows the results under different conditions, reported as relative light units (RLU), and the amount of enhancement relative to PPD + Fls.

[0154]

[0155] Example 5. Adsorption on filter paper

[0156] To determine whether the chemiluminescence enhancement achieved with polymeric cyclodextrin was due to micelle formation, the light intensity of the formulation in solution was compared with the light intensity of the same formulation adsorbed on filter paper.

[0157] To measure the light intensity in solution, 10 µL of alkaline phosphatase (AP) conjugate solution (3 ng ALP / mL in TRIS buffer, pH 9.0) was added to either 100 µL of LP530 or 100 µL of VPPD (0.3 mM) + Fls (0.3 mM) + bCDPol (0.8 mM) in AMP buffer at 37 °C. The light intensity was measured on a single-tube photometer (Turner TD-20 / 20, Turner Corporation, San Jose, California) after 10 minutes. The results are shown in Table 4 (“Solution” entry).

[0158]

[0159] To measure light intensity on filter paper, 5 µL of LP530 or 5 µL of the above VPPD+Fls+bCDPol solution was adsorbed onto room temperature filter paper (Mini Trans-Blot cell filter paper, BIO-RAD, Hercules, CA) at 37°C. Then, 3 µL of the AP conjugate solution (3 ng ALP / mL in TRIS buffer, pH 9.0) was added to the center of a 75 × 100 mm filter paper. After 10 minutes at room temperature, light intensity was measured using an Amersham Imager 600 (GE Life Sciences, Chicago, IL) with automatic exposure settings (which calculate the optimal exposure for a given light intensity).

[0160] Figure 7 The results are shown using Image Quant TL software v8.1 (GE). The light intensity data are presented in Table 5 (“Filter Paper” entry).

[0161]

[0162] The results of this example demonstrate that chemiluminescence is enhanced when VPPD is combined with Fls and polymeric cyclodextrin on a solid medium (e.g., filter paper) compared to when VPPD is combined with Fls and polymeric cyclodextrin in solution. In contrast, the formulation Lumi-Phos 530, known to form micellar structures, exhibits reduced chemiluminescence on a solid medium. The enhanced chemiluminescence observed in formulations containing polymeric cyclodextrin on a solid medium (which would disrupt most of the micellar structure) suggests that enhanced chemiluminescence does not require micellar formation. In contrast, the relatively reduced light intensity of Lumi-Phos 530 on filter paper is considered a result of a substantial reduction (or disruption) of the micellar structure.

[0163] Example 6. Adsorption on nitrocellulose membranes

[0164] To determine whether the chemiluminescence enhancement achieved with polymeric cyclodextrins could also be achieved on nitrocellulose membranes (such as those used in lateral flow immunoassays), the light intensity of the formulation in solution was compared with the light intensity of the same formulation adsorbed onto the nitrocellulose membrane. LP530 light intensity data in solution were used for comparison and were taken from Example 5.

[0165] To measure the light intensity on the nitrocellulose membrane, 5 µL of LP530 or 5 µL of the above VPPD+Fls+bCDPol solution was adsorbed onto a nitrocellulose membrane (Amersham Protran Premium 0.45 µm blot membrane, Cytiva, Marlborough, MA). Then, 3 µL of the AP conjugate solution (3 ng ALP / mL in TRIS buffer, pH 9.0) was added to the center of a 75 × 100 mm nitrocellulose membrane. After 10 minutes at room temperature, the light intensity was measured using an Amersham Imager 600 (GE Life Sciences, Chicago, Illinois) with automatic exposure settings.

[0166] Figure 8 The results are shown using Image Quant TL software v8.1 (GE). The light intensity data are presented in Table 6 (“Nitrocellulose Membrane” entry).

[0167]

[0168] The results of this example demonstrate that VPPD+Fls+bCDPol can be used to detect analytes of interest on nitrocellulose membranes.

[0169] Example 7. Adsorption on polyvinylidene fluoride (PVDF) membranes

[0170] To determine whether the chemiluminescence enhancement achieved with polymeric cyclodextrin could also be achieved on a PVDF membrane, the light intensity of the formulation in solution was compared with that of the same formulation adsorbed onto the PVDF membrane. LP530 light intensity data in solution, taken from Example 5, were used for comparison.

[0171] To measure the light intensity on the PVDF membrane, 5 µL of LP530 or 5 µL of the above VPPD+Fls+bCDPol solution was adsorbed onto the PVDF membrane (Amersham Hybond PVDF 0.2 µm imprint membrane, Sitervan, Marlborough, MA). Then, 3 µL of the AP conjugate solution (3 ng ALP / mL in TRIS buffer, pH 9.0) was added to the center of a 75 × 100 mm PVDF membrane. After 10 minutes at room temperature, the light intensity was measured using an Amersham Imager 600 (GE Life Sciences, Chicago, Illinois) with automatic exposure settings.

[0172] Figure 9The results are shown using Image Quant TL software v8.1 (GE). The light intensity data are presented in Table 7 (“PVDF film” entry).

[0173]

[0174] These results demonstrate that VPPD+Fls+bCDPol can be used to detect analytes of interest on PVDF membranes.

[0175] Example 8. Immunoassay

[0176] Immunoassays were performed to detect goat antigen (normal goat IgG control, R&D Systems, Minneapolis, MN). Magnetic particles (DYNABEADS MyOne epoxy, Thermo Fisher Scientific, Waltham, MA) were conjugated with capture antibodies (IgG fractionated monoclonal mouse anti-goat IgG, light chain specific, Jackson ImmunoResearch, West Grove, PA) and suspended in a TRIS-based diluent buffer for the particles.

[0177] Add 125 µL of particle diluent buffer and 25 µL of 1 mg / mL particles conjugated with the capture antibody to a 0.6 mL centrifuge tube. Then add 50 µL of goat antigen (in particle diluent buffer) (S1) or 50 µL of particle diluent buffer (to determine background, S0) to the tube. Finally, add particle diluent buffer containing 50 µL of 300 ng / mL mouse anti-goat alkaline phosphatase conjugate (alkaline phosphatase IgG fractionated monoclonal mouse anti-goat IgG, light chain specific, Jackson Institute for Immunology Laboratory, Sigrove, PA).

[0178] 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 three times with 0.5 mL of wash buffer (ACCESS Wash Buffer II (Beckman Coulter, Brea, CA)), 100 µL of chemiluminescent substrate was added. The chemiluminescent substrate was Lumi-Phos 530 (Lumigen, Southfield, Michigan) or formulation 1, which consisted of 0.8 mM PPD, Fls, and polymeric cyclodextrin (bCDpol).

[0179] Light output was measured after 15 minutes at 37°C using a Turner TD-20 / 20 single-tube photometer (Turner, San Jose, California). The results are shown in Table 8, providing a proof of concept that polymeric cyclodextrin-based formulations can be used to detect antigens in the context of immunoassays.

[0180]

[0181] Example 9. Lateral Flow Measurement

[0182] A two-step lateral flow assay will be performed to detect the antigen (e.g., normal goat IgG control, R&D Systems, Minneapolis, .M., .M., .M., R&D Systems, .M. ... The developed solution will include (1) a formulation comprising polymeric cyclodextrin, VPPD and Fls, (2) Lumi-Phos 530 (Lumigen, Southfield, Michigan) and a suitable positive control (e.g., APS5, Lumigen, Southfield, Michigan).

[0183] It is expected that when read on the detector after exposure to the developing solution, both the control line and the test line will be positive when a positive control developing solution is added. Based on the results of Example 6, it is expected that the light produced by the formulation comprising polymeric cyclodextrin, VPPD and Fls is greater than that produced by Lumi-Phos 530 in (2), indicating that (1) can produce significantly more light compared to (2) on a cellulose membrane.

[0184] While examples have been used to describe embodiments of the invention, those skilled in the art will understand that various changes can be made and equivalents can replace elements in the examples, and the examples are not intended to limit the scope of the invention, nor are they intended to limit the invention to the specific embodiments disclosed as examples.

Claims

1. A composition comprising: Donor chemiluminescent molecules; Energy acceptor molecules; and Host molecule, wherein the host molecule contains at least two guest molecules, The first guest molecule comprises a donor chemiluminescent molecule. The second guest molecule, which includes an energy acceptor molecule, When contained within the host molecule, the at least two guest molecules are structurally spaced apart to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor 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 suitable size and affinity for the encapsulation portion of the host molecule.

3. The composition according to any one of the preceding claims, wherein the host molecule and the at least two guest molecules form a combined inclusion complex; In the aforementioned composite inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule; and In the combined inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule.

4. The composition according to any one of the preceding claims, wherein the composition does not contain micelles.

5. The composition according to any one of the preceding claims, wherein the composition does not need to be suspended in a solution to generate an enhanced signal.

6. The composition according to any one of the preceding claims, wherein the host molecule comprises a dimer cyclodextrin, a polymeric cyclodextrin, a polymeric cyclodextrin molecule, and / or a combination thereof.

7. The composition according to any one of the preceding claims, wherein when contained in the host molecule, the distance between the chemiluminescent molecule and the energy acceptor molecule is 12 Å to 13.5 Å; 12 Å to 15 Å; 12 Å to 16.5 Å; 12 Å to 18 Å; 12 Å to 19.5 Å; alternatively 12 Å to 21 Å; alternatively 12 Å to 22.5 Å; 12 Å to 24 Å; 12 Å to 25.5 Å; and / or 12 Å to 50 Å.

8. The composition according to any one of the preceding claims, wherein, when contained in the host molecule, the chemiluminescent signal generated by the distance between the chemiluminescent molecule and the energy acceptor molecule is 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 according to any one of the preceding claims, wherein the host molecule comprises: Wherein R includes: OH, -O-Alk (containing alkyl), -O-PEG (containing PEG), -O(CH2)x-PR'3 (containing alkyl phosphocation), -O(CH2)x-NR'3 (containing alkyl ammonium), -O(CH2)x-(aryl)-CH2-NR'3 (containing alkyl ammonium), or -O(CH2)x-(aryl)-CH2-NR'3 (containing alkyl ammonium), or combinations thereof; and n is an integer of at least 2.

10. The composition of claim 8, wherein the average molecular weight of the host molecule comprises a range of n multiplied by 1,100 Daltons to n multiplied by 1,500 Daltons (n*1,100 to n*1,500 Da).

11. The composition according to any one of the preceding claims, wherein the donor chemiluminescent molecule comprises triggerable dioxane.

12. The composition of claim 11, wherein the triggerable dioxane comprises a removable protecting group trigger, a dioxane moiety, and an adamantyl group.

13. The composition of claim 11 or 12, wherein the triggerable dioxane comprises a triggerable group, the triggerable group comprising any portion that forms a phenolic ester when environmental conditions change or in the presence of a specific analyte or enzyme.

14. The composition according to any one of claims 11 to 13, wherein the trigger comprises -OH, -OP(O)(OR), -O acyl, -B(OH)2, -aryl-boronate, -β-D-galactoside, -OSiMe2tBu, -OSiR3 or -OR, wherein R comprises a group removable by a selectable analyte or by a selectable enzyme.

15. The composition according to any one of claims 11 to 14, wherein the triggerable dioxane comprises a triggerable phenoxy-dioxane having an electron-withdrawing or electron-accepting substituent at an ortho position of the phenoxy-dioxane.

16. The composition according to any one of claims 11 to 15, wherein the triggerable dioxane comprises 3-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)phenyl dihydrogen phosphate (PPD), 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD), or 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD) or a combination thereof.

17. The composition according to any one of the preceding claims, wherein the energy acceptor molecule comprises a fluorescent molecule, an anchor, and optionally a connector.

18. The composition of claim 17, wherein the fluorescent molecule comprises a fluorescent molecule with an excitation frequency in the range of 300 nm to 750 nm; in the range of 350 nm to 700 nm; in the range of 400 nm to 650 nm; in the range of 450 nm to 600 nm; or in the range of 500 nm to 550 nm.

19. The composition according to any one of claims 17 or 18, wherein the anchor comprises a group having a strong binding affinity to the host molecule, adapted to form an inclusion complex comprising the host molecule and the energy acceptor molecule.

20. The composition of claim 19, wherein the anchor comprises long-chain n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl and / or adamantyl.

21. The composition according to any one of claims 17 to 20, wherein the energy acceptor molecule comprises a connector, and wherein the connector connects the anchor and the fluorescent molecule.

22. The composition of claim 21, wherein the connector comprises a long-chain alkane, a long-chain alkyl ether, a long-chain alkyl ester, an acid anhydride, an isocyanate, carbodiimide, or glutaraldehyde or a combination thereof.

23. The composition according to any one of the preceding claims, wherein the donor chemiluminescent molecule is not covalently bound to the host molecule.

24. The composition according to any one of the preceding claims, wherein the energy acceptor molecule is not covalently bound to the host molecule.

25. The composition according to any one of the preceding claims, wherein the composition is water-soluble.

26. The composition according to any one of the preceding claims, wherein the composition generates a detectable signal when triggered.

27. The composition of claim 26, wherein the peak of the detectable signal is in the wavelength range of 300 nm to 850 nm; in the range of 350 nm to 700 nm; in the range of 400 nm to 650 nm; in the range of 450 nm to 600 nm; or in the range of 500 nm to 550 nm.

28. The composition according to any one of the preceding claims, wherein the active intermediate of the reaction between the donor chemiluminescent molecule and the energy acceptor molecule has an extended half-life compared to the half-life of the intermediate in the absence of recombination between the donor chemiluminescent molecule and the energy acceptor molecule with the host molecule.

29. The composition according to any one of the preceding claims, wherein the composition generates a detectable signal when the temperature of the composition is one of the following: The temperature falls within the range of 20 degrees Celsius to 80 degrees Celsius; the temperature falls within the range of 20 degrees Celsius to 70 degrees Celsius; the temperature falls within the range of 20 degrees Celsius to 60 degrees Celsius; the temperature falls within the range of 20 degrees Celsius to 50 degrees Celsius; the temperature falls within the range of 25 degrees Celsius to 40 degrees Celsius; or the temperature falls within the range of 35 degrees Celsius to 40 degrees Celsius.

30. The composition according to any one of the preceding claims, wherein the composition generates a detectable signal when the pressure is in the range of 0.8 atm to 1.2 atm; in the range of 0.9 atm to 1.1 atm; or 1 atm.

31. The composition according to any one of the preceding claims, wherein the composition generates the detectable signal when the pH of the composition is in the range of 2.0 to 13.0; in the range of 3.0 to 12.0; in the range of 4.0 to 11.0; in the range of 5.0 to 10.0; in the range of 6.0 to 9.0; or in the range of 6.0 to 8.

0.

32. The composition according to any one of the preceding claims, wherein when deposited on a solid medium and triggered, the composition generates a detectable signal.

33. The composition according to any one of the preceding claims, wherein the composition does not exhibit a relative attenuation of chemiluminescence when adsorbed on a filter medium and triggered, relative to when it is in solution and triggered.

34. A method of using the composition according to any one of claims 1 to 33.

35. A method for using the composition according to any one of claims 1 to 33 in an immunoassay.

36. A method for using the composition according to any one of claims 1 to 33 in a lateral flow measurement.

37. A method for using the composition according to any one of claims 1 to 33 in a protein blot assay.

38. A transient chemiluminescence enhancement system, comprising: Donor chemiluminescent molecules; Energy acceptor molecules; and Host molecule, wherein the host molecule contains at least two guest molecules, The first guest molecule comprises a donor chemiluminescent molecule. The second guest molecule, which includes an energy acceptor molecule, When contained within the host molecule, the at least two guest molecules are structurally spaced apart to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor 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 suitable size and affinity for the encapsulation portion of the host molecule.

40. The system according to any one of claims 38 to 39, wherein the host molecule and the at least two guest molecules form a combined inclusion complex; In the aforementioned composite inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule; and In the combined inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule.

41. The system according to any one of claims 38 to 40, wherein the composition does not contain micelles.

42. The system according to any one of claims 38 to 41, wherein the composition does not need to be suspended in a solution to generate an enhanced signal.

43. The system according to any one of claims 38 to 42, wherein the host molecule comprises dimer cyclodextrin, polymeric cyclodextrin, and / or polymeric cyclodextrin molecules.

44. The system according to any one of claims 38 to 43, wherein when contained in the host molecule, the distance between the chemiluminescent molecule and the energy acceptor molecule is 12 Å to 13.5 Å; 12 Å to 15 Å; 12 Å to 16.5 Å; 12 Å to 18 Å; 12 Å to 19.5 Å; 12 Å to 21 Å; 12 Å to 22.5 Å; 12 Å to 24 Å; 12 Å to 25.5 Å; or 12 Å to 50 Å.

45. The system according to any one of claims 38 to 44, wherein, when contained in the host molecule, the chemiluminescent signal generated by the distance between the chemiluminescent molecule and the energy acceptor molecule is 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 according to any one of claims 38 to 45, wherein the host molecule comprises: Wherein R includes: OH, -O-Alk (containing alkyl), -O-PEG (containing PEG), -O(CH2)x-PR'3 (containing alkyl phosphocation), -O(CH2)x-NR'3 (containing alkyl ammonium), -O(CH2)x-(aryl)-CH2-NR'3 (containing alkyl ammonium), or -O(CH2)x-(aryl)-CH2-NR'3 (containing alkyl ammonium), or combinations 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 1,100 Daltons to n multiplied by 1,500 Daltons (n*1,100 to n*1,500 Da).

48. The system according to any one of claims 38 to 47, wherein the donor chemiluminescent molecule comprises triggerable dioxane.

49. The system of claim 48, wherein the triggerable dioxane comprises a removable protecting group trigger, a dioxane moiety, and an adamantyl group.

50. The system of claim 48 or 49, wherein the triggerable dioxane comprises a triggerable group, wherein the trigger comprises any portion that forms a phenolic ester when environmental conditions change or in the presence of a specific analyte or enzyme.

51. The system according to any one of claims 48 to 50, wherein the trigger comprises -OH, -OP(O)(OR), -O acyl, -B(OH)2, -aryl-boronate, -β-D-galactoside, -OSiMe2tBu, -OSiR3 or -OR, wherein R comprises a group removable by a selectable analyte or by a selectable enzyme.

52. The system according to any one of claims 48 to 51, wherein the triggerable dioxane comprises a triggerable phenoxy-dioxane having an electron-withdrawing or electron-accepting substituent at an ortho position of the phenoxy-dioxane.

53. The system according to any one of claims 48 to 52, wherein the triggerable dioxane comprises 3-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)phenyl dihydrogen phosphate (PPD), 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-2-vinylphenyl dihydrogen phosphate (VPPD), or 5-(4'-methoxyspiro[adamantane-2,3'-[1,2]dioxane]-4'-yl)-3-methyl-2-vinylphenyl dihydrogen phosphate (VMPD) or a combination thereof.

54. The system according to any one of claims 48 to 53, wherein the energy acceptor molecule comprises a fluorescent molecule, an anchor, and optionally a connector.

55. The system of claim 54, wherein the fluorescent molecule comprises a fluorescent molecule with an excitation frequency in the range of 310 nm to 750 nm; in the range of 350 nm to 700 nm; in the range of 400 nm to 650 nm; in the range of 450 nm to 600 nm; or in the range of 500 nm to 550 nm.

56. The system according to any one of claims 54 or 55, wherein the anchor comprises a group having a strong binding affinity to the host molecule, adapted to form an inclusion complex comprising the host molecule and the energy acceptor molecule.

57. The system according to any one of claims 54 to 56, wherein the anchor comprises n-alkyl, cyclohexyl, biphenyl, naphthyl, tert-butyl and / or adamantyl.

58. The system according to any one of claims 54 to 57, wherein the energy acceptor molecule comprises a connector, and wherein the connector connects the anchor and the fluorescent molecule.

59. The system of claim 58, wherein the connector comprises a long-chain alkane, a long-chain alkyl ether, a long-chain alkyl ester, an acid anhydride, an isocyanate, carbodiimide, or glutaraldehyde or a combination thereof.

60. The system according to any one of claims 38 to 59, wherein the donor chemiluminescent molecule is not covalently bound to the host molecule.

61. The system according to any one of claims 38 to 59, wherein the energy acceptor molecule is not covalently bound to the host molecule.

62. The system according to any one of claims 38 to 61, wherein the composition is water-soluble.

63. The system according to any one of claims 38 to 62, wherein the composition generates a detectable signal when triggered.

64. The system according to any one of claims 38 to 63, wherein the peak of the detectable signal is in the wavelength range of 300 nm to 850 nm; in the range of 350 nm to 700 nm; in the range of 400 nm to 650 nm; in the range of 450 nm to 600 nm; or in the range of 500 nm to 550 nm.

65. The system according to any one of claims 38 to 64, wherein the active intermediate of the reaction between the donor chemiluminescent molecule and the energy acceptor molecule has an extended half-life compared to the half-life of the intermediate in the absence of recombination between the donor chemiluminescent molecule and the energy acceptor molecule with the host molecule.

66. The system according to any one of claims 38 to 65, wherein the composition generates a detectable signal when the temperature of the composition is in the following ranges: falling within the range of 20°C to 80°C; falling within the range of 20°C to 70°C; falling within the range of 20°C to 60°C; falling within the range of 20°C to 50°C; falling within the range of 25°C to 40°C; or falling within the range of 35°C to 40°C.

67. The system according to any one of claims 38 to 66, wherein the composition generates a detectable signal when the pressure is in the range of 0.8 atm to 1.2 atm; in the range of 0.9 atm to 1.1 atm; or 1 atm.

68. The system according to any one of claims 38 to 67, wherein the composition generates a triggerable chemiluminescence signal when the pH of the composition is in the range of 2.0 to 13.0; in the range of 3.0 to 12.0; in the range of 4.0 to 11.0; in the range of 5.0 to 10.0; in the range of 6.0 to 9.0; or in the range of 6.0 to 8.

0.

69. The system according to any one of claims 38 to 68, wherein the composition generates a detectable signal when deposited on a solid medium and triggered.

70. The system according to any one of claims 38 to 69, wherein the composition does not exhibit a relative attenuation of chemiluminescence when adsorbed on a filter medium and triggered relative to when it is in solution and triggered.

71. A method of using the system according to any one of claims 38 to 70.

72. A method for using the system according to any one of claims 38 to 70 in an immunoassay.

73. A method for using the system according to any one of claims 38 to 70 in lateral flow measurement.

74. A method for using the system according to any one of claims 38 to 70 in a protein blot assay.

75. A method for enhancing transient chemiluminescence, the method comprising: Provide host molecules, The donor chemiluminescent molecule recombines with the host molecule. This allows the energy receptor molecule to recombine with the host molecule.

76. The method of claim 75, further comprising: Configure the distance between the formed inclusions to generate a triggerable, detectable signal.

77. The method according to claim 75 or 76, wherein the host molecule comprises a dimer, a polymer, or a polymeric cyclodextrin molecule.

78. The method according to any one of claims 75 to 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 strong affinity for the encapsulation portion of the host molecule.

79. The method according to any one of claims 75 to 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. In the first inclusion complex, the host molecule spatially encloses at least a portion of the donor chemiluminescent molecule; and In the second inclusion complex, the host molecule spatially encloses at least a portion of the energy acceptor molecule.

80. The method according to any one of claims 75 to 79, wherein the method does not comprise: To form micelles or maintain micelle dynamic stability.

81. The method according to any one of claims 75 to 80, wherein the method does not comprise: A covalent bond is formed between the donor chemiluminescent molecule and the cyclodextrin molecule; or A covalent bond is formed between the energy acceptor molecule and the cyclodextrin molecule.

82. The method according to any one of claims 75 to 81, further comprising: Extend the half-life of the active intermediate in the reaction between the donor chemiluminescent molecule and the energy acceptor molecule.

83. The method according to any one of claims 75 to 82, further comprising: The distance between the donor chemiluminescent molecule that has compounded with the host molecule and the energy acceptor molecule is configured such that the chemiluminescent signal generated by the reaction between the donor chemiluminescent molecule that has compounded with the host molecule and the energy acceptor molecule is approximately inversely proportional to the sixth power of the distance between the donor chemiluminescent molecule that has compounded with the host molecule and the energy acceptor molecule.

84. The system according to any one of claims 75 to 83, wherein when contained in the host molecule, the distance between the chemiluminescent molecule and the energy acceptor molecule is 12 Å to 13.5 Å; 12 Å to 15 Å; 12 Å to 16.5 Å; 12 Å to 18 Å; 12 Å to 19.5 Å; 12 Å to 21 Å; 12 Å to 22.5 Å; 12 Å to 24 Å; 12 Å to 25.5 Å; or 12 Å to 50 Å.

85. A diagnostic testing system comprising: Test strip, the test strip comprising one or more test areas and one or more control areas, wherein the one or more test areas comprise: Donor chemiluminescent molecules; Energy acceptor molecules; and Host molecule, wherein the host molecule contains at least two guest molecules, The first guest molecule comprises a donor chemiluminescent molecule. The second guest molecule, which includes an energy acceptor molecule, When contained within the host molecule, the at least two guest molecules are structurally spaced apart to facilitate resonant energy transfer between the donor chemiluminescent molecule and the energy acceptor molecule.

86. The diagnostic testing system of claim 85, wherein the test strip supports lateral flow of fluid along a lateral flow direction, and wherein at least one test area includes an area 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.

87. An immunoassay composition comprising: Antibody, said antibody binding to a target analyte and conjugated to a triggering enzyme or substrate; and The composition according to any one of claims 1 to 33.

88. The immunoassay composition according to claim 86, further comprising the target analyte.

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