Chemiluminescent compounds for multiplexing

Abstract

To provide compounds, conjugates and methods that may be used to detect the presence of an analyte in a sample, such as a biological sample.SOLUTION: The invention provides compounds of formula (I), or salts thereof.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 883,926, filed Aug. 7, 2019, the contents of which are incorporated herein by reference.

[0002] The ability to multiplex and measure two or more analytes from one sample in a single test is highly sought after within the in vitro diagnostics market. Test multiplexing allows for increased throughput, faster time per result, and reduced consumables. There is also the potential to lower internal costs and improve overall profits. One method of discriminating between multiple signals in one test is via the emission wavelength of the reporter molecule. To achieve the wavelength shift using chemiluminescence, a triggerable chemiluminescent compound with a red-shifted emission wavelength is desirable. Summary of the Invention

[0003] (Brief Summary of the Invention) The present disclosure relates to a compound of formula (I), or a salt thereof

[0004] [ka] wherein X is -NH- or a diamine linker; Y is selected from nitrogen, oxygen, and sulfur; and when Y is nitrogen, R 1 is -SO2-A, A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl, and when Y is oxygen or sulfur, R 1 is absent, Q is -SO2- or -CO-, and L 1 and L 2 are each independently selected from alkylene and heteroalkylene; R 2is selected from -COOZ and -CN, Z is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, and heteroalkyl, R a , R b , R c , R d , R e , R f , R g , and R h are each independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl, and each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, heteroalkyl, alkylene, and heteroalkylene is independently optionally substituted with 1, 2, 3, 4, or 5 substituents.

[0005] The present disclosure relates to a conjugate of formula (II), or a salt thereof:

[0006] [ka] wherein X is -NH- or a diamine linker; Y is selected from nitrogen, oxygen, and sulfur; and when Y is nitrogen, R 1 is -SO2-A, A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl, and when Y is oxygen or sulfur, R 1 is absent, Q is -SO2- or -CO-, and L 1 is selected from alkylene and heteroalkylene; L 3 is the linker, Ra , R b , R c , R d , R e , R f , R g , and R h are each independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl, and the binding members are molecules capable of binding to a target analyte, and each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, alkylene, and heteroalkylene is independently optionally substituted with 1, 2, 3, 4, or 5 substituents.

[0007] The present disclosure further provides methods of detecting two or more analytes in a biological sample using the aforementioned conjugates. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows structures and fluorescence data for the 5- and 6-isomers of fluorescein attached to a substituted acridinium moiety via an acetamide linker. [Diagram 2] FIG. 1 shows structures and fluorescence data for the 5- and 6-isomers of fluorescein attached via a substituted acridinium moiety via a piperazine linker. [Diagram 3] FIG. 1 shows structures and fluorescence data for 5 / 6-carboxy and 2-carboxy isomers of rhodamine attached to a substituted acridinium moiety via various linkers. [Figure 4] FIG. 1 shows structures and emission data for substituted acridinium moieties linked to fluorophores via various attachment points. [Diagram 5]FIG. 1 shows structures and emission data for compounds in which fluorophores are attached to a substituted acridinium moiety via various linkers. [Figure 6] FIG. 1 shows the results of a cytomegalovirus IgG and IgM multiplex assay described in Example 78. [Figure 7] FIG. 1 shows the results of a multiplexed assay of HIV antigen and antibody combinations as described in Example 79. [Figure 8] FIG. 1 shows the results of a multiplexed Lyme Disease IgG and IgM assay described in Example 80. [Figure 9] FIG. 1 shows the results of a multiplexed assay of a combination of free T4 and thyroid stimulating hormone as described in Example 81. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Disclosed herein are compounds, conjugates and methods that can be used to detect the presence of an analyte in a sample, such as a biological sample. The compounds include an acridinium moiety and a fluorophore linked via a rigid diamine linker. When the chemiluminescence of the acridinium moiety is triggered, the light output can be shifted to the emission wavelength of the attached fluorophore. The compounds are conjugated to a molecule capable of specifically binding to the analyte of interest in the sample, so that the presence or absence of the analyte can be determined. The use of multiple conjugates with different fluorophores in a single assay can allow for the detection of more than one analyte from one sample in a single test, which can be particularly useful for in vitro diagnostics.

[0010] Chemiluminescence has been extensively studied since the mid-20th century. Enzyme-induced chemiluminescence, bioluminescence, peroxyoxylate chemicals, and acridinium chemicals are each examples of chemiluminescent systems defined by their ability to produce light through a chemical reaction. Research into chemiluminescence and bioluminescence has led to countless publications and patents, as well as a better understanding of fireflies and anglerfish (via bioluminescent bacteria), and commercial products such as chemical lights and immunoassays (Seliger et al., Proc. Natl. Acad. Sci. USA, 1961, 47, 1129-1134; Nealson et al., Microbiol. Rev. 1979, 43(4), 496-518; Rauhut, Acc. Chem. Res. 1969, 2(3), 80-87; Dodeigne et al., Talanta, 2000, 51, 415-439). The mechanism of chemiluminescence varies from one chemiluminescent system to another, but all theories result in an excited state molecule releasing a photon while relaxing to the ground state. The nature of the excited state molecule determines the emission wavelength of the emitted photon.

[0011] The ability to tune the emission wavelength of a chemiluminescent species is beneficial in several applications, including immunoassay multiplexing. A classic example of chemiluminescence with tunable emission wavelengths is chemiluminescence, where luminophores of various emission wavelengths can be used to generate a broad spectrum of chemiluminescent colors in an intermolecular process. By carefully selecting molecules that can be excited, the emission wavelength for a chemiluminescent system can be selected. Shifting emission can be achieved through chemiluminescent energy or electron transfer processes. These processes have been shown to function both intermolecularly and intramolecularly through a variety of hypothesized mechanisms, including Förster resonance energy transfer (FRET), Dexter electron transfer, chemiluminescent energy transfer (CRET), and variations of chemically initiated electron exchange luminescence (CIEEL). Examples of such intramolecular systems include BRET-based systems using enzymes that are fluorophore-tagged luciferases (Hiblot et al., Angew. Chem. Int. Ed. 2017, 56, 14556-14560), adamantyl dioxetane fluorophore constructs (Tseng et al., J. Biomed. Sci. 2015, 22(1), 4), acridinium-labeled quantum dots (Sklenarova et al., J. Lumin. 2017, 184, 235-241), and acridinium-labeled DNA systems (Browne et al., Anal. Chem. 2012, 84, 9222-9229). Using acridinium as a chemical initiator and an energy acceptor that intramolecularly links fluorophores, it may be possible to trigger energy or electron transfer events, producing emission wavelengths that are shifted from those of the acridinium / acridone chemiluminescence.

[0012] Chemiluminescence energy / electron transfer has been studied since the mid-1960s, and there are various hypotheses regarding the mechanism that causes the shifted emission (Phillips et al., Nature, 1967, 215, 1163-1165; Freed et al., J. Am. Chem. Soc. 1971, 93(9), 2097-2102; U.S. Patent No. 6,156,800). The prevailing theory in acridinium-initiated electron / energy transfer is that the length of the moiety linking the initiator (i.e., acridinium) to the acceptor (i.e., fluorophore) is the driving factor in shifted emission management. However, without wishing to be bound by theory, the inventors have gathered evidence that the orientation of the initiator (acridinium) and acceptor (fluorophore / luminophore) relative to each other may be an important driving factor in shifted emission efficiency. Compounds presented herein with a rigid diamine linker between the acridinium moiety and the fluorophore can achieve 100% shifted emission, i.e., the light output of the shifted emission is 100% of that expected from the acridinium alone, with little to no light observed in the narrower emission band in optimized systems. The requirement for orbital alignment and the observation of 100% shifted emission lends itself to the Dexter mechanism of electron transfer (Turro et al., Modern Molecular Photochemistry of Organic Molecules. University Science Books, Mill Valley, California, 2010; Dexter, J. Chem. Phys. 1953, 21, 836). The relative linker length may promote the correct orientation or allow high degrees of freedom to limit the proportion of molecules apart, which likely plays a role in the distance at which the fluorophore and initiator are in the correct orientation to facilitate transfer. However, it can be considered that the length of the linker is independent of the orientation: a long linker can fold / bend to produce the correct orientation, whereas a short linker may hold the two moieties in an unfavourable orientation. Thus, the length of the linker itself does not drive the shifted emission.Furthermore, the type of linker, the point of attachment of the fluorophore, and the point of attachment of the initiator each affect the moiety orientation and therefore can be important factors in the preparation of shifted emission chemiluminescent compounds.

[0013] definition As used herein, "Comprise(s), "include(s), "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0014] In the description of numerical ranges herein, each intervening number is expressly contemplated with the same precision, for example, in the range 6 to 9, the numbers 7 and 8 are expressly contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document including definitions will control. Preferred methods and materials are described below, however, methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0016] Definitions of certain functional groups and chemical terms are described in more detail below: In this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition, inside cover, and certain functional groups are generally defined as set forth therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, can be found in Sorrell, Organic Chemistry, 2nd Edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rd Edition, John Wiley & Sons, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987, the contents of each of which are incorporated herein by reference in their entirety.

[0017] As used herein, the term "alkyl" refers to an alkyl group having 1 to 16 carbon atoms (C 16 alkyl), e.g., 1 to 14 carbon atoms (C 14 Alkyl), 1 to 12 carbon atoms (C1 to C 12 Alkyl), 1 to 10 carbon atoms (C1 to C 10"alkyl" refers to a straight or branched saturated hydrocarbon chain containing 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0018] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2 to 16 carbon atoms and at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0019] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 16 carbon atoms and at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0020] As used herein, the term "alkylene" refers to an alkylene group having 1 to 10 carbon atoms (C 10It refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms (C1-C6 alkylene), for example. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, and -CH(CH3)CH2CH2CH2CH2-.

[0021] The term "alkoxy" as used herein refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0022] As used herein, the term "aryl" refers to a phenyl group or a bicyclic or tricyclic aromatic fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to a parent molecular moiety and fused to one phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to a parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryl include, but are not limited to, naphthyl. Representative examples of tricyclic aryl include, but are not limited to, anthracenyl and phenanthreneyl.

[0023] The term "arylalkyl," as used herein, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, phenylmethyl (i.e., benzyl) and phenylethyl.

[0024] The term "aryloxy" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0025] As used herein, the term "cycloalkyl" refers to a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls can be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[1.1.1]pentanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0026] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic carbocyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, and bicyclo[2.2.1]heptenyl.

[0027] The term "cycloalkylalkyl," as used herein, refers to a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an alkylene group, as defined herein. Representative examples of cycloalkylalkyl include, but are not limited to, cyclohexylmethyl.

[0028] As used herein, the term "diamine linker" refers to a linker moiety having an amine functional group (-NH- or -NR-) at each end. Diamine linkers can be linear, branched, or cyclic.

[0029] As used herein, the term “halogen” or “halo” means F, Cl, Br, or I.

[0030] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein, in which one or more hydrogen atoms are replaced by halogen.For example, 1, 2, 3, 4, 5, 6, 7 or 8 hydrogen atoms can be replaced by halogen, or all hydrogen atoms can be replaced by halogen.Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, perfluoroethyl, 2-fluoro-2-methylpropyl, and 3,3,3-trifluoropropyl.

[0031] The term "haloalkoxy" as used herein means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom. Representative examples of haloalkoxy include, but are not limited to, trifluoromethoxy.

[0032] As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which at least one carbon atom is replaced with a heteroatom, such as, for example, N, O, P, or S. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides.

[0033] As used herein, the term "heteroalkylene" refers to an alkylene group, as defined herein, in which at least one carbon atom has been replaced with a heteroatom, such as, for example, N, O, P, or S. Representative examples of heteroalkylene groups include polyethylene oxide and polypropylene oxide chains, polyethyleneimine groups, and the like.

[0034] As used herein, the term "heteroaryl" refers to an aromatic monocyclic ring or an aromatic bicyclic ring system or an aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. A bicyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring fused to a monocyclic aryl group as defined herein or a monocyclic heteroaryl group as defined herein. A tricyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring fused to two rings independently selected from a monocyclic aryl group as defined herein or a monocyclic heteroaryl group as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl.The monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0035] As used herein, the term "heteroarylalkyl" refers to a heteroaryl group, as defined herein, attached to the parent molecular moiety through an alkylene group, as defined herein.Representative examples of heteroarylalkyl include, but are not limited to, furan-3-ylmethyl, 1H-imidazol-2-ylmethyl, 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl, pyridin-3-ylmethyl, 6-chloropyridin-3-ylmethyl, pyridin-4-ylmethyl, (6-(trifluoromethyl)pyridin-3-yl)methyl, (6-(cyano)pyridin-3-yl)methyl, (2-(cyano)pyridin-4-yl)methyl, (5-(cyano)pyridin-2-yl)methyl, (2-(chloro)pyridin-4-yl)methyl, pyrimidin-5-ylmethyl, 2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

[0036] As used herein, the term "heterocycle" or "heterocyclic" refers to a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 7- and 8-membered ring contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, and piperidinyl. , pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms.Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of a bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0037] As used herein, the term "heterocyclylalkyl" refers to a heterocyclyl group, as defined herein, appended to the parent molecular moiety through an alkylene group, as defined herein. Representative examples of heterocyclylalkyl include, but are not limited to, piperidin-4-ylmethyl, piperazin-1-ylmethyl, 3-methyl-1-pyrrolidin-1-ylbutyl, (1R)-3-methyl-1-pyrrolidin-1-ylbutyl, (1S)-3-methyl-1-pyrrolidin-1-ylbutyl, and 3-morpholinopropyl.

[0038] As used herein, the term "hydroxy" refers to an --OH group.

[0039] As used herein, the term "hydroxyalkyl" refers to an alkyl group, as defined herein, substituted with at least one hydroxy group. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl, 3,4-dihydroxybutyl, and 5-hydroxypentyl.

[0040] In some cases, the number of carbon atoms in a group (e.g., alkyl, alkoxy, or cycloalkyl) is indicated by the prefix "Cx-Cy-," where x is the minimum number of carbon atoms in the group and y is the maximum number of carbon atoms in the group. Thus, for example, "C1-C3-alkyl" refers to an alkyl group containing from 1 to 3 carbon atoms.

[0041] The term "substituent" refers to a group that substitutes an atom of the indicated group.

[0042] When a group or moiety can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) hydrogens of the group designated in the phrase using "substituted" can be replaced with a selection of the designated group described or with suitable groups known to those of skill in the art (e.g., one or more of the groups described below). Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, arylalkyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, carboxy (-COOH), ketone, amide, carbamate, phosphoryl, selenyl, and acyl.

[0043] compound A compound of formula (I) or a salt thereof

[0044] [ka] wherein X is -NH- or a diamine linker; Y is selected from nitrogen, oxygen, and sulfur; and when Y is nitrogen, R 1 is -SO2-A, A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl, and when Y is oxygen or sulfur, R 1 is absent, Q is -SO2- or -CO-, and L 1 and L 2 are each independently selected from alkylene and heteroalkylene; R2 is selected from -COOZ and -CN, Z is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, and heteroalkyl, R a , R b , R c , R d , R e , R f , R g , and R h are each independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl, and each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, heteroalkyl, alkylene, and heteroalkylene are independently optionally substituted with 1, 2, 3, 4, or 5 substituents.

[0045] The X group is -NH- or a diamine linker. In some embodiments, X is -NH-. In some embodiments, X is a diamine linker. In some embodiments, the diamine linker can have the formula -NR'-L'-NR''-, where R' and R'' are each independently selected from hydrogen and methyl, and L' is selected from alkylene, heteroalkylene, cycloalkylene, and cycloalkenylene. In some embodiments, the diamine linker can be a cyclic diamine linker (e.g., a monocyclic or bicyclic diamine linker). In some embodiments, the diamine linker can be a rigid diamine linker. Exemplary rigid diamine linkers are:

[0046] [ka] Includes.

[0047] In some embodiments, X is

[0048] [ka] is selected from.

[0049] In some embodiments, X is

[0050] [ka] It is.

[0051] The Y group is selected from nitrogen, oxygen, and sulfur, and when Y is nitrogen, R 1 is -SO2-A, A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl, and when Y is oxygen or sulfur, R 1 does not exist.

[0052] In some embodiments, Y is nitrogen and R 1is -SO2-A. In some embodiments, A is aryl. In some embodiments, A is phenyl. In some embodiments, A is unsubstituted or substituted with 1, 2, 3, 4, or 5 substituents selected from C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, and amino. In some embodiments, A is phenyl substituted with one substituent selected from C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, and amino. In some embodiments, A is phenyl substituted with one substituent selected from C1-C4 alkyl. In some embodiments, A is phenyl substituted with one substituent selected from C1-C4 alkyl. In some embodiments, A is phenyl substituted with one methyl group. In some embodiments, A is p-tolyl.

[0053] R 2 is selected from -COOZ and -CN, and Z is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, and heteroalkyl. 2 is -COOZ. In some embodiments, Z is selected from hydrogen and C1-C4 alkyl. In some embodiments, Z is hydrogen.

[0054] In some embodiments, Q is -CO-. In some embodiments, Q is -SO2-.

[0055] L 1 and L 2 Each is independently selected from alkylene and heteroalkylene. 1 and L 2 are each independently C1-C4-alkylene. 1 is -CHCHCH-. In some embodiments, L 2 is -CH2CH2CH2-.

[0056] In some embodiments, R a , R b , R c , R d , R e , R f , R g , and R h are hydrogen.

[0057] In some embodiments, the compound is a compound of formula (Ia), or a salt thereof:

[0058] [ka] [wherein each R is independently selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl; m is 0, 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, 5, or 6.]

[0059] In some embodiments, m is 1 or 2. In some embodiments, m is 1. In some embodiments, m is 1 and R is C1-C4 alkyl. In some embodiments, m is 1 and R is methyl. In some embodiments, n is 3.

[0060] In some embodiments, the compound is a compound of formula (Ib), or a salt thereof:

[0061] [ka] It is.

[0062] Any reference herein to a compound of formula (I) should be construed as also being a reference to a compound of formula (Ia) or formula (Ib), whether or not explicitly stated.

[0063] In some embodiments, in any of the compounds of Formula (I), Formula (Ia), or Formula (Ib), the fluorophore is selected from fluoresceins, rhodamines, boron-dipyrromethenes, cyanines, oxazines, thiazines, coumarins, naphthalimides, rhodols, naphthalenes, squaraines, porphyrins, flavins, and lanthanide dyes.

[0064] Suitable fluorophores include QUASAR® dyes (available from Biosearch Technologies, Novato, Calif.), fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6-carboxyfluorescein (e.g., FAM), VIC, NED, carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TA MRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, SPECTRUM RED™, SPECTRUM GREEN™, cyanine dyes (e.g., CY-3™, CY-5™, CY-3.5™, CY-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), IR dyes (e.g., IRD40, IRD 700, IRD 800), and the like.Examples of other suitable fluorescent dyes that can be used as well as methods for linking or incorporating fluorescent dyes into oligonucleotides such as probes can be found in RP Haugland, "The Handbook of Fluorescent Probes and Research Chemicals", Publisher, Molecular Probes, Inc., Eugene, Oreg. (June 1992). Fluorescent dyes and labeling kits are commercially available, for example, from Amersham Biosciences, Inc. (Piscataway, NJ), Molecular Probes Inc. (Eugene, Oreg.), and New England Biolabs Inc. (Beverly, Mass.).

[0065] As one of ordinary skill in the art will appreciate, a fluorophore can be attached to a molecule via the reaction of two reactive moieties, one on the fluorophore and one on the remainder of the molecule. For example, many commercially available fluorophores are available with reactive functional groups, such as carboxylic acids, isocyanates, isothiocyanates, maleimides, or esters, such as succinimidyl, pentafluorophenyl, or tetrafluorophenyl esters. A fluorophore can be selected to contain a reactive group that reacts with a functional group on the remainder of the molecule. For example, a fluorophore isothiocyanate or a fluorophore succinimidyl ester can react with an amine group. It is understood that the term "fluorophore" as used in describing the molecules disclosed herein includes both the fluorescent moiety itself and any linking atom that serves to link the fluorescent moiety to the remainder of the molecule.

[0066] In some embodiments, the fluorophore is

[0067] [ka] Selected from TIFF2025076421000011.tif183166TIFF2025076421000012.tif67166.

[0068] For the compounds described herein, groups and substituents thereof may be selected in accordance with the permissible valences of atoms and substituents such that selection and substitution result in stable compounds that do not spontaneously undergo changes, such as by rearrangement, cyclization, elimination, and the like.

[0069] Compounds can exist as stereoisomers in which asymmetric or chiral centers exist. Stereoisomers are "R" or "S" depending on the configuration of the substituents around the chiral carbon atom. As used herein, the terms "R" and "S" are configurations as defined in the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45:13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, which are specifically included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds can be prepared by synthesis from commercially available starting materials containing asymmetric or chiral centers, or by preparation of racemic mixtures followed by resolution methods well known to those skilled in the art. These resolution methods are exemplified by (1) attachment of the mixture of enantiomers to a chiral auxiliary, separation of the resulting diastereomeric mixture by recrystallization or chromatography, and optional liberation of the optically pure product from the auxiliary, as described in Furniss, Hannaford, Smith, and Tatchell, Vogel's Textbook of Practical Organic Chemistry, 5th Edition (1989), Longman Scientific & Technical Ltd., Essex CM20 2JE, England; or (2) direct separation of the mixture of optical enantiomers on a chiral chromatographic column; or (3) fractional recrystallization methods.

[0070] It is to be understood that the compounds may have tautomeric forms and geometric isomers and these also form an aspect of the present invention.

[0071] The present disclosure also includes isotopically labeled compounds that are identical to those set forth in formula (I) except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that are suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Heavier isotopes, such as deuterium, i.e., but not limited to Cl. 2 Substitution with H can provide several advantages resulting from improved metabolic stability, for example increased in vivo half-life, and therefore may be preferred in some circumstances. The compounds can incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated into compounds of formula (I) are: 11 C. 13 N, 15 O, and 18 F. Isotopically labeled compounds of formula (I) may generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents.

[0072] The compounds disclosed herein can be in the form of salts, which can be prepared during the final isolation and purification of the compounds, or separately, for example, by reacting a basic group (e.g., an amino group) of the compound with a suitable acid, or an acidic group (e.g., a carboxylic acid group) of the compound with a suitable base.

[0073] Acid salts can be prepared during the final isolation and purification of the compound or separately by reacting a suitable group of the compound, such as an amino group, with a suitable acid. For example, the compound can be dissolved in a suitable solvent, such as but not limited to methanol or water, and treated with at least one equivalent of an acid, such as hydrochloric acid. The resulting salt can precipitate and be isolated by filtration and drying under reduced pressure. Alternatively, the solvent and excess acid can be removed under reduced pressure to obtain the salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloride, hydrobromide, sulfate, phosphate, and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides, and iodides, such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, m-butyl, stearyl, and the like.

[0074] Base addition salts can be prepared upon final isolation and purification of the disclosed compounds by reaction of the carboxyl group with a suitable base, for example, hydroxides, carbonates, or bicarbonates of metal cations such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or organic primary, secondary, or tertiary amines. Quaternary amine salts can be prepared, for example, from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0075] Compounds of formula (I) can be synthesized by a variety of methods, including that illustrated in Scheme 1, starting with the compound carboxypropylsulfopropyl-acridinium (CPSP-acridinium, 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridiniumcarboxamide) described by Adamczyk et al., J. Org. Chem., 1998, 63(16), 5636-5639.

[0076] [ka]

[0077] Those of skill in the art will appreciate that Scheme 1 is illustrative of the specific substituents (e.g., R 1 , R 2 , L 1 , L 2 The synthesis of several compounds having aryl groups (X, X, and Y groups) is illustrated, it being understood that compounds having other groups at the corresponding positions can be prepared in a similar manner.

[0078] Routine experimentation, including proper manipulation of the reaction conditions, reagents and sequences of the synthetic pathways, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at suitable points in the reaction sequence of the method, is within the scope of this disclosure. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art. Examples thereof can be found in PGM Wuts and TW Greene, Greene's book titled Protective Groups in Organic Synthesis (4th Edition), John Wiley & Sons, Inc., NY (2006), which is incorporated herein by reference in its entirety. The synthesis of the compounds of the present disclosure can be carried out by methods similar to those described in the synthetic schemes and specific examples described herein.

[0079] When an optically active form of a disclosed compound is required, it may be obtained by carrying out one of the procedures described herein using optically active starting materials (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a stereoisomeric mixture of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization or enzymatic resolution).

[0080] Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described above using a pure geometric isomer as a starting material, or by resolution of a mixture of geometric isomers of the compound or intermediates using standard procedures such as chromatographic separation.

[0081] The synthetic schemes and specific examples described are illustrative and should not be construed as limiting the scope of the invention, which is to be understood as being defined in the appended claims, all alternatives, modifications, and equivalents of the synthetic methods and specific examples are intended to be encompassed within the scope of the claims.

[0082] Conjugates Conjugates of formula (II)

[0083] [ka] wherein X is -NH- or a diamine linker; Y is selected from nitrogen, oxygen, and sulfur; and when Y is nitrogen, R 1 is -SO2-A, A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl, and when Y is oxygen or sulfur, R 1 is absent, Q is -SO2- or -CO-, and L 1 is selected from alkylene and heteroalkylene; L 3 is the linker, R a , R b , R c , R d , R e , R f , R g , and R h are each independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl, and the binding members are molecules capable of binding to a target analyte, and each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, alkylene, and heteroalkylene is independently optionally substituted with 1, 2, 3, 4, or 5 substituents.

[0084] X, Y, R 1 , A, L 1 , R a , R b , R c , R d , R e , R f , R g , R hThe groups, and fluorophores are the same as those described above for formula (I). Any group or combination of groups described above for compounds of formula (I) can also be included in compounds of formula (II).

[0085] In the compound of formula (II), L 3 is a linker. A wide variety of linkers can be used in the compounds of formula (II). In some embodiments, the linker can be a covalent bond. In some embodiments, the linker can be a C1-C 40 Alkylene linkers such as alkylene linkers, e.g., C1-C 30 , C1~C 20 , C1~C 12 , C1~C 10、 The linker can be a C1-C8, C1-C6, or C1-C4 alkylene linker. For example, the linker can be a C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 , or C 40 It can be an alkylene linker.

[0086] In some embodiments, the linker can be a heteroalkylene linker, such as a polyethylene glycol linker. Such linkers have the formula -(CH2CH2O) n1 In some embodiments, n1 is an integer from 1 to 20. For example, in some embodiments, n1 is an integer from 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4. In some embodiments, n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0087] In some embodiments, the linker can include an E moiety, where E is the product of a reaction between two reactive groups. For example, the E group can be an amide, ester, carbamate, triazole, sulfonamide, phosphoramide, phosphate, or sulfate.

[0088] A binding member is a molecule that can be used to detect an analyte of interest in the methods described herein. The terms "binding member", "specific binding partner" and "specific binding member" are used interchangeably herein and refer to one of two or more distinct molecules that specifically recognize another molecule compared to substantially less recognition of the other molecule. "Specifically binds" or "binding specificity" means that the binding member binds an analyte molecule with sufficient specificity to distinguish the analyte molecule from other components or contaminants of the sample. As will be understood by those skilled in the art, the appropriate specific binding member will be determined by the analyte being analyzed.

[0089] Binding members for a wide variety of target molecules are known or can be readily found or developed using known techniques. For example, when the target analyte is a protein, the binding members may include proteins, in particular antibodies or fragments thereof (e.g., antigen-binding fragments (Fab), Fab' fragments, F(ab')2 fragments), recombinant antibodies, chimeric antibodies, single chain Fvs ("scFvs"), single chain antibodies, single domain antibodies, such as camelid-derived variable heavy domains ("VHHs"; also referred to as "VHH fragments") (VHHs and methods for making them are described in Gottlin et al., Journal of Biomolecular Screening, 14:77-85 (2009)), recombinant VHH single domain antibodies, disulfide-linked Fvs ("sdFvs"), anti-idiotypic ("anti-Id") antibodies, and functionally active epitope-binding fragments of any of the above, full length polyclonal or monoclonal antibodies, antibody-like fragments, and other proteins, such as receptor proteins, protein A, or protein C. In embodiments where the analyte is a small molecule such as a steroid, bilin, retinoid, or lipid, the first and / or second binding member can be a scaffold protein (e.g., lipocalin) or a receptor. In some cases, the binding member for a protein analyte can be a peptide. For example, when the target analyte is an enzyme, suitable binding members can include enzyme substrates and / or enzyme inhibitors, which can be peptides, small molecules, and the like. In some cases, when the target analyte is a phosphorylated species, the binding member can include a phosphate binder. For example, the phosphate binder can include a metal ion affinity medium (see, for example, U.S. Pat. No. 7,070,921 and U.S. Patent Application Publication No. 2006 / 0121544). In other embodiments, the binding member can be a vitamin, a nutrient, a nutrient metabolite, a nucleic acid, a carbohydrate, a dendrimer, a dendritic structure, a glycoprotein, an antigen, a receptor, an enzyme, a pharmaceutical (e.g., an antibiotic), or a drug of abuse.

[0090] In certain cases, the specific binding member can be an aptamer, such as those described in U.S. Patent Nos. 5,270,163; 5,475,096; 5,567,588; 5,595,877; 5,637,459; 5,683,867; and 5,705,337. As used herein, the term "aptamer" refers to a nucleic acid or peptide molecule that can bind to a preselected target, including small molecules, proteins, and peptides, especially with high affinity and specificity. Nucleic acid aptamers (e.g., single-stranded DNA molecules or single-stranded RNA molecules) can be developed to capture virtually any target molecule. Aptamers typically bind target molecules with very high affinity in a highly specific, conformation-dependent manner, although aptamers with lower binding affinity can also be selected. Aptamers can distinguish target analyte molecules based on very slight structural differences, such as the presence or absence of methyl or hydroxyl groups, and certain aptamers can distinguish between D- and L-enantiomers and diastereomers. Aptamers can bind small molecule targets, including drugs, metal ions, and organic dyes, peptides, biotin, and proteins. Aptamers can retain functional activity after biotinylation, fluorescein labeling, and when attached to glass surfaces and microspheres.

[0091] Nucleic acid aptamers are oligonucleotides and can be single-stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotides or oligoribonucleotides. A "modified" oligodeoxynucleotide or oligoribonucleotide refers to a nucleotide with a covalently modified base and / or sugar. For example, modified nucleotides include nucleotides in which the sugar is covalently attached to a low molecular weight organic group other than the hydroxyl group at the 3' position and other than the phosphate group at the 5' position. Such modified nucleotides can also include 2'-substituted sugars, such as 2'-O-methyl; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halo or 2-azido-ribose, carbocyclic sugar analogs, anomeric sugars; epimeric sugars, such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, and sedoheptulose.

[0092] Peptide aptamers can be designed to interfere with protein interactions. Peptide aptamers can be based on protein scaffolds to which variable peptide loops are attached, thereby constraining the conformation of the aptamer. In some cases, the scaffold portion of the peptide aptamer is derived from bacterial thioredoxin A (TrxA).

[0093] When the analyte is a carbohydrate, suitable binding members include, for example, antibodies, lectins, and selectins. As will be appreciated by those of skill in the art, any molecule that can specifically bind to the analyte of interest can potentially be used as a binding member.

[0094] In some embodiments, the conjugate comprises an additional specific binding member that can serve as a carrier moiety for the dye construct. The additional specific binding member can be covalently linked to any of the specific binding members described above, or non-covalently linked to a compound, such as a lysosome, a hydrogel, or a dendrimer that has the dye inserted within its cavity.

[0095] In certain embodiments, suitable analyte / binding member complexes may include, but are not limited to, antibody / antigen, antigen / antibody, receptor / ligand, ligand / receptor, protein / nucleic acid, enzyme / substrate and / or inhibitor, carbohydrate (including glycoproteins and glycolipids) / lectin and / or selectin, protein / protein, protein / small molecule, and the like.

[0096] method The present disclosure provides methods for detecting one or more analytes of interest in a biological sample using the conjugates described herein.

[0097] Analytes of interest The terms "analyte," "target analyte," and "analyte of interest" are used interchangeably and refer to the analyte to be measured by the methods disclosed herein. As will be understood by one of skill in the art, any analyte that can be specifically bound by a binding member (e.g., a first specific binding member and a second specific binding member) can be detected and optionally quantified using the methods of the present disclosure.

[0098] In some embodiments, the analyte can be a biomolecule. Non-limiting examples of biomolecules include macromolecules such as proteins, lipids, and carbohydrates. In certain cases, the analyte can include hormones, antibodies, growth factors, cytokines, enzymes, receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, and creatine kinase), toxins, drugs (e.g., drugs of addiction), and metabolic agents (e.g., vitamins). Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, and the like.

[0099] In certain embodiments, the analyte may be a post-translationally modified protein (e.g., a phosphorylated, methylated, glycosylated protein) and the corresponding binding member (described above) may be an antibody specific for the post-translational modification. The modified protein may be bound to a first binding member immobilized to a solid support. The first binding member binds to the modified protein but not to the unmodified protein. In other embodiments, the first binding member may bind to both the unmodified and modified protein and the second binding member may be specific for the post-translationally modified protein.

[0100] In some embodiments, the analyte can be a cell, such as, for example, a circulating tumor cell, a pathogenic bacterial cell, or a fungal cell, hi other embodiments, the analyte can be a virus (e.g., a retrovirus, a herpes virus, an adenovirus, a lentivirus, a filovirus (Ebola), a hepatitis virus (e.g., A, B, C, D, and E), or a human papilloma virus (HPV)).

[0101] A non-limiting list of analytes that may be analyzed according to the present disclosure includes thyroglobulin, prolactin, Aβ42 amyloid beta-protein, fetuin-A, tau, secretogranin II, prion protein, alpha-synuclein, tau protein, neurofilament light chain, parkin, PTEN-induced putative kinase 1, DJ-1, leucine-rich repeat kinase 2, mutant ATP13A2, apoH, ceruloplasmin, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), transthyretin, vitamin D binding protein, proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (semen), p14 endocan fragment, serum, ACE2, autoantibodies to CD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, osteopontin, human epididymis protein 4 (HE4), alpha-fetoprotein (AFP), albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18 (IL-18), kidney injury molecule-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), LMP1, BARF1, IL-8, carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, LZTS1, alpha-amylase, carcinoembryonic antigen (CEA), CA12 5, interleukin-8 (IL-8), thioredoxin, beta-2 microglobulin, tumor necrosis factor-alpha receptor, CA15-3, follicle-stimulating hormone (FSH), luteinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39, amphiregulin, dUTPase, secreted gelsolin (pGSN), PSA (prostate-specific antigen), thymosin β15, insulin, plasma C-peptide, glycosylated hemoglobin (HBA1c), C-reactive protein (CRP), interleukin-6 (IL-6), ARHGDIB (RhoGDP-dissociation inhibitor 2), CFL1 (cofilin-1), PFN1 (profilin-1), GSTP1 (glutathione S-transferase P), S100A11 (protein S100-A11), PRDX6 (peroxiredoxin-6), HSPE1 (10 kDa heat shock protein, mitochondrial l), LYZ (lysozyme C precursor), GPI (glucose-6-phosphate isomerase), HIST2H2AA (histone H2A type 2-A), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (basement membrane-specific heparan sulfate proteoglycan core protein precursor), LGALS3BP (galectin-3-binding protein precursor), CTSD (cathepsin D precursor), APOE (apolipoprotein E precursor), IQGAP1 (Ras GTPase activating-like protein IQGAP1), CP (ceruloplasmin precursor), and IGLC2 (IGLC1 protein), PCDGF / GP88, EGFR, HER2, MUC4, IGF-IR, p27(kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1 (3-phosphoinositide-dependent protein kinase-1), TSC1, TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEK mitogen-activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) II alpha 170kDa, FRAP1, NRG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1, F-oxo3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1, PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA), leptin (LEP), receptor for advanced glycation end products (AGER or RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14 molecule (CD14), ferritin (FTH1), insulin-like growth factor binding protein 1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular cell adhesion molecule 1 (VCAM1) and VonWillebrand factor (VWF), myeloperoxidase (MPO), IL1α, TNFα, perinuclear antineutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilms' tumor-1 protein, aquaporin-1, MLL3, AMBP, VDAC1, E. coli enterotoxin (heat-labile exotoxin, heat-stable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxin, Shiga toxin, Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxins A and B, These include glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), S100B, neurofilament light polypeptide (NF-L), tau, p-tau, amyloid beta 40 and 42, neuron-specific enolase (NSE), brain natriuretic peptide (BNP), N-terminal (NT)-prohormone BNP (NT-proBNP), CA19-9, placental growth factor (PlGF), sFlt-1, opioids, tacrolimus, and protein induced by vitamin K deficiency-II (PIVKA-II).

[0102] Other examples of analytes include drugs of abuse (e.g., cocaine), protein biomarkers (nucleolin, nuclear factor-κB essential modulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoforms, immunoglobulin μ heavy chain (IGHM), immunoglobulin E, αvβ3 integrin, α-thrombin, HIV gp120, NF-κB, E2F transcription factors, HER3, plasminogen activator inhibitor, tenascin-C, CXCL12 / SDF-1, prostate-specific membrane antigen (PSMA), and HGC-27; cells (including but not limited to non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM, acute myeloid leukemia (AML) cells (HL60), small cell lung carcinoma (SCLC) cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells, HER-2-overexpressing human breast cancer cells, SK-BR-3, pancreatic cancer cells (Mia-PaCa-2); and infectious agents (Mycobacterium tuberculosis tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella O8, and Salmonella enteritidis.

[0103] sample The terms "sample", "test sample", and "biological sample" are used interchangeably herein and refer to a fluid sample that contains or is suspected of containing an analyte of interest. In some cases, a sample can include a liquid, a flowable particulate solid, or a fluid suspension of solid particles. In certain embodiments, a sample can be a liquid sample or a liquid extract of a solid sample. In some cases, a sample can be processed prior to the analysis described herein. For example, a sample can be separated or purified from its source prior to the analysis. However, in certain embodiments, an unprocessed sample containing an analyte can be assayed directly. A sample can be derived from any suitable source. For example, a sample source can be synthetic (e.g., generated in a laboratory), environmental (e.g., air, soil, fluid samples such as water supplies, etc.), animal (e.g., mammalian), plant, or any combination thereof. In certain examples, the sample is a human body substance (e.g., bodily fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, tissue, or organ). Tissues can include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, cardiac muscle tissue, brain tissue, bone marrow, cervical tissue, skin, etc. In certain cases, the source of the sample can be an organ or tissue, such as a biopsy sample, which can be solubilized by tissue disintegration / cell lysis.

[0104] In some cases, the fluid sample may be diluted prior to use in the assay. For example, in embodiments where the source of the analyte molecules is a human bodily fluid (e.g., blood, serum), the fluid may be diluted with an appropriate solvent (e.g., a buffer such as PBS buffer). The fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more prior to use.

[0105] In some cases, the sample may undergo pre-analysis treatment, as described above. Pre-analysis treatment may provide additional functionality, such as non-specific protein removal and / or effective, yet inexpensively feasible, mixing functionality. Common methods of pre-analysis treatment may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art. In some cases, the fluid sample may be concentrated prior to use in an assay. For example, in embodiments where the sample is a human body fluid (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or combinations thereof. The fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more prior to use.

[0106] solid support In certain embodiments, one or more compounds of formula (II) are immobilized on a solid support. The terms "solid phase" or "solid support" are used interchangeably herein and refer to any material that can be used to attach, attract, and / or immobilize one or more specific binding members. For example, the specific binding members can be part of a conjugate of formula (II) disclosed herein. Any solid support known in the art can be used in the methods described herein, including, but not limited to, solid supports in the form of planar substrates or beads made of polymeric materials. In certain embodiments, the beads can be particles, e.g., microparticles. The terms "beads" and "particles" are used interchangeably herein and refer to a substantially spherical solid support. The terms "microparticles" and "microbeads" are used interchangeably herein and refer to microbeads or microparticles that can reside or be anchored in a series of wells, such as a series of wells in a detection module. The microparticles or microbeads can contain at least one compound of formula (II) that contains at least one specific binding member that binds to an analyte of interest. When more than one analyte of interest is to be detected, the method may include one microparticle containing two or more different compounds of Formula (II) that contain first and second specific binding members that bind to a first analyte, and a second microparticle that contains third and fourth specific binding members that bind to a second analyte, etc.

[0107] In some embodiments, the microparticles can be about 0.1 nm to about 10 microns, about 50 nm to about 5 microns, about 100 nm to about 1 micron, about 0.1 nm to about 700 nm, about 500 nm to about 10 microns, about 500 nm to about 5 microns, about 500 nm to about 3 microns, about 100 nm to 700 nm, or about 500 nm to 700 nm. For example, the microparticles can be about 4 to 6 microns, about 2 to 3 microns, or about 0.5 to 1.5 microns. Particles less than about 500 nm can be referred to as "nanoparticles." Thus, the microparticles can optionally be nanoparticles of about 0.1 nm to about 500 nm, about 10 nm to about 500 nm, about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm.

[0108] In certain embodiments, the beads may be magnetic beads or particles. The magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, NiO / Fe. Exemplary ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO.Fe2O3). The beads may have a magnetic solid core surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer around a non-magnetic core. The solid support on which the binding member (e.g., a compound of formula (II)) is immobilized may be stored in dry form or in liquid. The magnetic beads may be subjected to a magnetic field before or after contacting the sample with the magnetic beads on which the binding member is immobilized.

[0109] The solid support can be contacted with a quantity of sample using any suitable method known in the art. As used herein, the term "contacting" refers to any type of combinatorial action that brings the binding member immobilized on the solid support into sufficient proximity with the analyte of interest in the sample such that a binding interaction occurs if the analyte of interest specific for the binding member is present in the sample. Contacting can be accomplished in a variety of ways, including combining the sample with microparticles or exposing the target analyte to the microparticles containing the binding member by introducing the microparticles in close proximity to the analyte. Contacting can be repeated as many times as necessary.

[0110] Whatever method is used, the solid support is contacted with a quantity of sample under conditions whereby one or more analytes, if present in the sample, bind to at least one specific binding member (e.g., a portion of a conjugate of formula (II) disclosed herein) immobilized on the surface of the solid support (e.g., microparticle). In one embodiment, contact between the solid support and the sample volume is maintained (i.e., incubated) for a time sufficient to allow binding interactions between the specific binding members and the analytes to occur. In one embodiment, the sample volume is incubated with the solid support for at least 30 seconds and no more than 10 minutes. For example, the sample may be incubated with the solid support for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 minutes. In one embodiment, the sample may be incubated with the microparticle for about 2 minutes. Additionally, the incubating may be performed in a binding buffer that promotes specific binding interactions, such as, for example, albumin (e.g., BSA), non-ionic detergents (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). The binding affinity and / or specificity of the specific binding members may be manipulated or altered in the assay by changing the binding buffer. In some embodiments, the binding affinity and / or specificity may be increased or decreased by changing the binding buffer. Other conditions of the binding interaction, such as, for example, temperature and salt concentration, may be determined empirically or based on manufacturer's instructions. For example, the contacting may be performed at room temperature (21° C. to 28° C., e.g., 23° C. to 25° C.), 37° C., or 4° C.

[0111] In one embodiment, the solid support desirably comprises a multiplicity (e.g., 2 or more, 50 or more, 100 or more, 1,000 or more, or 5,000 or more) of specific binding members that bind to analytes of interest immobilized on its surface. After a sufficient incubation time between the solid support and the sample as discussed above, one or more analytes of interest present in the sample are desirably captured on the surface of the solid support via the specific binding members immobilized on the surface of the solid support. As used herein, the term "immobilization" refers to a stable association of a binding member with the surface of the solid support.

[0112] As discussed above, the methods disclosed herein are suitable for detecting two or more different analytes. Thus, in some embodiments, the method may include capturing a second, third, fourth or subsequent analyte of interest on a surface of a second, third, fourth or subsequent solid support, where (i) each of the first, second, third, fourth and subsequent analytes are different from each other, and (ii) the second, third, fourth or subsequent solid support comprises one or more specific binding members immobilized on its surface that bind to the second, third, fourth or subsequent analytes. The method may further include reacting the captured second, third, fourth or subsequent analytes with a second, third, fourth or subsequent conjugate, where the second, third, fourth or subsequent conjugate is labeled with a fluorophore and comprises a specific binding member that binds to the second, third, fourth or subsequent analyte, where each fluorophore is different.

[0113] In certain embodiments, the solid support may also include a protective, blocking, or passivation layer that can eliminate or minimize non-specific adhesion of non-capture components (e.g., analyte molecules, binding members) to the binding surface during the assay, which may lead to false positive signals or loss of signal during detection. In certain embodiments, examples of materials that may be utilized to form the passivation layer include, but are not limited to, polymers that block non-specific binding of proteins (e.g., polyethylene glycol); naturally occurring proteins (e.g., serum albumin and casein); surfactants (e.g., zwitterionic surfactants, sulfobetaine); naturally occurring long chain lipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.

[0114] In certain embodiments, a specific binding member (e.g., a compound of Formula (II) containing a specific binding member) may be attached to a solid support via linkage, functionalization, or modification of the support and / or binding member that may include any moiety that facilitates attachment of the binding member to the support. The linkage between the binding member and the support may include one or more chemical or physical (e.g., non-specific attachment via van der Waals forces, hydrogen bonding, electrostatic interactions, hydrophobic / hydrophilic interactions, etc.) bonds and / or chemical spacers that effect such bonds. Certain embodiments utilize binding members that are proteins or polypeptides, and a number of techniques can be used to attach the polypeptides to a wide variety of solid supports (see, e.g., U.S. Pat. No. 5,620,850; and Heller, Acc. Chem. Res., 23:128 (1990)).

[0115] In some embodiments, the binding affinity between the analyte molecule and the binding member should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specifically bound molecules or particles. In some cases, e.g., for the detection of certain biomolecules, the binding constant of an analyte molecule to its complementary binding member is at least about 10 4 ~about 10 6 M-1 , at least about 10 5 ~about 10 9 M -1 , at least about 10 7 ~about 10 9 M -1 , about 10 9 M -1 It can be more than or even more.

[0116] multiplexing In some embodiments, the method involves determining the presence and / or concentration of an analyte in a sample. In this regard, the method may include contacting a biological sample with at least one first specific binding member and at least one second specific binding member, where each of the at least one first specific binding member and the at least one second specific binding member specifically binds to the analyte of interest, thereby generating one or more first complexes comprising the first specific binding member-analyte-second specific binding member, where the second specific binding member comprises any one of the conjugates described above. In such embodiments, the method further includes detecting the presence or absence of a signal from the second specific binding member, where detection of the signal indicates that the analyte is present in the sample and the absence of a signal indicates that the analyte is not present in the sample. In each of these embodiments, the specific binding member may be part of a compound of formula (II).

[0117] In certain embodiments, the methods may also be used to determine the presence and / or concentration of multiple different analytes present in a sample (i.e., multiplexing). In this regard, the disclosed methods may include two or more specific binding members and solid supports (e.g., 2, 3, 4, 5 or more) that detect two or more (e.g., 2, 3, 4, 5 or more) target analytes in a sample, referred to herein as a "multiplexed immunoassay" or "multiplexed assay." Each of the specific binding members binds to a different analyte, and each specific binding member and / or solid support (e.g., microparticle) may include a different detectable label.

[0118] In some embodiments, the disclosure provides a method of detecting two or more analytes of interest in a biological sample, comprising the steps of: (a) simultaneously or sequentially contacting the biological sample with: (i) at least one first specific binding member that binds to a first analyte of interest to form at least one first complex; and (ii) at least one second specific binding member that binds to a second analyte of interest to form at least one second complex, wherein each of the first and second specific binding members comprises any one of the conjugates described above, and wherein the fluorophores of the conjugates on each of the first and second specific binding members are selected from the group consisting of fluorophores, ... and (b) detecting the presence or absence of a signal from each of the first and second specific binding members, where (i) detection of a signal from the first specific binding member indicates the presence of a first analyte in the sample and the absence of a signal from the first specific binding member indicates the absence of the first analyte in the sample, and (ii) detection of a signal from the second specific binding member indicates the presence of a second analyte in the sample and the absence of a signal from the second specific binding member indicates the absence of the second analyte in the sample.

[0119] In other embodiments, the disclosure provides a method of detecting two or more analytes of interest in a biological sample, comprising the steps of: (a) contacting the biological sample with at least one first specific binding member and at least one second specific binding member, where each of the at least one first specific binding member and the at least one second specific binding member specifically binds to a first analyte of interest, thereby generating one or more first complexes comprising first specific binding member-first analyte-second specific binding member, where the second specific binding member comprises any one of the conjugates described above; and (b) simultaneously or sequentially contacting the biological sample with at least one third specific binding member and at least one fourth specific binding member, where each of the at least one third specific binding member and the at least one fourth specific binding member specifically binds to a second analyte of interest. and (c) detecting the presence or absence of a signal from each of the second and fourth specific binding members, wherein (i) detection of a signal from the second specific binding member indicates the presence of the first analyte in the sample and the absence of a signal from the second specific binding member indicates the absence of the first analyte in the sample, and (ii) detection of a signal from the fourth specific binding member indicates the presence of the second analyte in the sample and the absence of a signal from the fourth specific binding member indicates the presence of the second analyte in the sample.

[0120] In certain embodiments, the methods described herein may be used to detect more than two analytes of interest. For example, when a biological sample contains three analytes of interest, the method may include the steps of simultaneously or sequentially contacting the biological sample with at least one fifth specific binding member and at least one sixth specific binding member, each of which specifically binds to a third analyte of interest, thereby generating one or more third complexes comprising fifth specific binding member-third analyte-sixth specific binding member, the sixth specific binding member comprising any one of the conjugates described above, wherein the fluorophores of the conjugates in each of the second, fourth and sixth specific binding members are different, and contacting each of the second, fourth and sixth specific binding members with at least one sixth specific binding member. The method may further comprise the step of detecting the presence or absence of a signal from the second specific binding member, where (i) detection of a signal from the second specific binding member indicates the presence of the first analyte in the sample and the absence of a signal from the second specific binding member indicates the absence of the first analyte in the sample, (ii) detection of a signal from the fourth specific binding member indicates the presence of the second analyte in the sample and the absence of a signal from the fourth specific binding member indicates the presence of the second analyte in the sample and (iii) detection of a signal from the sixth specific binding member indicates the presence of a third analyte in the sample and the absence of a signal from the sixth specific binding member indicates the presence of the third analyte in the sample.

[0121] After reaction of one or more captured analytes with a conjugate described herein, any specific binding member (e.g., antibody or antibody fragment) or component of the conjugate that is not bound to the captured analyte may be removed, followed by an optional washing step. Any unbound antibody, antibody fragment, or component of the conjugate may be separated from the complex by any suitable means, such as droplet actuation, electrophoresis, electrowetting, dielectrophoresis, electrostatic actuation, electric field mediated, electrode mediated, capillary forces, chromatography, centrifugation, aspiration, or surface acoustic wave (SAW) based washing methods.

[0122] It is recognized that the capture of analytes in different conformations and the above complex formation methods are within the scope of the present disclosure.In fact, the above various solid support components, specific binding members, conjugates, and fluorophores can be arranged or utilized in any suitable combination, conformation, or format.For example, the disclosed methods can be performed in one-step, delayed one-step, or two-step format.The assay reagents (e.g., microparticles, conjugates, fluorophores) can be premixed or added sequentially as needed.

[0123] Detection and quantification of analytes The presence or amount of an analyte of interest present in a sample may be determined (e.g., quantified) using any suitable method known in the art. Such methods include, but are not limited to, immunoassays. Any suitable immunoassay may be utilized, such as sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays), competitive inhibition immunoassays (e.g., forward and reverse), chemiluminescence immunoassays, competitive binding assays, heterogeneous assays, and capture on the fly assays. Immunoassay components and techniques that may be used in the disclosed methods are further described, for example, in International Patent Application Publication Nos. 2016 / 161402 and 2016 / 161400. The method may involve single molecule counting. In one aspect, the assay used is in a clinical chemistry format.

[0124] As discussed herein, the disclosed compounds include an acridinium moiety and a fluorophore linked via a rigid diamine linker. Thus, upon chemiluminescence triggering of the acridinium moiety, the light output can be shifted to the emission wavelength of the attached fluorophore. The use of acridinium compounds as detectable labels in homogeneous chemiluminescent assays has been described, for example, in Adamczyk et al., Bioorg. Med. Chem. Lett. 16:1324-1328(2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4:2313-2317(2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14:3917-3921(2004); and Adamczyk et al., Org. Lett. 5:3779-3782(2003)). In one embodiment, chemiluminescence triggering of the acridinium moiety involves the addition of hydrogen peroxide to the biological sample prior to the detection step. Hydrogen peroxide may be provided or supplied to the biological sample prior to, simultaneously with, or after the addition of the specific binding member comprising the conjugate. The source of hydrogen peroxide may be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, a solution of hydrogen peroxide may simply be added to the biological sample.

[0125] In other embodiments, the fluorophores of the conjugates of each of the first, second, third, fourth, fifth or further specific binding members are different. Any suitable fluorophore known in the art and described herein can be attached to the disclosed compounds. The fluorescent signal from each specific binding member can be visualized and identified using any suitable device known in the art, including, but not limited to, a photomultiplier tube, a photodiode array, or a charge-coupled device camera. In some embodiments, these devices can be equipped with filters that can distinguish between one wavelength at a time.

[0126] In some embodiments, concentrations of an analyte in a sample that can be substantially accurately determined are less than about 5000 fM (femtomolar), less than about 3000 fM, less than about 2000 fM, less than about 1000 fM, less than about 500 fM, less than about 300 fM, less than about 200 fM, less than about 100 fM, less than about 50 fM, less than about 25 fM, less than about 10 fM, less than about 5 fM, less than about 2 fM, less than about 1 fM, less than about 500 aM (attomolar), less than about 100 aM, less than about 10 aM, less than about 5 aM, less than about 1 aM, less than about 0.1 aM, less than about 500 zM (zeptomolar), less than about 100 zM, less than about 10 zM, less than about 5 zM, less than about 1 zM, less than about 0.1 zM, or less. For example, the concentration of the analyte in a sample that can be substantially accurately determined is between about 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM, between about 100 fM and about 1 zM, between about 100 aM and about 0.1 zM, or in a range defined by any two of the above values.

[0127] In some embodiments, the lower detection limit (e.g., the lowest concentration of the analyte that can be determined in solution) is about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM, about 500 aM (attomolar), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM, about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM or less.

[0128] The upper detection limit (e.g., the upper concentration of the analyte that can be determined in solution) can be at least about 100 fM, at least about 1000 fM, at least about 10 pM (picomolar), at least about 100 pM, at least about 100 pM, at least about 10 nM (nanomolar), at least about 100 nM, at least about 1000 nM, at least about 10 μM, at least about 100 μM, at least about 1000 μM, at least about 10 mM, at least about 100 mM, at least about 1000 mM, or more.

[0129] In some cases, the presence and / or concentration of an analyte in a sample can be detected rapidly, usually in less than about 1 hour, for example, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, or 30 seconds.

[0130] The disclosed methods may include a quality control component. "Quality control components" include, but are not limited to, calibrators, controls, and sensitivity panels, in the context of the immunoassays and kits described herein. "Calibrators" or "standards" can be used (e.g., one or more, e.g., multiple) to establish a calibration (standard) curve for interpolating the concentration of an analyte, e.g., an antibody. Alternatively, a single calibrator can be used that is close to a reference level or control level (e.g., a "low," "medium," or "high" level). Multiple calibrators (i.e., more than one calibrator or calibrator(s) with varying amounts) can be used together to comprise a "sensitivity panel." A calibrator is optionally part of a set of calibrators, where each calibrator differs from the other calibrators in the set, for example, by concentration or detection method (e.g., colorimetric or fluorescent detection).

[0131] Variations on the disclosed method The disclosed method can be adapted as necessary for other methods for analyzing analytes. Examples of well-known variations include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays), immunoassays involving enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA)), competitive inhibition immunoassays (e.g., forward and reverse), enzyme-amplified immunoassay technology (EMIT), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture-on-the-fly assays. In some instances, the following description may overlap with the above methods, and in others, the following description may indicate alternatives.

[0132] Immunoassays The analytes of interest and / or peptides or fragments thereof may be analyzed using an immunoassay. Any immunoassay may be utilized. The immunoassay may be an enzyme-linked immunoassay (ELISA), a competitive inhibition assay, such as a forward or reverse competitive inhibition assay, or a competitive binding assay. In some embodiments, a detectable label (e.g., one or more fluorescent labels) is attached to the capture antibody and / or the detection antibody.

[0133] Heterogeneous formats may be used. For example, after a sample is obtained from a subject, a first mixture is prepared. The mixture contains the sample to be evaluated for an analyte of interest and a first specific binding member, where the first specific binding member and any analyte of interest contained in the sample form a first specific binding member-analyte of interest complex. Preferably, the first specific binding member is an antibody or fragment thereof against the anti-analyte of interest. The order in which the sample and the first specific binding member are added to form the mixture is not important. Preferably, the first specific binding member is immobilized on a solid phase. The solid phase used in the immunoassay (with the first specific binding member, optionally with the second specific binding member) can be any solid phase known in the art, such as, but not limited to, magnetic particles, beads, nanobeads, microbeads, nanoparticles, microparticles, membranes, scaffold molecules, films, filter paper, discs, or chips (e.g., microfluidic chips).

[0134] After a mixture containing the first specific binding member-analyte of interest complex is formed, unbound analyte of interest is removed from the complex using any technique known in the art. For example, unbound analyte of interest may be removed by washing. However, desirably, the first specific binding member is present in excess of any analyte of interest present in the sample, such that all analytes of interest present in the sample are bound by the first specific binding member.

[0135] After any unbound analyte of interest is removed, a second specific binding member is added to the mixture to form a first specific binding member-analyte of interest-second specific binding member complex. The second specific binding member is preferably an anti-analyte of interest (e.g., an antibody) that binds to an antigenic determinant on the analyte of interest that is different from the antigenic determinant on the analyte of interest bound by the first specific binding member. Furthermore, preferably, the second specific binding member is labeled with or contains a detectable label (e.g., a detectable label, a tag attached by a cleavable linker).

[0136] The use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. Antibodies are immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, latex particles or surface-modified latex particles, polymers or polymer films, plastics or plastic films, planar substrates, microfluidic surfaces, pieces of solid substrate material, and the like.

[0137] Sandwich immunoassay Sandwich immunoassays measure the amount of antigen between two layers of antibodies (i.e., a capture antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody). The capture antibody and the detection antibody bind to different antigenic determinants on the antigen, e.g., the analyte of interest. Desirably, binding of the capture antibody to the antigenic determinant does not interfere with binding of the detection antibody to the antigenic determinant. Monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich immunoassays.

[0138] Typically, at least two antibodies are used to separate and quantify an analyte of interest in a sample. More specifically, at least two antibodies bind to certain antigenic determinants of the analyte of interest or fragments of the analyte of interest forming an immune complex called a "sandwich". One or more antibodies may be used to capture the analyte of interest in the sample (these antibodies are often referred to as the "capture" antibody(ies)), and one or more antibodies with a detectable label (e.g., fluorescent label, tag attached by a cleavable linker) that also bind to the analyte of interest (these antibodies are often referred to as the "detection" antibody(ies)) may be used to complete the sandwich. In some embodiments, an aptamer may be used as the second binding member. In a sandwich assay, the binding of an antibody to its antigenic determinant is desirably not diminished by the binding of any other antibody in the assay to its respective antigenic determinant. In other words, the antibodies are selected so that one or more first antibodies are contacted with a sample suspected of containing an analyte of interest that does not bind to all or a portion of the antigenic determinant recognized by a second, or subsequent, antibody, thereby interfering with the ability of one or more second detection antibodies to bind to the analyte of interest.

[0139] In one embodiment, a sample suspected of containing an analyte of interest may be contacted with at least one capture antibody(ies) and at least one detection antibody simultaneously or sequentially. In a sandwich assay format, a sample suspected of containing an analyte of interest (e.g., a membrane-bound analyte of interest, a soluble analyte of interest, a membrane-bound fragment of an analyte of interest, a fragment of a soluble analyte of interest, a variant of an analyte of interest (membrane-bound or soluble analyte of interest), or any combination thereof) is first contacted with at least one capture antibody that specifically binds to a particular antigenic determinant under conditions that allow for the formation of an antibody-analyte of interest complex. If more than one capture antibody is used, multiple capture antibody-analyte of interest complexes are formed. In a sandwich assay, an antibody, preferably at least one capture antibody, is used in molar excess of the maximum amount of analyte of interest or fragment of analyte of interest expected in the sample.

[0140] Optionally, at least one capture antibody can be bound to a solid support prior to contacting the sample with the at least one first capture antibody, which facilitates separation of the antibody-analyte of interest complex from the sample. Any solid support known in the art can be used, including, but not limited to, solid supports made of polymeric materials, such as planar substrates or in the form of beads. The antibody(ies) can be bound to the solid support by adsorption, by covalent attachment using chemical coupling agents, or by other means known in the art, provided that such attachment does not interfere with the ability of the antibody to bind the analyte of interest or a fragment of the analyte of interest. Furthermore, if necessary, the solid support can be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, azide, alkynyl, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0141] After a sample suspected of containing an analyte of interest is contacted with at least one capture antibody, the sample is incubated to allow for the formation of a capture antibody(ies)-analyte of interest complex. The incubation can be carried out at a pH of about 4.5 to about 10.0, at a temperature of about 2° C. to about 45° C., and for a period of at least about 1 minute to about 18 hours, about 2 to 6 minutes, or about 3 to 4 minutes.

[0142] After the formation of the capture antibody(ies)-analyte of interest complex, the complex is then contacted with at least one detection antibody (under conditions that allow the formation of the capture antibody(ies)-analyte of interest-detection antibody(ies) complex). If the capture antibody-analyte of interest complex is contacted with more than one detection antibody, then a capture antibody(ies)-analyte of interest-detection antibody(ies) detection complex is formed. Similar to the capture antibody, when at least one detection (and subsequent) antibody contacts the capture antibody-analyte of interest complex, the formation of the capture antibody(ies)-analyte of interest-detection antibody(ies) complex requires a period of incubation under similar conditions as described above. Preferably, at least one detection antibody contains a detectable label (e.g., a fluorescent label, a tag attached by a cleavable linker). The detectable label can be bound to at least one detection antibody before, simultaneously with, or after the formation of the capture antibody(ies)-analyte of interest-detection antibody(ies) complex. Any detectable label known in the art can be used, for example, fluorescent labels known in the art, such as those discussed herein.

[0143] The order in which the sample and specific binding member(s) are added to form the assay mixture is not important. If the first specific binding member is detectably labeled (e.g., fluorescently labeled), a detectably labeled first specific binding member-analyte of interest complex is formed. Alternatively, if a second specific binding member is used and the second specific binding member is detectably labeled (e.g., fluorescently labeled), a first specific binding member-analyte of interest-second specific binding member detectably labeled complex is formed. Any unbound specific binding members, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0144] A signal is then generated that indicates the presence of the analyte of interest or a fragment thereof. Based on parameters of the signal generated, the amount of the analyte of interest in the sample can be quantified. Optionally, a standard curve can be generated using serial dilutions or solutions of known concentrations of the analyte of interest by mass spectrometry, gravimetry, and other techniques known in the art.

[0145] Forward competitive inhibition In the forward competition format, an aliquot of a known concentration of a labeled analyte of interest (e.g., an analyte bearing a fluorescent label) is used to compete with the analyte of interest in the sample for binding to the analyte of interest antibody.

[0146] In a forward competitive assay, an immobilized specific binding member (e.g., an antibody) can be contacted sequentially or simultaneously with the sample and a labeled analyte of interest, a fragment of the analyte of interest, or a variant thereof of the analyte of interest. The analyte of interest, a fragment of the analyte of interest, or a variant thereof of the analyte of interest can be labeled with any detectable label, including a detectable label consisting of a tag attached with a cleavable linker. In this assay, the antibody can be immobilized to a solid support. Alternatively, the antibody can be coupled to another antibody, e.g., an anti-species antibody, that is immobilized to a solid support, e.g., a microparticle or a planar substrate.

[0147] Reverse competition assay In a reverse competitive assay, the immobilized analyte of interest can be contacted, either sequentially or simultaneously, with the sample and at least one labeled antibody. The analyte of interest can be bound to a solid support, such as those solid supports discussed above in connection with the sandwich assay format.

[0148] One-step immunoassay or "capture on the fly" In a capture-on-the-fly immunoassay, the solid substrate is pre-coated with a fixative. The capture agent, analyte, and detection agent are added together to the solid substrate, followed by a washing step before detection. The capture agent may bind the analyte and may include a ligand for the fixative. The capture agent and detection agent may be an antibody or any other moiety capable of capture or detection as described herein or known in the art. The ligand may include a peptide tag and the fixative may include an anti-peptide tag antibody. Alternatively, the ligand and fixative may be any pair of agents capable of binding together as used in a capture-on-the-fly assay (e.g., specific binding pairs are known in the art). More than one analyte may be measured. In some embodiments, the solid substrate may be coated with an antigen and the analyte being analyzed is an antibody.

[0149] In certain other embodiments, a one-step immunoassay or "capture on the fly" uses a solid support (e.g., a microparticle) pre-coated with an immobilizing agent (e.g., biotin, streptavidin) and at least a first specific binding member and a second specific binding member (acting as a capture reagent and a detection reagent, respectively). The first specific binding member comprises a ligand of the immobilizing agent (e.g., if the immobilizing agent on the solid support is streptavidin, the ligand on the first specific binding member can be biotin) and also binds to the analyte of interest. The second specific binding member comprises a detectable label and binds to the analyte of interest. The solid support and the first and second specific binding members can be added (sequentially or simultaneously) to the sample. The ligand on the first specific binding member binds to the immobilizing agent on the solid support to form a solid support / first specific binding member complex. Any analyte of interest present in the sample binds to the solid support / first specific binding member complex to form a solid support / first specific binding member / analyte complex. The second specific binding member binds to the solid support / first specific binding member / analyte complex and the detectable label is detected. An optional washing step may be used prior to detection. In some embodiments, more than one analyte may be measured in a one-step assay. In some other embodiments, more than two specific binding members may be used. In some other embodiments, multiple detectable labels may be added. In some other embodiments, multiple analytes of interest may be detected.

[0150] The capture on the fly assay can be performed in a variety of formats, as described herein and known in the art, for example, the format can be a sandwich assay, such as those described above, or it can be a competitive assay, which can use a single specific binding member, or other known variations.

[0151] Combination Assays In a combinatorial assay, a solid substrate, such as a microparticle, is co-coated with an antigen and an antibody to capture the antibody and antigen, respectively, from a sample. A solid support may be co-coated with two or more different antigens to capture two or more different antibodies from a sample. A solid support may be co-coated with two or more different antibodies to capture two or more different antigens from a sample.

[0152] Additionally, the methods described herein may use blocking agents to prevent specific or non-specific binding reactions (e.g., HAMA problems) among the assay compounds. Once the agent (and optionally any control) is immobilized on the support, the remaining binding sites of the agent may be blocked on the support. Any suitable blocking reagent known to those skilled in the art may be used. For example, bovine serum albumin ("BSA"), a phosphate buffered saline ("PBS") solution of casein in PBS, Tween 20™ (Sigma Chemical Company, St. Louis, Mo.), or other suitable detergents, as well as other blocking reagents, may be used.

[0153] As will be apparent from the present disclosure, the methods disclosed herein, including variations, may be used to diagnose a disease, disorder, or condition in a subject suspected of having the disease, disorder, or condition. For example, sample analysis may be useful for detecting disease markers, e.g., cancer markers, cardiac condition markers, toxins, pathogens, e.g., viruses, bacteria, or portions thereof. The methods may also be used to measure analytes present in a biological sample. The methods may also be used in blood screening assays to detect target analytes. Blood screening assays may be used to screen the blood supply.

[0154] Device for analyzing an analyte The methods described herein can be carried out using any device suitable for analyzing the analyte, a variety of devices being known in the art, such as peristaltic pump systems (e.g., peristaltic pump systems available from FISHERBRAND™ Variable-Flow Peristaltic Pumps, ThermoFisher Scientific, Waltham, MA; and MilliporeSigma, Burlington, MA), automated / robotic sample delivery systems (commercially available, for example, from Hamilton Robotics, Reno, NV; and ThermoFisher Scientific, Waltham, MA), microfluidic devices, droplet-based microfluidic devices, digital microfluidic devices (DMF), surface acoustic wave-based microfluidic (SAW) devices, or electrowetting-on-dielectric (EWOD) digital microfluidic devices (e.g., Peng et al., Lab Chip, 14(6):1117-1122 (2014); and Huang et al., PLoS ONE, 10(5):e0124196 (2015)), as well as other automated systems, such as KINGFISHER™ instruments (ThermoFisher Scientific, Waltham, MA), ARCHITECT™ Analyzers (Abbott, Abbott Park, IL), and other automated instruments known in the art.

[0155] In one embodiment, the methods described herein may be performed using a microfluidic device, such as a digital microfluidic (DMF) device. Any suitable microfluidic device known in the art may be used to perform the methods described herein, such as those described in, for example, International Application Nos. 2007 / 136386, 2009 / 111431, 2010 / 040227, 2011 / 137533, 2013 / 066441, 2014 / 062551, 2014 / 066704, and U.S. Patent No. 8,287,808. In certain cases, the device may be a lab-on-a-chip device in which analyte analysis may be performed on droplets of a sample containing or suspected of containing the analyte.

[0156] In one embodiment, at least two steps (e.g., two, three, or all steps) of the methods described herein are performed in a digital microfluidic device. The terms "digital microfluidics (DMF)", "digital microfluidic module (DMF module)", or "digital microfluidic device (DMF device)" are used interchangeably herein to refer to a module or device that utilizes digital or droplet-based microfluidic technology to manipulate discrete and small amounts of liquids in the form of droplets. Digital microfluidics uses the principles of emulsion science to create fluid-fluid dispersions (mainly water-in-oil emulsions) within channels, allowing the generation of monodisperse droplets / bubbles with low polydispersity. Digital microfluidics is based on the micromanipulation of discrete fluid droplets within a reconfigurable network. Complex instructions can be programmed by combining basic operations such as droplet formation, movement, splitting, and merging.

[0157] Digital microfluidics operates on discrete fluid volumes that can be manipulated by binary electrical signals. Using droplets of discrete unit volume, microfluidic operations can be defined as a series of repeated elementary operations, i.e., the movement of one unit of fluid over one unit of distance. Droplets can be formed using the surface tension properties of liquids. The actuation of droplets is based on the presence of electrostatic forces generated by electrodes placed under the bottom surface on which the droplets are located. Different types of electrostatic forces can be used to manage the shape and movement of the droplets. One technique that can be used to create the aforementioned electrostatic forces is based on dielectrophoresis due to the difference in dielectric constant between the droplet and the surrounding medium, and can utilize a high frequency AC electric field. Another technique that can be used to create the aforementioned electrostatic forces is based on electrowetting, which relies on the dependence of the surface tension between a liquid droplet present on a surface and the surface on the electric field applied to the surface.

[0158] In another embodiment, the methods described herein can be performed with a surface acoustic wave (SAW)-based microfluidic device as a front-end assay processing method. As used herein, the term "surface acoustic wave (SAW)" generally refers to an acoustic wave propagating in a direction along a surface. A "traveling surface acoustic wave" (TSAW) can couple the surface acoustic wave into a liquid. In some embodiments, the coupling can be in the form of penetration or leakage of the surface acoustic wave into the liquid. In other embodiments, the surface acoustic wave is a Rayleigh wave (see, e.g., Oliner, AA (ed.), Acoustic Surface Waves. Springer (1978)). The propagation of the surface acoustic wave can be performed by a variety of different means and using different materials, including the generation of an electric potential by a transducer, e.g., a series or a plurality of electrodes, or by flowing the surface acoustic wave into the liquid.

[0159] In some embodiments, the DMF or SAW devices are fabricated by roll-to-roll based printed electronics methods, examples of such devices are described in International Patent Application Publication Nos. WO 2016 / 161402 and WO 2016 / 161400.

[0160] Many of the above devices allow for the detection of a single molecule of an analyte of interest. Other devices and systems known in the art that allow for single molecule detection of one or more analytes of interest can also be used in the methods described herein. Such devices and systems include, for example, Quanterix SIMOA™ (Lexington, MA) technology, Singulex's Single Molecule Counting (SMC™) technology (Alameda, CA, see, for example, U.S. Patent No. 9,239,284), and devices described, for example, in U.S. Patent Application Publication Nos. 2017 / 0153248 and 2018 / 0017552.

[0161] Kits and Cartridges Also provided herein are kits for use in carrying out the above methods. The kits may be used with any of the above devices. The instructions included in the kit may be affixed to the packaging material or may be included as a package insert. The instructions may be, but are not limited to, written or printed. Any medium capable of storing such instructions and communicating the instructions to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions.

[0162] The kit may include a cartridge that includes a microfluidic module. In some embodiments, the microfluidic module may be integrated into the cartridge. The cartridge may be disposable. The cartridge may include one or more reagents useful for practicing the methods disclosed above. The cartridge may include one or more containers that hold the reagents as one or more separate compositions, or, optionally, as an admixture where compatibility of the reagents allows. The cartridge may also include other material(s) that may be desirable from a user's perspective, such as buffer(s), diluent(s), standard(s) (e.g., calibrators and controls), and / or any other material useful for processing the sample, washing, or performing any other step of the assay. The cartridge may include one or more specific binding members as described above.

[0163] The kit may further include a reference standard for quantifying the analyte of interest. The reference standard may be used to establish a standard curve for interpolating and / or extrapolating the concentration of the analyte of interest. The kit may include reference standards that vary in concentration level. For example, the kit may include one or more reference standards at high, medium, or low concentration levels. With regard to the range of concentrations for the reference standard, this may be optimized for each assay. Exemplary concentration ranges for the reference standards include, for example, about 10 fg / mL, about 20 fg / mL, about 50 fg / mL, about 75 fg / mL, about 100 fg / mL, about 150 fg / mL, about 200 fg / mL, about 250 fg / mL, about 500 fg / mL, about 750 fg / mL, about 1000 fg / mL, about 10 pg / mL, about 20 pg / mL, about 50 pg / mL, about 75 pg / mL, about 100 pg / mL, about 150 pg / mL, about 200 pg / mL, about 250 pg / mL, about 500 pg / mL, about 750 pg / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 12.5ng / mL, approximately 15ng / mL, approximately 20ng / mL, approximately 25ng / mL, approximately 40ng / mL, approximately 45ng / mL, approximately 50ng / mL, approximately 55ng / mL, approximately 60ng / mL, approximately 75ng / mL, approximately 80ng / mL, approximately 85ng / mL, approximately 90ng / mL, approximately 95ng / mL, approximately 100ng / mL, approximately 125ng / mL, approximately 150ng / mL, approximately 165ng / mL, approximately 175ng / mL, approximately 200ng / mL, approximately 225ng / mL, approximately 250ng / mL, approximately 27 5ng / mL, approximately 300ng / mL, approximately 400ng / mL, approximately 425ng / mL, approximately 450ng / mL, approximately 465ng / mL, approximately 475ng / mL, approximately 500ng / mL, approximately 525ng / mL, approximately 550ng / mL, approximately 575ng / mL, approximately 600ng / mL, approximately 700ng / mL, approximately 725ng / mL, approximately 750ng / mL, approximately 765ng / mL, approximately 775ng / mL, approximately 800ng / mL, approximately 825ng / mL, approximately 850ng / mL, approximately 875ng / mL, Approximately 900ng / mL, approximately 925ng / mL, approximately 950ng / mL, approximately 975ng / mL, approximately 1000ng / mL, approximately 2μg / mL, approximately 3μg / mL, approximately 4μg / mL, approximately 5μg / mL, approximately 6μg / mL, approximately 7μg / mL, approximately 8μg / mL, approximately 9μg / mL, approximately 10μg / mL, approximately 20μg / mL, approximately 30μg / mL, approximately 40μg / mL, approximately 50μg / mL, approximately 60μg / mL, approximately 70μg / mL, approximately 80μg / mL, approximately 90μg / mL, approximately 100μg / mL, approximately Including, but not limited to, about 200 μg / mL, about 300 μg / mL, about 400 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, about 2000 μg / mL, about 3000 μg / mL, about 4000 μg / mL, about 5000 μg / mL, about 6000 μg / mL, about 7000 μg / mL, about 8000 μg / mL, about 9000 μg / mL, or about 10000 μg / mL.

[0164] The kit may include reagents for labeling the specific binding member, reagents for detecting the specific binding member and / or for labeling the analyte, and / or reagents for detecting the analyte. The kit may also include components that direct cleavage of the tag, such as a cleavage-mediating reagent. For example, the cleavage-mediating reagent may include a reducing agent, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). The specific binding members, calibrators, and / or controls may be provided in separate containers or may be pre-dispensed into a suitable assay format or cartridge.

[0165] The kit may also include quality control components (e.g., sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well known in the art and described in the insert sheets for immunodiagnostic products. Sensitivity panel members are optionally used to establish assay performance characteristics and are useful indicators of kit reagent integrity and assay standardization.

[0166] The kit may optionally also include other reagents required to perform the diagnostic assay or to facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents. Other components, such as buffers and solutions for isolating and / or processing the test sample (e.g., pretreatment reagents), may also be included in the kit. The kit may further include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit may further include reagents suitable for reconstituting the lyophilized components. One or more of the components may be in liquid form.

[0167] The various components of the kit may optionally be provided in suitable containers as needed. The kit may further include containers for holding or storing samples (e.g., containers or cartridges for urine, saliva, plasma, cerebrospinal fluid, or serum samples, or suitable containers for storing, transporting, or processing tissue to produce tissue aspirates). Where appropriate, the kit may optionally contain reaction vessels, mixing vessels, and other components that facilitate preparation of reagents or test samples. The kit may also include one or more sample collection / acquisition devices to aid in obtaining the test sample, such as various blood collection / transfer devices (e.g., microsampling devices, microneedles, or other minimally invasive, painless blood collection methods; blood collection tube(s); lancets; capillary blood collection tubes; other single finger prick blood collection methods; buccal swabs, nasal / throat swabs; 16 gauge or other size needles, circular blades for punch biopsy (e.g., 1-8 mm, or other suitable size), scalpels or lasers (e.g., especially handheld), syringes, sterile containers, or cannulas for obtaining, storing, or aspirating tissue samples). The kit may include one or more devices to aid in joint aspiration, cone biopsy, punch biopsy, fine needle aspiration biopsy, image-guided percutaneous needle aspiration biopsy, bronchoaveolar lavage, endoscopic biopsy, endoscopic biopsy, and laproscopic biopsy. EXAMPLES

[0168] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0169] Reagents used in the following examples were purchased from commercial sources and used as received unless otherwise indicated.

[0170] [Example 1]

[0171] [ka]

[0172] 1.0 g (1.7 mmol) of CPSP-acridinium (J. Org. Chem., 1998, vol. 63, pp. 5636-5639) was treated with 2 mL of [COCl]2 (23 mmol) in 25 mL of methylene chloride (DCM), followed by the addition of 5 μL of dimethylformamide. The slurry was stirred at room temperature for 2 h to give a yellow solution. After this time, the volatile components were removed from the reaction on a rotary evaporator under vacuum to give the diacid chloride as a yellow sticky foam. The residue was redissolved in DCM (25 mL). A saturated aqueous solution of potassium bifluoride (15 mL) was prepared and added to the DCM solution. The two-phase system was stirred vigorously for 2 h. After this time, the upper aqueous phase of the reaction was removed by pipette and the lower DCM layer was evaporated on a rotary evaporator under vacuum. The resulting yellow solid was suspended in water (about 25 mL) and filtered through a Buchner funnel. The solid was washed with a small amount of cold water (about 65 mL). Yield 1.08 g of a yellow solid. MS(M+): C 28 H 28 Calculated for FN2O7S2+: Exact mass: 587.13; Molecular weight: 587.66. UPLC / MS observed value 587.39.

[0173] [Example 2]

[0174] [ka]

[0175] A 25 mL round bottom flask equipped with a magnetic stir bar and nitrogen inlet was charged with 0.1 g (0.17 mmol) of the product of Example 1, DCM (10 mL), then 0.14 g (1.7 mmol) of piperazine was added in one portion to the yellow slurry resulting in a clear solution. The reaction was stirred at room temperature for 5.5 days. After this time a milky white slurry was obtained. The reaction was evaporated to dryness under vacuum and the solid was dissolved in water (5 mL), methanol (5 mL) and 1N HCl (2 mL). The resulting solution was purified by reverse phase HPLC using a YMC ODS AQ 50 x 250 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 70 mL / min), ACN / H2O / H2O to 0.5 TFA was used. The product-containing fractions were combined and the volatile components were removed on a rotary evaporator in vacuo at 30° C., followed by high vacuum at room temperature for 18 h. Yield: 0.163 g of a yellow glass (title compound as the TFA salt). MS (M+): C 32 H 37 Calculated for N4O7S2+: Exact mass: 653.21; Molecular weight: 653.79. UPLC / MS observed 653.33.

[0176] [Example 3]

[0177] [ka]

[0178] Using the same procedure outlined for the preparation of Example 2, the title compound was prepared utilizing 0.1 g (0.17 mmol) of the product of Example 1, DCM (5 mL) and 0.057 mL (0.85 mmol) of ethylenediamine (EDA). Yield 0.027 g of a yellow film (title compound as the TFA salt). MS (M+): C 30 H 35 Calculated for N4O7S2+: Exact mass: 627.1942; Molecular weight: 627.7510. UPLC / MS observed 627.43.

[0179] [Example 4]

[0180] [ka]

[0181] Using the same procedure outlined for the preparation of Example 2, the title compound was prepared utilizing 0.026 g (0.044 mmol) of the product of Example 1, DCM (5 mL) and 0.1 mL (0.45 mmol) of 4,7,10-trioxa-1,13-tridecanediamine. Yield 0.018 g of a yellow film (title compound as the TFA salt). MS (M+): C 38 H 51 N4O 10 Calculated for S2+: Exact mass: 787.3041; Molecular weight: 787.9618. UPLC / MS observed 787.53.

[0182] [Example 5]

[0183] [ka]

[0184] Using the same procedure outlined for the preparation of Example 2, the title compound was prepared utilizing 0.03 g (0.051 mmol) of the product of Example 1, DCM (1 mL) and 0.1 g (0.57 mmol) of 1,8-bis(methylamino)-3,6-dioxaoctane. Yield 0.016 g of a yellow film (title compound as the TFA salt). MS (M+): C 36 H 47 Calculated for N4O9S2+: Exact mass: 743.2779; Molecular weight: 743.9092. UPLC / MS observed value 743.39.

[0185] [Example 6]

[0186] [ka]

[0187] A 5 mL round bottom flask equipped with a magnetic stir bar and nitrogen inlet was charged with 0.015 g (0.026 mmol) of the product of Example 1, DMF (1 mL), N,N-diisopropylethylamine (DIEA) (0.34 mL, 2 mmol), and then (1S,4S)-(+)-2,5-diazabicyclo[2.2.1]heptane dihydrobromide (0.14 g, 0.52 mmol) was added in one portion. The reaction was stirred at room temperature for 2 days. The entire solution was purified by reversed phase HPLC using a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5 TFA. The product-containing fractions were combined and the volatile components were removed on a rotary evaporator in vacuo at 30° C., followed by high vacuum at room temperature for 18 h. Yield 0.0084 g of a yellow film (title compound as the TFA salt). MS(M+): C 33 H 37 Calculated for N4O7S2+: Exact mass: 665.2098; Molecular weight: 665.7989. UPLC / MS observed value 665.20.

[0188] [Example 7]

[0189] [ka]

[0190] A 5 mL round bottom flask equipped with a magnetic stir bar and nitrogen inlet was charged with 0.015 g (0.026 mmol) of the product of Example 1, DCM (0.5 mL), DIEA (0.17 mL, 1 mmol), and then (cis-racemic 0-tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (0.055 g, 0.26 mmol) was added in one portion to the yellow slurry. The reaction was stirred at room temperature for 18 hours. Using a stream of nitrogen, the reaction was evaporated to dryness and then dissolved in a small amount of MeOH. YMC ODS AQ with a Waters Separations 2000 system monitoring at 254 nm was used. The entire solution was purified by reversed-phase HPLC using a 30 x 150 mm I.D. steel column. Recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5 formic acid. The product-containing fractions were combined and the volatile components were removed on a rotary evaporator in vacuum at 30°C, followed by high vacuum at room temperature for 18 h. Yield 0.0205 g of a yellow film (Boc-protected amine intermediate). MS (M+): C 39 H 47 Calculated for N4O9S2+: Exact mass: 779.2779; Molecular weight: 779.9413. UPLC / MS observed value 779.16.

[0191] A 4 mL vial equipped with a magnetic stir bar was charged with the Boc protected amine intermediate and DCM (0.5 mL). Trifluoroacetic acid (TFA) (0.5 mL) was added and the mixture was stirred at RT for 1 h. The reaction was evaporated to dryness overnight using a stream of nitrogen. The crude product was dissolved in a small amount of MeOH. The entire solution was purified by reverse phase HPLC using a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5 TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C. followed by high vacuum at room temperature for 18 h. Yield 0.0175 g of a yellow film (title compound as TFA salt). MS(M+):C 34 H 39 Calculated for N4O7S2+: Exact mass: 679.2255; Molecular weight: 679.8255. UPLC / MS observed value 679.24.

[0192] [Example 8]

[0193] [ka]

[0194] Using the same procedure outlined for the preparation of Example 7, the title compound was prepared utilizing 0.015 g (0.026 mmol) of the product of Example 1, 5-Boc-octahydro-pyrrolo[3,4-c]pyridine (0.01 g, 0.044 mmol), DCM (0.5 mL for amine coupling and 0.5 mL for the deprotection step), DIEA (for amine coupling, 0.17 mL, 1 mmol), and TFA (for Boc deprotection, 0.5 mL). Yield 0.0074 g of a yellow film (Boc-protected amine intermediate). MS (M+): C 40 H 49Calculated for N4O9S2+: Exact mass: 793.2935; Molecular weight: 793.9679. UPLC / MS observed value 793.20. Yield 0.0077 g of a yellow film (title compound as the TFA salt). MS(M+):C 35 H 41 Calculated for N4O7S2+: Exact mass: 693.2411; Molecular weight: 693.8521. UPLC / MS observed value 693.20.

[0195] [Example 9]

[0196] [ka]

[0197] A 5 mL round bottom flask equipped with a magnetic stir bar and nitrogen inlet was charged with 0.015 g (0.026 mmol) of the product of Example 1, DCM (0.5 mL) and DIEA (0.17 mL, 1 mmol). trans-1,2-diaminocyclohexane was added in one portion to the yellow slurry. The reaction was stirred at room temperature for 18 hours. The reaction was evaporated to dryness using a stream of nitrogen and then dissolved in a small amount of MeOH. All solutions were purified by reversed phase HPLC using a YMC ODS AQ 30 x 150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The product-containing fractions were combined and the volatile components were removed on a rotary evaporator in vacuo at 30° C., followed by high vacuum at room temperature for 18 h. Yield 0.010 g of a yellow film (title compound as the TFA salt). MS (M+): C 34 H 41 Calculated for N4O7S2+: Exact mass: 681.2411; Molecular weight: 681.8414. UPLC / MS observed 681.27.

[0198] [Example 10]

[0199] [ka]

[0200] Using the same procedure outlined for the preparation of Example 9, the title compound was prepared utilizing 0.015 g (0.026 mmol) of the product of Example 1, DCM (0.5 mL), DIEA (0.17 mL, 1 mmol) and (+-)-trans-1,2-diaminocyclohexane (0.029 g, 0.26 mmol). Yield 0.0154 g of a yellow film (title compound as the TFA salt). MS (M+): C 34 H 41 Calculated for N4O7S2+: Exact mass: 681.2411; Molecular weight: 681.8414. UPLC / MS observed 681.34.

[0201] [Example 11]

[0202] [ka]

[0203] Using the same procedure outlined for the preparation of Example 9, the title compound was prepared utilizing 0.015 g (0.026 mmol) of the product of Example 1, DCM (0.5 mL), DIEA (0.17 mL, 1 mmol) and (S,S)-(+)-n,N'-dimethyl-1,2-cyclohexanediamine (0.037 g, 0.26 mmol). Yield 0.0056 g of a yellow film (title compound as the TFA salt). MS (M+): C 36 H 45 Calculated for N4O7S2+: Exact mass: 709.2724; Molecular weight: 709.8946. UPLC / MS observed value 709.27.

[0204] [Example 12]

[0205] [ka]

[0206] A 5 mL round bottom flask equipped with a magnetic stir bar and nitrogen inlet was charged with a mixture of 0.005 g (0.0065 mmol) of the product of Example 2, DMF (0.5 mL) and 0.01 g (0.021 mmol) of (5) 6-Carboxyfluorescein-NHS ester, followed by DIEA (0.05 mL, 0.28 mmol). The reaction was stirred at room temperature for 2.5 days. A few drops of water were added and the mixture was stirred at room temperature for 30 min. The reaction was diluted with MeOH (2 mL) and purified by reversed phase HPLC using a YMC ODS AQ 30 x 150 mm steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5 TFA. The product-containing fractions were combined, the volatile components were removed on a rotary evaporator under vacuum at 30° C., and dried under high vacuum (1 mmHg) for 2 hours. Yield: 0.0012 g of a yellow film (title compound). MS (M+): C 53 H 47 N4O 13 Calculated for S2+: Exact mass: 1011.2576; Molecular weight: 1012.0887. UPLC / MS observed value 1011.39.

[0207] A 5 mL round bottom flask equipped with a magnetic stir bar and nitrogen inlet was charged with 0.012 g of the product from the above step, DMF (0.5 mL), and pyridine (0.5 mL, 0.62 mmol). Pentafluorophenyl trifluoroacetate (0.05 mL, 0.3 mmol) was then added to the mixture in one portion and the reaction was stirred at room temperature for 1 h. The volatile components were removed from the mixture under vacuum and the residue was triturated 5× with 1:1 ether-hexanes and traces of volatile components were removed at high vacuum (1 mmHg) for 2 h. Yield 0.008 g of a yellow film (title compound, R=-O-pentafluorophenyl). MS (M+): C 59 H 46 F5N4O 13Calculated for S2+: Exact mass: 1177.2417; Molecular weight: 1178.1370. UPLC / MS observed 1177.21. The product was split into two equal portions for subsequent reactions and conjugations.

[0208] 0.004 g of the pentafluorophenyl ester product from the last step was dissolved in DCM (0.5 mL). Azido-dPEG3-amine (0.1 g, 0.45 mmol) in DCM (0.5 mL) was then added dropwise and the reaction mixture was stirred at room temperature for 1 h. Volatile components were removed from the reaction mixture under a stream of nitrogen for 18 h. The reaction mixture was purified by reversed-phase HPLC by diluting with MeOH (1 mL) and water (1 mL) and eluting on a YMC ODS AQ 30 x 150 mm steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed is 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The product-containing fractions were combined, the volatile components were removed on a rotary evaporator under vacuum at 30° C., and dried at high vacuum (1 mmHg) for 18 hours. Yield 0.007 g of a yellow film (title compound, R=-O-PEG-azide). MS(M+):C 61 H 63 N8O 15 Calculated for S2+: Exact mass: 1211.3849; Molecular weight: 1212.3270. UPLC / MS observed 1211.47.

[0209] [Example 13]

[0210] [ka]

[0211] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing a mixture of 0.039 g (0.049 mmol) of the product of Example 3, DMF (2.0 mL), 0.028 g (0.06 mmol) of (5) 6-carboxyfluorescein-NHS ester, and DIEA (0.1 mL, 0.6 mmol). Yield 0.008 g of a yellow film (title compound). MS (M+): C 51 H 45 N4O 13 Calculated for S2+: Exact mass: 985.2419; Molecular weight: 986.0515. UPLC / MS observed 985.49.

[0212] [Example 14]

[0213] [ka]

[0214] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.01 g (0.013 mmol) of the product of Example 2, DMF (0.25 mL), 0.03 g (0.055 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.025 mL, 0.055 mmol). Yield 0.0018 g of a yellow film (title compound). MS (M+): C 53 H 47 N4O 13 Calculated for S2+: Exact mass: 1011.2576; Molecular weight: 1012.0887. UPLC / MS observed value 1011.38.

[0215] [Example 15]

[0216] [ka]

[0217] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.01 g (0.013 mmol) of the product of Example 2, DMF (0.25 mL), 0.03 g (0.055 mmol) of 6-carboxyfluorescein-PFP ester (obtained from 6-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.025 mL, 0.055 mmol). Yield 0.0029 g of a yellow film (title compound). MS (M+): C 53 H 47 N4O 13 Calculated for S2+: Exact mass: 1011.2576; Molecular weight: 1012.0887. UPLC / MS observed value 1011.45.

[0218] [Example 16]

[0219] [ka]

[0220] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing a mixture of 0.01 g (0.013 mmol) of the product of Example 2, DMF (0.25 mL), 0.011 g (0.021 mmol) of (5) 6-TAMRA-NHS ester, and DIEA (0.025 mL, 0.055 mmol). The individual product isomers were separated during purification. Yield, fraction 9 to isomer A: 0.002 g of a purple film (title compound). MS (M+): C 57 H 57 NO 11 Calculated for S2+: Exact mass: 1065.3521; Molecular weight: 1066.2255. UPLC / MS observed 1065.55 (weak); M++ 533.45 (strong). Yield: 0.002 g of isomer B (title compound) from fraction 10. 57 H 57 NO 11Calculated for S2+: Exact mass: 1065.3521; Molecular weight: 1066.2255. UPLC / MS observed 1065.48 (weak); M++ 533.45 (strong).

[0221] [Example 17]

[0222] [ka]

[0223] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.01 g (0.011 mmol) of the product of Example 4, DMF (0.25 mL), 0.014 g (0.026 mmol) of 6-carboxyfluorescein-PFP ester (prepared from 6-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.025 mL, 0.055 mmol). Yield 0.005 g of a yellow film (title compound). MS (M+): C 59 H 61 N4O 16 Calculated for S2+: Exact mass: 1145.3518; Molecular weight: 1146.2623. UPLC / MS observed 1145.30.

[0224] [Example 18]

[0225] [ka]

[0226] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.0049 g (0.0057 mmol) of the product of Example 5, DMF (0.25 mL), 0.01 g (0.016 mmol) of Rhodamine B-PFP ester (prepared from Rhodamine B and pentafluorophenyl trifluoroacetate) and DIEA (0.025 mL, 0.055 mmol). Yield 0.0016 g of a purple film (title compound). MS (M+): C 64 H75 NO 11 Calculated for S2+: Exact mass: 1167.49; Molecular weight: 1168.45. UPLC / MS observed 1167.61.

[0227] [Example 19]

[0228] [ka]

[0229] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.0042 g (0.0054 mmol) of the product of Example 6, DMF (0.2 mL), 0.008 g (0.017 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0048 g of an orange-yellow film (title compound). MS (M+): C 54 H 47 N4O 13 Calculated for S2+: Exact mass: 1023.2576; Molecular weight: 1024.0994. UPLC / MS observed 1023.22.

[0230] [Example 20]

[0231] [ka]

[0232] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.0045 g (0.005 mmol) of the product of Example 7, DMF (0.2 mL), 0.008 g (0.017 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0033 g of an orange-yellow film (title compound). MS (M+): C 55 H 49 N4O 13 Calculated for S2+: Exact mass: 1037.2732; Molecular weight: 1038.1260. UPLC / MS observed 1037.18.

[0233] [Example 21]

[0234] [ka]

[0235] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.0038 g (0.0042 mmol) of the product of Example 8, DMF (0.2 mL), 0.008 g (0.017 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0023 g of an orange-yellow film (title compound). MS (M+): C 54 H 47 N4O 13 Calculated for S2+: Exact mass: 1051.2889; Molecular weight: 1052.1526. UPLC / MS observed 1051.30.

[0236] [Example 22]

[0237] [ka]

[0238] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.005 g (0.0063 mmol) of the product of Example 9, DMF (0.2 mL), 0.008 g (0.017 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0042 g of a yellow film (title compound). MS (M+): C 55 H 51 N4O 13 Calculated for S2+: Exact mass: 1039.2889; Molecular weight: 1040.1419. UPLC / MS observed 1039.29.

[0239] [Example 23]

[0240] [ka]

[0241] The title compound was prepared using the same procedure outlined for the preparation of Example 12 utilizing 0.0057 g (0.0072 mmol) of the product of Example 10, DMF (0.2 mL), 0.008 g (0.017 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0024 g of a yellow film (title compound). MS (M+): C 55 H 51 N4O 13 Calculated for S2+: Exact mass: 1039.2889; Molecular weight: 1040.1419. UPLC / MS observed 1039.21.

[0242] [Example 24]

[0243] [ka]

[0244] The title compound was prepared using the same procedure outlined for the preparation of Example 12 utilizing 0.003 g (0.0036 mmol) of the product of Example 11, DMF (0.2 mL), 0.008 g (0.017 mmol) of 5-carboxyfluorescein-PFP ester (obtained from 5-carboxyfluorescein and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0006 g of a yellow film (title compound). MS (M+): C 57 H 55 N4O 13 Calculated for S2+: Exact mass: 1067.32; Molecular weight: 1068.20. UPLC / MS observed 1067.14.

[0245] [Example 25]

[0246] [ka]

[0247] Using the same procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.006 g (0.008 mmol) of the product of Example 2, DMF (0.2 mL), 0.008 g (0.013 mmol) of Rhodamine B-PFP ester (prepared from Rhodamine B and pentafluorophenyl trifluoroacetate) and DIEA (0.01 mL, 0.06 mmol). Yield 0.0031 g of a purple film (title compound). MS (M+): C 60 H 65 Calculated for N6O9S2+: Exact mass: 1077.42; Molecular weight: 1078.33. UPLC / MS observed 1077.51.

[0248] [Example 26]

[0249] [ka]

[0250] CP-acridine methyl ester (J. Org. Chem., 1998, vol. 63, pp. 5636-5639) (0.012 g, 0.025 mmol) and 5-(iodoacetamido)fluorescein (0.015 g, 0.029 mmol) were mixed in a 5 mL round bottom flask equipped with a nitrogen inlet. Without solvent, the flask was heated in an oil bath at 160-170° C. for 15 min. LCMS after this showed a complex mixture with both starting materials as well as the title compound present as components. The reactants were dissolved in DMF / MeOH / water (approximately 0.5 mL each) and purified by reversed phase HPLC using a YMC ODS AQ 30×150 mm steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% formic acid. Volatiles were removed on a rotary evaporator under vacuum at 30°C and dried under high vacuum (1 mmHg) for 24 hours. Yield 0.0007 g of a yellow film (title compound). MS (M+): C 48 H 38 N3O 11 Calculated for S+: Exact mass: 864.2222; Molecular weight: 864.8933. UPLC / MS observed 864.43.

[0251] [Example 27]

[0252] [ka]

[0253] Using the same procedure outlined for the preparation of Example 26, the title compound was prepared utilizing 0.012 g (0.025 mmol) of CP-acridinium methyl ester and 0.006 g (0.012 mmol) of 6-(iodoacetamido)fluorescein. Yield 0.0011 g of a yellow film (title compound). MS (M+): C 48 H38 N3O 11 Calculated for S+: Exact mass: 864.2222; Molecular weight: 864.8933. UPLC / MS observed 864.51.

[0254] [Example 28]

[0255] [ka]

[0256] The compound is

[0257] [ka] Prepared from.

[0258] SPCN (0.048 g), (Organic Letters, 2003, Vol. 5(No. 21), pp. 3779-379) was dissolved in 0.5 mL of DMF. 0.128 mL of DIEA was added, followed by PyAOP (0.032 g). The reaction was stirred at ambient temperature for 5 min (preactivation). 0.032 g of 5-acetamidoaminofluorescein (5-AAF) (Chemistry of Materials, 1992, Vol. 4(No. 4), pp. 879-84) was dissolved in 1 mL of DMF and 0.064 mL of DIEA. The 5-AAF solution was added to the SPCN solution. After 18 h, the reaction was treated with 3 mL of water. The solution was purified by HPLC by injecting it directly onto a YMC ODS-AQ column (40×100). Eluted with a gradient of 5-40% acetonitrile over 70 min at 45 mL / min (mobile phase ACN / H2O / H2O to 0.5% TFA). Fractions containing product were frozen and lyophilized. Yield 0.026 g (title compound). MS consistent with the title compound.

[0259] [Example 29]

[0260] [ka]

[0261] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.01 g of the product of Example 2, DMF (0.5 mL), 0.005 g (0.012 mmol) of BODIPY™ 493 / 503 NHS ester (ThermoFisher) and DIEA (0.01 mL, 0.06 mmol). The reaction was stirred overnight. Yield 0.0021 g of a red film (title compound). MS (M+): C 48 H 54 Calculated for BF2N6O8S2+; Exact mass: 955.3500; Molecular weight: 955.9203. UPLC / MS observed 955.38.

[0262] [Example 30]

[0263] [ka]

[0264] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.014 g (0.018 mmol) of the product of Example 2, DMF (0.5 mL), 0.005 g (0.011 mmol) of BDP558 / 568 NHS ester (Lumiprobe) and DIEA (0.01 mL, 0.06 mmol). The reaction was stirred overnight. Yield 0.0033 g of a purple film (title compound). MS (M+): C 48 H 48 Calculated for BF2N6O8S3+; Exact mass: 981.2751; Molecular weight: 981.9323. UPLC / MS observed 981.33.

[0265] [Example 31]

[0266] [ka]

[0267] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.03 g (0.039 mmol) of the product of Example 2, DMF (1 mL), 0.01 g (0.025 mmol) of BDP FL NHS ester (Lumiprobe) and DIEA (0.02 mL, 0.12 mmol). The reaction was stirred overnight. Yield 0.0026 g of a red film (title compound). MS (M+): C 46 H 50 Calculated for BF2N6O8S2+; Exact mass: 927.3187; Molecular weight: 927.8663. UPLC / MS observed 927.52.

[0268] [Example 32]

[0269] [ka]

[0270] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.03 g (0.039 mmol) of the product of Example 2, DMF (1 mL), 0.014 g (0.027 mmol) of BDP TR NHS ester (Lumiprobe) and DIEA (0.02 mL, 0.12 mmol). The reaction was stirred overnight. Yield 0.019 g of a blue film (title compound). MS (M+): C 53 H 50 Calculated for BF2N6O9S3+; Exact mass: 1059.2857; Molecular weight: 1060.0023. UPLC / MS observed 1059.26.

[0271] [Example 33]

[0272] [ka]

[0273] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.005 g (0.0069 mmol) of Alexa Fluor 532 carboxylic acid, 0.0029 g of HBTU (0.0076 mmol), DMSO (0.5 mL), and DIEA (0.05 mL, 0.3 mmol). The reaction was stirred at room temperature for 15 min, and then a DMSO solution (0.5 mL) containing the product of Example 2 (0.015 g, 0.020 mmol) was added. The reaction was stirred overnight. The crude reaction mixture was diluted with MeOH and water. All solutions were purified by reverse phase HPLC by eluting with a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield 0.0025 g of a red film. MS (M+): C 62 H 64 NO 15 Calculated for S4: Exact mass: 1260.3312; Molecular weight: 1261.4610. UPLC / MS observed 1262.42.

[0274] [Example 34]

[0275] [ka]

[0276] Using a similar procedure outlined for the preparation of Example 33, the title compound was prepared utilizing 0.012 g (0.016 mmol) of the product of Example 2, DMSO (1 mL), 0.005 g (0.0059 mmol) of Alexa Fluor 488 carboxylic acid, 0.0025 g (0.0066 mmol) of HBTU, and DIEA (0.05 mL, 0.3 mmol). Yield: 0.002 g of a red film (title compound 5(6)-mixed isomers). MS (M+): C53 H 48 NO 17 Calculated for S4; Exact mass: 1168.1959; Molecular weight: 1169.2320. UPLC / MS observed 1169.28.

[0277] [Example 35]

[0278] [ka]

[0279] Using a similar procedure outlined for the preparation of Example 33, the title compound was prepared utilizing 0.0085 g (0.011 mmol) of the product of Example 2, DMSO (1 mL), 0.005 g (0.005 mmol) of Alexa Fluor 568 carboxylic acid, 0.0021 g (0.0055 mmol) of HBTU, and DIEA (0.05 mL, 0.3 mmol). Yield 0.0025 g of a purple film (title compound 5(6)-mixed isomers). MS(M+):C 65 H 64 NO 17 Calculated for S4; Exact Mass: 1328.3211; Molecular Weight: 1329.4920. UPLC / MS Observed 1330.24.

[0280] [Example 36]

[0281] [ka]

[0282] A 20 mL reaction vial equipped with a magnetic stir bar was charged with 0.075 g (0.17 mmol) of methyl-4-carboxy-silyl rhodamine (Angew. Chemi. Int. Ed., 2018, vol. 57, pp. 2436-2440) and aqueous HCl (1 mL, 6 M). The contents were heated to 90° C. for 1 h. The mixture was cooled to room temperature and then diluted with a 4:1 CHCl3:methanol solvent mixture. The organic layer was washed with water and then with brine before drying over sodium sulfate. The solvent was removed under vacuum. The crude solid was dissolved in MeOH and water. The entire solution was purified by reverse phase HPLC by eluting with a YMC ODS AQ 50×250 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 70 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator under vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield 0.054 g of a blue film. MS (M+): C 26 H 29 Calculated for N2O2Si+; Exact Mass: 429.1993; Molecular Weight: 429.6145. UPLC / MS Observed 429.19.

[0283] [Example 37]

[0284] [ka]

[0285] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.025 g (0.033 mmol) of the product of Example 2, DMF (1 mL), 0.009 g (0.015 mmol) of 4-carboxy-SiR-PFP ester (Example 37 and pentafluorophenyl trifluoroacetate) and DIEA (0.1 mL, 0.6 mmol). Yield 0.004 g of a blue film (title compound). MS (M+): C 58 H64 N6O8S2Si 2+ Calculated for; Exact Mass: 1064.3985; Molecular Weight: 1065.3879. UPLC / MS Observed 1064.44 (weak); M++ 532.46 (strong).

[0286] [Example 38]

[0287] [ka]

[0288] A 20 mL reaction vial equipped with a magnetic stir bar was charged with 0.315 g (0.84 mmol) of 5-carboxyfluorescein and oleum (5 mL, 30% free SO3 based) and heated to 90° C. for 1 h. The reaction mixture was cooled to room temperature and then carefully added to a beaker containing ice, followed by the addition of KCl (1 g), resulting in a yellow precipitate. The solid was filtered, washed with cold water and acetone, and dried under high vacuum for 18 h. The solid was used in the next step without further purification. Yield 0.250 g of a yellow solid. MS(M-):C 21 H 11 O 13 Calculated for S2-; Exact mass: 534.9647; Molecular weight: 535.4265. UPLC / MS observed value 534.93.

[0289] [Example 39]

[0290] [ka]

[0291] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.009 g (0.012 mmol) of the product of Example 2, DMF (0.5 mL), 0.009 g (0.015 mmol) of 5-carboxy-4',5'-disulfofluorescein-PFP ester (Example 38 and pentafluorophenyl trifluoroacetate) and DIEA (0.05 mL, 0.3 mmol). Yield 0.007 g of a yellow film. MS(M-):C 53 H 45 N4O 19 S4 - Calculated for; Exact Mass: 1169.1566; Molecular Weight: 1170.1925. UPLC / MS Observed 1169.99.

[0292] [Example 40]

[0293] [ka]

[0294] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.013 g (0.04 mmol) of fluorescein, 0.014 g of HBTU (0.037 mmol), DMSO (1 mL), and DIEA (0.1 mL, 0.6 mmol). The reaction was stirred at 45° C. for 60 min. The solution was then cooled to room temperature before adding a DMSO solution (0.5 mL) containing the product of Example 2 (0.04 g, 0.052 mmol). The reaction was stirred overnight. The crude reaction mixture was diluted with MeOH and water. All solutions were purified by reversed-phase HPLC by eluting with a YMC ODS AQ 50×250 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 70 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield 0.002 g of a red film. MS (M+): C52 H 47 N4O 11 Calculated for S2+; Exact Mass: 967.2677; Molecular Weight: 968.0845. UPLC / MS Observed 967.32 (weak); M++ 484.38 (strong).

[0295] [Example 41]

[0296] [ka]

[0297] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.015 g (0.020 mmol) of the product of Example 2, DMF (0.5 mL), 0.008 g (0.016 mmol) of Rhodamine 19-NHS ester (Rhodamine 19 and TSTU) and DIEA (0.05 mL, 0.3 mmol). Yield: 0.002 g of a red film. MS (M+): C 58 H 62 N6O9S2 2+ Calculated for; Exact Mass: 1050.4009; Molecular Weight: 1051.2859. UPLC / MS Observed 1049.31 (weak); M++ 525.46 (strong).

[0298] [Example 42]

[0299] [ka]

[0300] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.0065 g (0.0085 mmol) of the product of Example 2, DMF (0.4 mL), 0.002 g (0.003 mmol) of Atto 700 NHS-ester, and DIEA (0.05 mL, 0.3 mmol). Yield: 0.003 g of a green film. MS (M+): C 62 H 70 N7O 12Calculated for S3+; Exact Mass: 1200.4239; Molecular Weight: 1201.4585. UPLC / MS Observed 1200.56 (weak); M++ 600.92 (strong).

[0301] [Example 43]

[0302] [ka]

[0303] A 20 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.2 g (0.30 mmol) of IR780 iodide, DMF (2 mL), and a solution of methylamine in THF (3 mL, 2 M). It was heated to 80° C. for 1 h, during which the color of the solution changed from green to blue. The reaction mixture was cooled to room temperature and the product was triturated in diethyl ether. The product was used in the next step without further purification. Yield: 0.160 g of blue powder. MS (M+): calculated for formula: C 37 H 48 N3+; Exact mass: 534.3843; Molecular weight: 534.8115. UPLC / MS observed 534.37.

[0304] [Example 44]

[0305] [ka]

[0306] A 20 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.025 g (0.038 mmol) of the product of Example 43, DCM (10 mL), and 0.033 g (0.114 mmol) of triphosgene. The reaction mixture was cooled to 0° C. in an ice bath before adding 0.3 mL of DIEA. Stirring was continued for 1 h before removing the solvent under vacuum. The crude material was then charged with 0.040 g (0.052 mmol) of the product of Example 2, DMF (1 mL), and DIEA (0.1 mL, 0.6 mmol). The reaction mixture was stirred at room temperature for 36 h. The crude reaction mixture was diluted with MeOH and water. The entire solution was purified by reversed phase HPLC by eluting with a YMC ODS AQ 50×250 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient (flow rate 70 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield 0.004 g of a green film. MS (M+): C 70 H 83 N7O8S2 2+ Calculated for; Exact Mass: 1213.5734; Molecular Weight: 1214.5939. UPLC / MS Observed 1212.50 (weak); M++ 607.05 (strong).

[0307] [Example 45]

[0308] [ka]

[0309] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.0165 g (0.021 mmol) of the product of Example 2, DMF (1 mL), 0.008 g (0.015 mmol) of Lucifer Yellow VS dilithium salt, and DIEA (0.05 mL, 0.3 mmol). Yield: 0.009 g of yellow powder. MS(M-): C52 H 49 NO 17 S5 - Calculated for; Exact Mass: 1189.1763; Molecular Weight: 1190.2895. UPLC / MS Observed 1189.42.

[0310] [Example 46]

[0311] [ka]

[0312] A 4 mL reaction vial equipped with a magnetic stir bar was charged with 0.110 g (0.30 mmol) of Lucifer Yellow anhydrous, 0.123 g (1.65 mmol) of glycine, and an aqueous solution of sodium acetate (3 mL, 1 M). The mixture was heated to 90° C. and stirred overnight. The crude reaction mixture was diluted with MeOH and water. The entire solution was purified by reversed-phase HPLC by eluting on a YMC ODS AQ 50×250 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 70 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield: 0.120 g of yellow powder. MS(M-): C 14 H9N2O 10 Calculated for S2; Exact mass: 428.9704; Molecular weight: 429.3505. UPLC / MS observed value 429.05.

[0313] [Example 47]

[0314] [ka]

[0315] Using a similar procedure outlined for the preparation of Example 12, the title compound was prepared utilizing 0.085 g (0.11 mmol) of the product of Example 2, DMF (1 mL), 0.040 g (0.076 mmol) of the product of Example 46-NHS ester (Example 46 and TSTU) and DIEA (0.17 mL, 1 mmol). Yield: 0.018 g yellow powder. MS(M-): C 46 H 43 NO 16 Calculated for S4-, exact mass: 1063.1624, molecular weight: 1064.1165. UPLC / MS observed value: 1063.24.

[0316] [Example 48]

[0317] [ka]

[0318] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.013 g (0.012 mmol) of the product of Example 47, 0.0055 mg (0.018 mmol) of TSTU, DMSO (0.5 mL), and DIEA (0.05 mL, 0.3 mmol). The mix was stirred at room temperature for 1 h and then diluted in a small amount of ACN. The entire solution was purified by reversed-phase HPLC by eluting on a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.05% formic acid. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield: 0.008 mg of a yellow film. MS(-): C 50 H 46 N7O 18 Calculated for S4-; Exact mass: 1160.1788; Molecular weight: 1161.1895. UPLC / MS observed 1160.28.

[0319] [Example 49]

[0320] [ka]

[0321] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.006 g (0.0052 mmol) of the product of Example 48, 0.020 g (0.062 mmol) of amino-dPEG® 4-t-butyl ester, DMF (0.5 mL), and DIEA (0.1 mL, 0.6 mmol). The mixture was stirred for 1 h and then diluted in a small amount of ACN. The entire solution was purified by reversed-phase HPLC by eluting on a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% formic acid. The product-containing fractions were combined and the volatile components were removed on a rotary evaporator in vacuo at 30° C. followed by high vacuum at room temperature for 18 h. The purified material was transferred to a 4 mL reaction vial equipped with a stir bar and dissolved in 1 mL of DCM and 1 mL of TFA. The mixture was stirred for 1 h and then the solvent was removed on a rotary evaporator in vacuo at 30° C. followed by high vacuum at room temperature for 18 h. No further purification was required. Yield: 0.0088 g yellow film. MS(-): C 57 H 64 N7O 21 Calculated for S4-; Exact mass: 1310.3044; Molecular weight: 1311.4075. UPLC / MS observed 1310.82.

[0322] [Example 50]

[0323] [ka]

[0324] Using a similar procedure outlined for the preparation of Example 48, the title compound was prepared utilizing 0.0088 g (0.0067 mmol) of the product of Example 49, 0.003 g (0.010 mmol) of TSTU, DMF (0.5 mL), and DIEA (0.05 mL, 0.3 mmol). After purification and evaporation, 10% of the material was hydrolyzed back to the carboxylic acid form. Yield: 0.006 g. MS(-): C 61 H 67 N8O 23 Calculated for S4-; Exact mass: 1407.3207; Molecular weight: 1408.4805. UPLC / MS observed 1408.50.

[0325] [Example 51]

[0326] [ka]

[0327] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.007 g (0.0072 mmol) of the product of Example 29, 0.0026 g (0.017 mmol) of EDC, 0.0036 g (0.017 mmol) of N-hydroxysulfosuccinimide sodium salt, DMF (0.5 mL), and DIEA (0.01 mL, 0.06 mmol). The reaction was stirred overnight and then diluted in a small amount of ACN. All solutions were purified by reversed-phase HPLC by eluting on a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.05% formic acid. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield: 0.0025 g. MS(+): C 52 H 56 BF2N7O 13Calculated for S3; Exact mass: 1131.3159; Molecular weight: 1132.0428. UPLC / MS observed (MF) +1112.20.

[0328] [Example 52]

[0329] [ka]

[0330] Using a similar procedure outlined for the preparation of Example 51, the title compound was prepared utilizing 0.009 g (0.0085 mmol) of the product of Example 32, 0.0026 g (0.017 mmol) of EDC, 0.0036 g (0.017 mmol) of N-hydroxysulfosuccinimide sodium salt, DMF (0.5 mL), and DIEA (0.01 mL, 0.06 mmol). Yield: 0.0013 g. MS(+): C 57 H 52 BF2N7O 14 Calculated for S4; Exact mass: 1235.2516; Molecular weight: 1236.1248. UPLC / MS observed (MF) +1216.40.

[0331] [Example 53]

[0332] [ka]

[0333] A 100 mL RB flask equipped with a stir bar and nitrogen inlet was charged with propargyl triflate (J.Org Chem., 1977, vol. 42, pp. 3109-3113) (20.98 mmol) and CHCl (25 mL). To this solution was added 2,6-di-tert-butylpyridine (6.96 mL, 31.45 mmol), followed by acridine (J.Org Chem., 1998, vol. 63, pp. 5636-5639) (1.00 g, 2.10 mmol) and stirred for 18 h. The mixture was concentrated in vacuo. The residue was purified by reverse phase HPLC using a gradient method of 10%-90% acetonitrile / H0 containing 0.5% TFA. The desired fractions were collected, pooled, frozen, and lyophilized to yield 1.213 g of the title compound as a yellow solid (quantitative yield). Yield: 1.213 g of a yellow solid. MS(+): C 29 H 27 N2O5S + Calculated for; Exact mass: 515.6; Molecular weight: 515.6. UPLC / MS observed (M)+514.85.

[0334] [Example 54]

[0335] [ka]

[0336] A 50 mL Rb flask equipped with a stir bar and nitrogen inlet was charged with the product of Example 53 (0.014 g, 0.027 mmol), 5-azidofluorescein (J. Am. Chem. Soc. 2012, 134, 17428-17431) (0.010 g, 0.027 mmol) and a solution of DMF:HO (2 mL, 1:1). To this mixture was added a solution of copper(II) sulfate (0.001 g, 0.001 mmol) in HO (100 μL), followed by a solution of sodium ascorbate (0.001 g, 0.005 mmol) in HO (100 μL) and stirred for 18 h. The mixture was purified by reverse phase HPLC using a gradient of 10%-90% acetonitrile / HO containing 0.5% TFA. The desired fractions were collected, frozen and lyophilized to give 14 mg of the title compound (58%). Yield: 0.014 g. MS(+): C 49 H 39 N5O 11 S + Calculated for; Exact mass: 888.23; Molecular weight: 888.92. UPLC / MS observed (M)+888.46.

[0337] [Example 55]

[0338] [ka]

[0339] A 25 mL RB flask equipped with a stir bar and nitrogen inlet was charged with CPSP (0.020 g, 0.034 mmol), HBTU (0.014 g, 0.037 mmol), HOBt (0.005 g, 0.037 mmol) and DMF (2 mL). To this mixture was added DIEA (0.030 mL, 0.171 mmol) and stirred for 30 min. To this mixture was added 4'-aminomethylfluorescein (U.S. Pat. No. 4,510,251, 1985) (0.034 g, 0.094 mmol) and stirred for 18 h. The mixture was concentrated in vacuo. The residue was purified by reverse phase HPLC using a gradient method of 10%-90% acetonitrile / H2O with 0.5% TFA. The desired fractions were collected, frozen and lyophilized to give 0.010 g of the title compound as a yellow-orange solid (32%). Yield: 0.010 g of a yellow-orange solid. MS(+): C 49 H 41 N3O 12 S2 + Calculated for; Exact mass: 927.21; Molecular weight: 928.00. UPLC / MS observed (M)+928.50.

[0340] [Example 56]

[0341] [ka]

[0342] A 25 mL RB flask equipped with a stir bar and nitrogen inlet was charged with CPSP (0.050 g, 0.086 mmol), HBTU (0.036 g, 0.094 mmol), and HOBt (0.013 g, 0.094 mmol) and DMF (2 mL). To this mixture was added DIEA (0.074 mL, 0.428 mmol) and the reaction was stirred for 30 min. To this mixture was added 5-aminomethylfluorescein (Bioconjugate Chem., 1992, vol. 3, pp. 430-431) (0.034 g, 0.094 mmol) and stirred for 18 h. The mixture was concentrated in vacuo. The residue was purified by reverse phase HPLC using a gradient of 10%-90% acetonitrile / H2O containing 0.5% TFA. The desired fractions were collected and lyophilized to give 0.027 g of the title compound as a yellow-orange solid (34%). Yield: 0.027 g of a yellow-orange solid. MS(+): C 49 H 41 N3O 12 S2 + Calculated for; Exact mass: 927.21; Molecular weight: 928.00. UPLC / MS observed (M+H)+929.45.

[0343] [Example 57]

[0344] [ka]

[0345] In a 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet, 0.007 g (0.0072 mmol) of the product of Example 2, 0.003 g (0.003 mmol) of DTBTA-Eu 3+(Inorg. Chem., 2006, vol. 4, pp. 4088-4096), DMF (0.5 mL), and DIEA (0.01 mL, 0.06 mmol) were charged. The reaction was stirred overnight and then diluted in a small amount of ACN / H2O. The entire solution was purified by reversed-phase HPLC by eluting on a YMC ODS AQ 30 x 150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed is 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.05 formic acid. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30°C followed by high vacuum at room temperature for 18 h. Yield 0.002 g pale yellow powder. MS (M+): C 72 H 65 ClEuN 13 O 15 S2 4+ Calculated for; Exact Mass: 1603.3043; Molecular Weight: 1603.9198. UPLC / MS Observed 1604.65.

[0346] [Example 58]

[0347] [ka]

[0348] Using a similar procedure outlined for the preparation of Example 49, the title compound was prepared utilizing 0.011 g (0.0095 mmol) of the product of Example 48, 0.045 g (0.090 mmol) of amino-dPEG® 8-t-butyl ester, DMF (0.5 mL), and DIEA (0.1 mL, 0.6 mmol). Yield: 0.006 g of a yellow film. MS(-): C 65 H 81 N7O 25 Calculated for S4-; Exact mass: 1487.4165; Molecular weight: 1488.6270. UPLC / MS observed 1487.71.

[0349] [Example 59]

[0350] [ka]

[0351] Using a similar procedure outlined for the preparation of Example 48, the title compound was prepared utilizing 0.006 g (0.0067 mmol) of the product of Example 58, 0.002 g (0.0067 mmol) of TSTU, DMF (0.5 mL), and DIEA (0.03 mL, 0.17 mmol). Yield: 0.004 g MS(-): C 69 H 84 N8O 27 Calculated for S4-; Exact mass: 1584.4329; Molecular weight: 1585.7000. UPLC / MS observed 1584.75.

[0352] [Example 60]

[0353] [ka]

[0354] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.006 g (0.0052 mmol) of the product of Example 48, 0.025 g (0.25 mmol) of 3-azido-1-propanamine, DMF (0.5 mL), and DIEA (0.1 mL, 0.6 mmol). The mixture was stirred for 1 h and then diluted in a small amount of ACN. The entire solution was purified by reversed-phase HPLC by eluting on a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% formic acid. The product-containing fractions were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield: 0.003 g of a yellow film. MS(-): C 49H 50 N 10 O 15 S4 - Calculated for; Exact mass: 1145.2267; Molecular weight: 1146.2265. UPLC / MS observed 1145.63.

[0355] [Example 61]

[0356] [ka]

[0357] Using a similar procedure outlined for the preparation of Example 60, the title compound was prepared utilizing 0.006 g (0.0052 mmol) of the product of Example 48, 0.030 g (0.076 mmol) of azido-dPEG® 7-amine, DMF (0.5 mL), and DIEA (0.1 mL, 0.6 mmol). Yield: 0.004 g of a yellow film. MS(-): C 62 H 76 N 10 O 22 Calculated for S4; Exact mass: 1440.4018; Molecular weight: 1441.5780. UPLC / MS observed 1440.82.

[0358] [Example 62]

[0359] [ka]

[0360] Using a similar procedure outlined for the preparation of Example 60, the title compound was prepared utilizing 0.0075 g (0.0065 mmol) of the product of Example 48, 0.020 g (0.076 mmol) of MPS-EDA (Quanta Biodesign), DMF (0.5 mL), and DIEA (0.1 mL, 0.6 mmol). Yield: 0.002 g of a yellow film. MS(-): C 55 H 54 N9O 18 S4 -Calculated for; Exact mass: 1256.2475; Molecular weight: 1257.3225. UPLC / MS observed value: 1256.53

[0361] [Example 63]

[0362] [ka]

[0363] The title compound was prepared using a similar procedure outlined for the preparation of Example 60 utilizing 0.006 g (0.0052 mmol) of the product of Example 48, 0.005 g (0.0067 mmol) of 2-(6-aminohexanamido)-thyroxine (Bioconjugate Chem., 1997, Vol. 8, pp. 133-145), DMF (0.5 mL), and DIEA (0.01 mL, 0.06 mmol). Yield: 0.005 g of a yellow film. MS(-): C 67 H 65 I3N8O 20 S4 - Calculated for; Exact mass: 1810.0332; Molecular weight: 1811.2464. UPLC / MS observed value: 1810.59 (weak); M 2- 904.99 (strong).

[0364] [Example 64]

[0365] [ka]

[0366] A 4 mL reaction vial equipped with a magnetic stir bar and nitrogen inlet was charged with 0.004 g (0.0025 mmol) of the product of Example 59, 0.0082 g (0.013 mmol) of thyroxine, DMF (0.5 mL), and DIEA (0.01 mL, 0.06 mmol). The mixture was stirred for 1 h and then diluted in a small amount of ACN. The entire solution was purified by reversed-phase HPLC by eluting on a YMC ODS AQ 30×150 mm I.D. steel column with a Waters Separations 2000 system monitored at 254 nm. The recorder chart speed was 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used with the mobile phase ACN / H2O / H2O to 0.5% formic acid. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30° C., followed by high vacuum at room temperature for 18 h. Yield: 0.002 g of a yellow film. MS(-): C 80 H 90 I3N8O 28 S4 - Calculated for; Exact mass: 2119.1887; Molecular weight: 2120.5820. UPLC / MS observed M 2- 1059.82

[0367] [Example 65]

[0368] [ka]

[0369] The title compound was prepared by treating a solution of Example 50 (0.0018 g, 0.0013 mmol) in DMF (0.25 mL) with Biotin-dPEG7-NH2 (Quanta BioDesign catalog #10826, 0.030 g) in DMF (1 mL). The reaction was stirred at room temperature for 1 h. The resulting solution was purified by reverse phase HPLC using a YMC ODS AQ 30 x 150 mm I.D. steel column with a Waters Separations2000 system monitored at 254 nm. The recorder chart speed is 5 mm / min. A manual step gradient method (flow rate 40 mL / min) was used from ACN / H2O / H2O to 0.5 TFA. The fractions containing the product were combined and the volatile components were removed on a rotary evaporator in vacuum at 30°C followed by high vacuum at room temperature for 18 h. Yield 0.0024 g of a yellow film. MS(-):C 83 H 112 N 11 O 29 S5 - Calculated for; Exact mass: 1886.6236; Molecular weight: 1888.16. UPLC / MS observed 1887.59.

[0370] [Example 66] Chemiluminescence Data Protocol for the measurement of the full chemiluminescence spectrum in the visible wavelength range. Equipment: Andor Shamrock 303i imaging spectrometer, ruling grating at 50 lines / mm, 600 nm blaze, MgF2 coated aluminum, 100 μm entrance slit. Andor iXon EM +512x512 CCD camera, model DU-897E-CSO-#BV, 550nm AR coated back-illuminated sensor. The CCD detector chip is 16μm 2E2V Tech CCD97 with electron multiplying readout with 100% chromaticity. Thermoelectric cooling was to -70°C. For maximum sensitivity, pixel (column) binning along a vertical line (image of a slit) was selected, covering most of the chip's range. Using the spectrometer's software, the detection wavelength was calibrated with several mercury lines of an Ar-Hg pen lamp, and the spectral dispersion obtained at the detector was approximately 1 nm / pixel. Integration was 5 seconds, which is usually about 5 decay lifetimes of chemiluminescence. Software: Andor Solis for Spectroscopy: X3964, version 4.3. Reagents: Architect pre-trigger solution, 6E23-65 with detergent, acid, and hydrogen peroxide; Architect trigger solution, 6C55-60 with detergent and base. Method: A Hi-Tech Rapid Kinetics Accessory, model SFA-11, was used to mix the solutions in the chamber for less than 20 ms according to the user manual. Software data collection was triggered by a hotkey and two 2.5 mL syringes were manually pressed to achieve a 50:50 mix in the cuvette. The delay from the start of integration to mixing was expected to be less than 0.5 seconds. The cuvette was oriented to provide a path length of 2 mm. Samples were typically tested at 500 nanomolar concentrations as determined by UV absorbance at the appropriate wavelength for each fluorophore.

[0371] Protocol for luminometer plate reader measurements of chemiluminescence at multiple wavelengths. Equipment: Berthold Mithras LB940 microplate reader; optical filters, Semrock Brightline single-band bandpass, multilayer dielectric, 442 / 46 nm, 531 / 46 nm; white 96-well plate, Microfluor I, Thermo 6905. Software: Mikrowin 2000 v. 4.41. Reagents: Architect pre-trigger solution, 6E23-65 with detergent, acid, and hydrogen peroxide; Architect trigger solution, 6C55-60 with detergent and base. 50 μL of test compound in Architect pre-trigger solution was placed in the wells of a 96-well plate, loading a separate well for each wavelength measurement. Methods: Samples were typically tested at 20-200 pM concentrations as determined by absorbance at the appropriate wavelength for each fluorophore. In the luminometer, the appropriate wavelength optical filter was selected for readout. 75 μL of Architect trigger solution was injected into each well immediately prior to detection. Light counts were measured by a photomultiplier tube over 10 seconds at 0.1 second intervals. Readings were measured in triplicate. The results of the assay are presented in Table 1.

[0372] [Table 1] TIFF2025076421000082.tif168166

[0373] [Example 67] Fluorophore attachment points and linker lengths were tested using acetamide linkers and isolated 5- and 6-carboxy isomers of fluorescein. The data shown in Figure 1 demonstrate that the shifted emission is determined by the fluorophore attachment point, which can lead to differences in the overall directionality of the two species or aggregation of species, altering the ability to shift emission in short linker configurations.

[0374] The 5 and 6 carboxy isomers of fluorescein were further tested using the piperazine linker. The data are shown in FIG. 2. Shifted emission was observed with near 100% efficiency, but differences in intensity were observed between the 5 and 6 isomeric moieties. The differences in intensity could be due to hindrance of the chemical reaction leading to chemiluminescence, unfavorable orientation possibly leading to quenching or non-radiative decay pathways, or aggregation of the compounds leading to changes in the absorbance / emission profile. These results indicate that the choice of fluorophore attachment point is a critical factor for shifted emission.

[0375] Fluorophore attachment points and linker lengths were also tested for emission efficiency using both the 5 / 6 carboxyrhodamine dye mix and the 2 carboxyrhodamine dye. The data are shown in Figure 3. The 5 / 6 carboxyrhodamines showed efficient shifted emission, while the 2 carboxyrhodamines showed efficient shifted emission in most situations, although there were some differences depending on the type of linker. For example, 2-carboxyrhodamine B showed efficient stable, shifted emission when attached to acridinium via a dimethyl-PEG(2)-diamine linker, but the same 2-carboxyrhodamine B showed an increase in acridone emission levels within the measurement interval when attached to acridinium via a piperazine linkage. These findings suggest that the construct may not be stable under the triggering conditions utilized. In contrast, 2 carboxyrhodamine 6G appeared to produce stable shifted emission when attached to acridinium via a piperazine linkage, although only 90% shifted emission and 10% blue light were observed.

[0376] The initiator attachment point was tested by varying the location of the fluorophore between the sulfopropyl and carboxypropyl moieties of carboxypropylsulfopropylacridinium. Attachment to the carboxypropyl group positions the fluorophore on the leaving group of the acridinium / acridone molecule. Thus, upon triggering, the fluorophore dissociates from the resulting acridone moiety. Two fluorescein compounds were attached to the acridinium via either the xanthene ring attachment point or the phenyl ring attachment point to test two different molecular orientations. Emission was measured with a luminometer equipped with 442 nm and 531 nm filters. The data are shown in Figure 4. The fluorescein compounds prepared with carboxypropyl initiator attachment failed to show shifted emission and produced similar wavelength light as the acridinium control. Modification of the carboxypropyl with the preferred piperazine linkage was also tried, resulting in emission similar to the acridinium control. FIG. 4 shows that for the selection of carboxypropyl compounds prepared, the light output and distribution in each filter channel matched that of the acridinium control compound.

[0377] Diamine linkers of various lengths and rigidities were used to test linker type and linker length. Rigid linkers can hold the initiator and acceptor in a preferred orientation for shifted emission, while longer linkers can have the flexibility to bend and wrap around into a preferred orientation. The data are shown in FIG. 5. Shifted emission was observed with near 100% efficiency for each of the compounds. However, differences in intensity were observed for the ethylenediamine linkers. The differences in intensity could be due to hindrance of the chemical reaction leading to chemiluminescence, or unfavourable orientation possibly leading to quenching or non-radiative decay pathways. These data illustrate that linker selection can be an important factor for shifted emission.

[0378] This example demonstrates that several structural elements are important in developing chemiluminescent acridinium compounds with shifted wavelength emission. The stability of the fluorophore to triggering conditions is of significant importance. For example, the linking of cyanine and silicon rhodamine dyes to acridinium leads to a short shifted emission followed by acridone emission, indicating possible instability of the structure in triggering matrices. Water solubility is another element required to function in aqueous-based applications such as immunoassays. Overall, the selection of linker length, fluorophore attachment point, and initiator attachment promotes shifted emission. Without wishing to be limited by theory, these three criteria appear to determine the orientation of the fluorophore and initiator relative to each other and therefore the efficiency of the shifted emission.

[0379] [Example 68] HIV p24mAb-acridinium-lucifer yellow conjugate. A stock solution of the compound of Example 48 was prepared by reconstituting the dry powder in dimethylsulfoxide (DMSO). Two 100x dilutions of the stock solution were prepared using pH 5.5 MES buffer. Concentrations were determined by reading absorbance at 370 nm using a Cary60 UV-Vis spectrophotometer.

[0380] Approximately 0.3 mg of HIV p24 mAb was added to 35 μL of 10 mM phosphate buffered saline (PBS) and the pH was adjusted using 5 μL of spiking buffer (250 mM PBS, pH 8 containing 7.5% CHAPS) to achieve a final reaction pH of 7.5 and a final CHAPS concentration of 0.5% in a separate reaction vessel. The vessel was protected from light, and a stock solution of the compound of Example 48 was added to the reaction vessel to achieve a molar input ratio of 6, 9, or 12 relative to the moles of mAb. The reaction vessel was vortexed briefly and then incubated statically overnight for approximately 20 hours while protected from light. After this, the reaction vessel was centrifuged to separate insoluble aggregates, and the protein remaining in the supernatant was purified by HPLC on a TSKGel G3000SWxl column with a mobile phase of 10 mM PBS pH 6.3. A flow rate of 1 mL / min was used and the eluate was monitored at 280 nm, 370 nm, and 431 nm with a photodiode array detector. Label concentrations of protein and Example 48 were determined by UV-Vis (280 and 370 nm, respectively). Label to protein incorporation ratios (IR) were determined by dividing the molar concentration of Example 48 to the molar concentration of HIV mAb. Final IR values ​​of 2.0, 2.5, and 3.0 were achieved for molar input ratios of 1:6, 1:9, and 1:12, respectively. Protein conjugates were stored at 2-8°C protected from light until use.

[0381] Label to protein incorporation ratios were determined by dividing the corrected A280 concentration (A280 absorbance minus the A280 contribution of acridinium) by the A370 absorbance of acridinium. Protein conjugates were stored at 2-8 °C until use.

[0382] [Example 69] Anti-human IgM mAb-acridinium-lucifer yellow conjugate. A stock solution of the compound of Example 48 was prepared by reconstituting the dry powder in DMSO. Two 100x dilutions of the stock solution were prepared using pH 5.5 MES buffer. Concentrations were determined by reading absorbance at 370 nm using a Cary60 UV-Vis spectrophotometer.

[0383] Approximately 0.3 mg of anti-human IgM mAb was added to 35 μL of 10 mM phosphate buffered saline (PBS) and the pH was adjusted using 5 μL of spiking buffer (250 mM PBS, pH 8, containing 7.5% CHAPS) to achieve a final reaction pH of 7.5 and a final CHAPS concentration of 0.5%. The vessel was protected from light and a stock solution of the compound of Example 48 was added to achieve a molar input ratio of 8.5 to moles of mAb. The reaction vessel was vortexed briefly and then incubated statically for 5 hours while protected from light. After this, the reaction vessel was centrifuged to separate insoluble aggregates and the protein remaining in the supernatant was purified by HPLC on a TSKGel G3000SWxl column with a mobile phase of 10 mM PBS, pH 6.3. A flow rate of 1 mL / min was used and the eluent was monitored at 280 nm, 370 nm, and 431 nm with a photodiode array detector. Labeling concentrations of protein and Example 48 were determined by UV-Vis (280 and 370 nm, respectively). Label to protein incorporation ratios (IR) were determined by dividing the molar concentration of Example 48 to the molar concentration of HIV mAb. A final IR value of 2.6 was achieved for a 1:8.5 molar input ratio. Protein conjugates were stored at 2-8°C protected from light until use.

[0384] [Example 70] Anti-human IgG mAb-Acridinium-Fluorescein Conjugate. A stock solution of the active ester compound of Example 12 was prepared by reconstituting the dry powder in DMSO to 5 mg / mL dry weight.

[0385] In separate reaction vessels, approximately 2 mg of anti-human IgG antibody was added to approximately 890 μL of 10 mM phosphate buffered saline pH 8.0. The vessels were protected from light, and the active ester of Example 12 solution was added to each reaction vessel to achieve a molar input ratio of 3, 5, or 7 relative to the moles of mAb. The reaction vessels were briefly vortexed and then incubated statically overnight for approximately 16 hours while protected from light. After this, the reaction vessels were centrifuged to separate insoluble aggregates, and the protein remaining in the supernatant was desalted using a PD10 G25 desalting column with a mobile phase of 10 mM PBS pH 6.3. Triggerable counts were measured with a Mithras LB 940 luminometer by adding 70 ng / mL conjugate to the Architect pre-trigger and trigger. The protein conjugates were stored at 2-8° C. while protected from light until use.

[0386] [Example 71] HIV p24 mAb-Acridinium-Fluorescein Conjugate. A 10 mg / mL stock solution of DBCO-PEG-NHS (Click Chemistry Tools A134) was prepared by reconstituting the dry powder in dimethyl sulfoxide (DMSO). HIV p24 mAb was desalted using a zeba spin column and the antibody concentration was determined by UV-Vis absorbance at 280 nm. The reaction vessel was protected from light and DBCO solution was added to achieve a molar input ratio of 8 to moles of mAb. The reaction vessel was vortexed briefly and then incubated statically overnight (approximately 20 hours). The resulting solution was purified by HPLC. The DBCO antibody concentration was again determined by UV-Vis absorbance at 280 nm. A stock solution of the azide compound of Example 12 was prepared at 3.2 μM dry weight in DMSO. The DBCO antibody was reacted with the Example 12 azide by incubating with 50 μL of DBCO antibody solution with 50 μL of Example 12 azide solution in a reaction vessel at room temperature overnight (20 hours) protected from light. The label to protein incorporation ratio (IR) was determined by dividing the molar concentration of Example 12 to the molar concentration of HIV mAb. A final IR value of approximately 2.0 was achieved. The protein conjugate was stored at 2-8° C. protected from light until use.

[0387] [Example 72] HIV p24 mAb-acridinium-BODIPY493 conjugate. A stock solution of the compound of Example 51 was prepared by reconstituting the dry powder in DMSO. Two 100x dilutions of the stock solution were prepared using pH 5.0 MES buffer. Concentrations were determined by reading absorbance at 370 nm using a Cary60 UV-Vis spectrophotometer.

[0388] Approximately 0.3 mg of HIV p24 mAb was added to approximately 40 μL of 10 mM phosphate buffered saline (PBS) in separate reaction vessels. The vessels were protected from light, and a stock solution of the compound of Example 51 was added to each reaction vessel to achieve a molar input ratio of 5, 10, or 15 relative to the moles of mAb. The reaction vessels were vortexed briefly and then incubated statically overnight for approximately 16 hours while protected from light. After this, the reaction vessels were centrifuged to separate insoluble aggregates, and the protein remaining in the supernatant was purified by HPLC on a TSKGel G3000SWxl column with a mobile phase of 10 mM PBS pH 6.3. Using a flow rate of 1 mL / min, the eluate was monitored at 280 nm, 370 nm, and 431 nm with a photodiode array detector. Protein and Example 51 label concentrations were determined by UV-Vis (280 and 370 nm, respectively). Label to protein incorporation ratios (IR) were determined by dividing the molar concentration of Example 51 relative to the molar concentration of HIV mAb. Soluble conjugate aggregates produced IR values ​​of 8.8, 7.9, and 8.4 for 1:5, 1:10, and 1:15 molar input ratios, respectively, representing saturation points for the IR with the input ratios investigated. Protein conjugates were stored at 2-8°C protected from light until use.

[0389] [Example 73] HIV p24 mAb-acridinium-BODIPY Texas Red (TR) conjugate. A stock solution of the compound of Example 52 was prepared by reconstituting the dry powder in DMSO. Two 100x dilutions of the stock solution were prepared using pH 5.5 MES buffer. Concentrations were determined by reading absorbance at 370 nm using a Cary 60 UV-Vis spectrophotometer.

[0390] Approximately 0.3 mg of HIV p24 mAb was added to approximately 7.5 μL of 10 mM phosphate buffer in separate reaction vessels. The vessels were protected from light, and a stock solution of the compound of Example 52 was added to each reaction vessel to achieve a molar input ratio of either 1:10. DMSO was added, increasing the volume to 30% of the reaction volume, to aid in solubilization of the compound of Example 52. The final reaction volume was 25 μL. The reaction vessels were briefly vortexed and then incubated statically overnight for approximately 16 hours, protected from light. After this, the reaction vessels were centrifuged to separate insoluble aggregates, and the protein remaining in the supernatant was purified by HPLC on a TSKGel G3000SWxl column with a mobile phase of 10 mM PBS pH 6.3. Using a flow rate of 1 mL / min, the eluent was monitored at 280 nm, 370 nm, and 431 nm with a photodiode array detector. Soluble aggregates were observed and isolated for further studies. Protein conjugates were stored at 2-8 °C protected from light until use.

[0391] [Example 74] HIV p24 mAb-PEG-acridinium-lucifer yellow conjugate. A stock solution of the compound of Example 50 was prepared by reconstituting the dry powder in DMSO. Two 100x dilutions of the stock solution were prepared using pH 5.5 MES buffer. Concentrations were determined by reading absorbance at 370 nm using a Cary60 UV-Vis spectrophotometer.

[0392] Approximately 0.3 mg of HIV p24 mAb was added to 35 μL of 10 mM phosphate buffered saline (PBS) and the pH was adjusted using 5 μL of spiking buffer (250 mM PBS, pH 8 containing 7.5% CHAPS) to achieve a final reaction pH of 7.5 and a final CHAPS concentration of 0.5%. The vessel was protected from light and the compound from the stock solution of Example 50 was added to the reaction vessel to achieve a molar input ratio of 20 relative to moles of mAb. The reaction vessel was vortexed briefly and then incubated statically overnight for approximately 16 hours while protected from light. After this, the reaction vessel was centrifuged to separate insoluble aggregates and the protein remaining in the supernatant was purified by HPLC on a TSKGel G3000SWxl column with a mobile phase of 10 mM PBS pH 6.3. A flow rate of 1 mL / min was used and the eluent was monitored at 280 nm, 370 nm, and 431 nm with a photodiode array detector. Protein and Example 50 label concentrations were determined by UV-Vis (280 and 370 nm, respectively). Label worth protein incorporation ratio (IR) was determined by dividing the molar concentration of Example 50 to the molar concentration of HIV mAb. A final IR value of 4.0 was achieved for a 1:20 molar input ratio. Protein conjugates were stored at 2-8°C protected from light until use.

[0393] [Example 75] Anti-Human IgG MAB-Lucifer Yellow-CPSP-PEG4 Acridinium Conjugate. A stock solution of Lucifer Yellow-CPSP-PEG4 active ester (Example 50) was prepared by reconstituting the dry powder in DMSO to 9.3 mg / mL.

[0394] Approximately 1 mg of anti-human IgG mAb was dialyzed against 50 mM potassium phosphate 150 mM potassium chloride pH 8.0 at a ratio of 0.2 L / mL. After dialysis, 0.7 mg of antibody was added to 60 μL of potassium phosphate buffer, pH 8.0, containing cyclodextrin (30% in reaction) in a reaction vessel protected from light. Lucifer Yellow-CPSP-PEG4 Acridinium solution was added to the reaction vessel to achieve a molar input ratio of 10 to moles of mAb. The reaction vessel was vortexed briefly and incubated statically overnight for approximately 22 hours while protected from light. The reaction vessel was centrifuged to separate insoluble aggregates and the remaining supernatant was purified via SEC-HPLC on a G3000 column with a mobile phase of 10 mM PBS pH 6.3. The conjugate IR was determined via UV-VIS and A280 and A370 were measured. The protein conjugate was stored at 2-8 °C.

[0395] [Example 76] Anti-TSHMAB-Lucifer Yellow-CPSP-PEG4 Acridinium Conjugate. A stock solution of Lucifer Yellow-CPSP-PEG4 active ester (Example 50) was prepared by reconstituting the dry powder in DMSO to 9.3 mg / mL.

[0396] Approximately 3 mg of anti-TSH mAb was desalted on a Zeba desalting column into phosphate buffer pH 8.0. After desalting, 2.6 mg of antibody was added to 200 μL of phosphate buffer containing cyclodextrin (30% in reaction), pH 8.0, in a reaction vessel protected from light. Lucifer Yellow-CPSP-PEG4 Acridinium solution was added to the reaction vessel to achieve a molar input ratio of 7.5 to moles of mAb. The reaction vessel was vortexed briefly and incubated statically overnight for approximately 18 hours, protected from light. The reaction vessel was centrifuged to separate insoluble aggregates, and the remaining supernatant was purified via SEC on a Sephacryl S-300 column with a mobile phase of 10 mM PBS pH 6.3. The conjugate IR was determined via UV-VIS, measuring A280 and A370. The protein conjugate was stored at 2-8°C.

[0397] [Example 77] Anti-NGAL mAb Biotin-Acridinium-Lucifer Yellow (LY). Stock solutions of biotin active ester (as purchased) and acridinium lucifer yellow (Example 48) were prepared by separately reconstituting the dry powders in DMSO to 10 mg / mL dry weight.

[0398] Approximately 200 μg of anti-NGAL IgG antibody was added to approximately 100 μL of 10 mM phosphate buffered saline pH 8.0. The vessel was protected from light and a solution of active ester of biotin was added to achieve a 5-fold molar input ratio relative to the moles of mAb. The reaction vessel was vortexed briefly and then incubated statically overnight for approximately 16 hours while protected from light. The solution was then loaded onto a desalting column (Zeba spin desalting column, Thermo Scientifics). The concentration of the labeled antibody was determined by measuring the absorption spectrum at A280 nm. The extinction coefficient for A280 was 1.45 / mg / mL. The purified protein was then reacted with active ester of acridinium-Lucifer Yellow at a molar ratio of 1:0.5 (mAb:acridinium-LY) for another 16 hours. The amount of acridinium-LY used for labeling was intentionally kept low. It is preferable to remove unreacted acridinium-LY with a separate desalting column, but the product can also be used without further purification. The protein conjugate was stored at 2-8 °C, protected from light, until use.

[0399] [Example 78] Evaluation of multiplex assay - Cytomegalovirus (CMV) IgG and IgM assays. CMV IgG and IgM antibody detection kits (Total CMV) were assembled by diluting anti-human IgG antibody-acridinium-fluorescein conjugate (70ng / mL, Example 70) for CMV IgG antibody detection and anti-human IgM antibody-acridinium conjugate (25ng / mL) for CMV IgM antibody detection in Architect CMV IgG conjugate diluent containing MES buffer. The experimental conjugate bottles were combined with CMV microparticles marketed by Abbott and assay specific diluent (ASD) (Abbott list number 6C15). Microparticle processing was performed using a 96-well plate on a KingFisher instrument and luminescence readings were performed on a Mithras LB 940 luminometer. Briefly, a 96-well plate was prepared with microparticles, ASD, and sample in column 1 and incubated with shaking for approximately 18 minutes. Columns 2-4 were filled with 200 μL wash buffer to wash the particles three times after sample incubation. The microparticles were transferred to column 5, which contained the conjugate, and incubated for 4 minutes. The microparticles were washed three more times using 200 μL wash buffer in columns 6-8. Finally, the microparticles were transferred to column 9, which contained 100 μL Architect pre-trigger, and incubated for 5 minutes. After incubation, 33 μL of the reaction mixture was transferred in triplicate to a new 96-well plate and placed in a Mithras LB 940 luminometer. The injector on the luminometer was programmed to dispense the Architect trigger into each well, followed by 10 seconds of chemiluminescence collection with or without wavelength filters. Triplicate reaction wells were used and read with no filter, a green filter, and a blue filter. A 442 / 46 nm filter was used to capture blue light and a 531 / 46 nm filter was used to capture green light. Relative light unit (RLU) readings for each well were generated by summing the total light output for the first 3 seconds of the reading window.

[0400] A multiplex test was performed in which a CMV IgG only sample (Architect CMV positive control) was combined with a sample containing only CMV IgM at known relative amounts. Samples containing IgM and IgG at ratios of 0:100, 25:75, 50:50, 75:25, and 100:0 were created. Signals generated with no filter, green filter, and blue filter were processed and analyzed. The results, shown in Figure 6, demonstrate that the assembled reagent kit can distinguish mixed IgM and IgG signals in a single sample.

[0401] [Example 79] Multiplexed Assay Evaluation - HIV Antigen and Antibody Combination Assay. HIV antigen and antibody detection kits (HIV Combo) were assembled by diluting HIV p24 mAb-Acridinium-Fluorescein conjugate (125ng / mL, Example 71) for HIV antigen detection and HIV antigen-Acridinium conjugate (50ng / mL) for HIV antibody detection in Architect HIV Combo conjugate diluent containing phosphate buffer, bovine serum albumin, and surfactant. Experimental conjugate bottles were combined with HIV Combo microparticles marketed by Abbott and assay specific diluent (Abbott list number 2P36). Assay testing was performed on an Abbott Architect automated immunoassay analyzer modified with a two-channel optical configuration. Briefly, a dual photomultiplier tube (PMT) assembly was constructed where a dichroic mirror with a 500 nm wavelength cutoff was used to reflect low wavelength light (blue) to a vertical PMT while high wavelength light (green) was passed through the mirror to a second PMT. Appropriate filters were placed behind the dichroic mirror to further filter the light before it reached each PMT. The Architect instrument hardware was used to read the output from the reflected (blue) PMT while a separate counter module and laptop computer interface was used to collect the signal from the in-line (green) PMT. Custom IDL code was developed to automatically process the signal from the in-line PMT. Assay testing using CMIA technology to perform a 2-step immunoassay was performed using the commercially available Architect HIV Combo assay file. Briefly, the sample, ARCHITECT wash buffer, assay diluent, and microparticles were combined in the first step. HIV p24 antigen and HIV antibodies present in the sample bind to the HIV antigen and HIV p24 mAb coated microparticles. After washing, an acridinium-labeled conjugate is added and binds to the HIV p24 antigen and HIV antibodies captured on the microparticles.After another wash cycle, pre-trigger and trigger solutions are added to the reaction mixture to induce a chemiluminescent signal, which is measured as relative light units (RLU).

[0402] A multiplexed test was performed in which normal human plasma was spiked with elevated or reduced levels of HIV antibodies and HIV p24 antigen. Samples were created containing 400, 300, 200, 100, and 0 pg / mL p24 antigen in combination with 0, 45, 90, 135, and 180 ng / mL anti-HIV antibodies. The samples represented mixture ratios with standardized sample amounts of 0:100, 25:75, 50:50, 75:25, and 100:0 percent. The signals generated in both data channels were processed and analyzed. The results, shown in Figure 7, demonstrated that the combined reagent kit and two channel PMT setup can distinguish mixed antigen and antibody signals in a single sample.

[0403] [Example 80] Evaluation of multiplexed assays - Lyme Disease IgG and IgM assays. Lyme Disease IgG and IgM antibody detection kits (total Lyme) were assembled by preparing anti-human IgG antibody-acridinium-lucifer yellow conjugate solutions (25 ng / mL, Example 69) for Lyme IgG antibody detection and anti-human IgM antibody-acridinium conjugate solutions (15 ng / mL) for Lyme IgM antibody detection in Lyme IgG conjugate diluent (containing MES, detergent, and protein stabilizer). The kits consisted of experimental conjugates, microparticles coated with recombinant antigens derived from the variable major protein-like sequence expression (VlsE) of Borrelia burgdorferi, and assay specific diluent at pH 7.5. Assay testing was performed on an Abbott ARCHITECT® automated immunoassay analyzer modified with a two-channel optical configuration. Briefly, a dual photomultiplier tube (PMT) assembly was constructed where a dichroic mirror with a 500 nm wavelength cutoff was used to reflect low wavelength light (blue) to the vertical PMT while high wavelength light (green) was passed through the mirror to the second PMT. Appropriate filters were placed behind the dichroic mirror to further filter the light before it reached each PMT. The Architect instrument hardware was used to read the output from the reflected (blue) PMT while a separate counter module and laptop computer interface was used to compile the signal from the in-line (green) PMT. A custom computer program (IDL code) was developed to automatically process the signal from the in-line PMT. Assay testing using CMIA technology to perform a 2-step immunoassay was performed using the assay file. Briefly, the sample, ARCHITECT® wash buffer, assay diluent, and microparticles were combined in the first step. Human anti-Lyme IgG and IgM antibodies present in the sample bind to the Lyme antigen coated microparticles. After washing, an anti-human acridinium-labeled conjugate is added and allowed to bind to the human antibodies captured on the microparticles.After another wash cycle, pre-trigger and trigger solutions are added to the reaction mixture to generate a chemiluminescent signal, which is measured as relative luminescence units (RLU).

[0404] A multiplexed test was performed in which a Lyme IgG only sample was combined with a sample containing Lyme IgM only in a 1:1 ratio, and the results of the mixed sample were compared to those of the single component samples. The signals generated in the green and blue channels, respectively, were processed and analyzed. The results, shown in Figure 8, demonstrated that the assembled reagent kit can distinguish the mixed IgM and IgG signals in a single sample.

[0405] [Example 81] Combined Free T4 and Thyroid Stimulating Hormone Assay - A Free T4 and Thyroid Stimulating Hormone (TSH) detection kit (FT4 / TSH) was assembled by preparing a T3-acridinium-lucifer yellow conjugate solution (750 ng / mL, Example 64) for T4 detection in ARCHITECT®-Free T4 Conjugate Diluent containing detergent and MES buffer. A microparticle bulk solution was created by combining anti-T4 antibody-coated microparticles with anti-TSH antibody-coated microparticles in ARCHITECT®-Free T4 Microparticle Diluent containing Tris buffer, bovine serum albumin, and detergent. Experimental T4 conjugate and FT4 / TSH microparticle bottles were combined with Abbott's marketed TSH conjugate (anti-TSH antibody labeled with acridinium) and an assay specific diluent consisting of Tris buffer, pH 7.4. Assay testing was performed on an Abbott ARCHITECT® automated immunoassay analyzer modified with a two-channel optical configuration. Briefly, a dual photomultiplier tube (PMT) assembly was constructed in which a dichroic mirror with a 500 nm wavelength cutoff was used to reflect low wavelength light (blue) to a vertical PMT while high wavelength light (green) was mirrored to a second PMT. Appropriate filters were placed behind the dichroic mirror to further filter the light before it reached each PMT. Hardware in the ARCHITECT® instrument was used to read the output from the reflected (blue) PMT while a separate counter module and laptop computer interface were used to collect the signal from the in-line (green) PMT. A custom computer program (IDL code) was developed to automatically process the signals from the in-line PMT. Assay testing was performed using CMIA technology and a 4-bottle assay file with different steps of adding conjugate reagents to sequentially create 1-step and 2-step immunoassays.Briefly, sample, ARCHITECT® wash buffer, assay diluent, microparticles, and experimental T4 conjugates were combined in the first step. T4 competes with T3 acridinium-lucifer yellow conjugate in the sample for binding to anti-T4 microparticles, and TSH binds to anti-TSH coated microparticles in the sample. After washing, acridinium-labeled anti-TSH antibody conjugate is added and binds to TSH captured on the microparticles. After another wash cycle, pre-trigger and trigger solutions are added to the reaction mixture to promote a chemiluminescent signal that is measured as relative luminescence units (RLU).

[0406] Assay performance was measured by the shape of the calibration curves for free T4 and TSH and the ability to read single component controls (FT4 calibrator levels used were 0, 0.5, 1, 2, 3.5, and 6 ng / dL. TSH calibrator levels used were 0, 0.5, 2, 10, 40, and 100 mIU / L). Signals generated in both data channels were processed and analyzed. The results, shown in Figure 9 and Table 2, demonstrate that the assembled reagent kit and two channel PMT settings are capable of calibrating and reading free T4 and TSH controls within the specification limits.

[0407] [Table 2]

Claims

1. A compound of formula (I) or a salt thereof 【Chemistry 1】 [In the formula, X is -NH- or a diamine linker; Y is selected from nitrogen, oxygen, and sulfur; When Y is nitrogen, R １ is -SO ２ -A, where A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl; When Y is oxygen or sulfur, R １ does not exist, Q is -SO ２ - or -CO-, L １ and L ２ each is independently selected from alkylene and heteroalkylene; R ２ is selected from -COOZ and -CN; Z is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, and heteroalkyl; R ａ , R ｂ , R ｃ , R ｄ , R ｅ , R ｆ , R ｇ , and R ｈ are hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ Alkoxy, C １ ~C ４ Haloalkyl, C １ ~C ４ independently selected from haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl; Each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, aryloxy, heteroalkyl, alkylene, and heteroalkylene is independently optionally substituted with 1, 2, 3, 4, or 5 substituents.

2. X, 【Chemistry 2】 2. The compound according to claim 1, or a salt thereof, wherein the diamine linker is selected from the group consisting of:

3. X, 【Chemistry 3】 3. The compound according to claim 1 or 2,

4. The compound or salt thereof according to any one of claims 1 to 3, wherein Y is nitrogen.

5. A is unsubstituted or C １ ~C ４ Alkyl, C １ ~C ４ Alkoxy, C １ ~C ４ Haloalkyl, C １ ~C ４ 5. The compound according to claim 4, which is aryl substituted with 1, 2, 3, 4, or 5 substituents selected from haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl, or a salt thereof.

6. Q is -SO ２ The compound according to any one of claims 1 to 5, or a salt thereof, wherein

7. R ２ The compound according to any one of claims 1 to 6, or a salt thereof, wherein is -COOZ.

8. Z is hydrogen and C １ ~C ４ The compound according to any one of claims 1 to 7, wherein the aryl group is selected from alkyl.

9. L １ and L ２ Each independently C １ ~C ４ -alkylene.

10. R ａ , R ｂ , R ｃ , R ｄ , R ｅ , R ｆ , R ｇ , and R ｈ The compound according to any one of claims 1 to 9, wherein each is hydrogen.

11. Formula (Ia) 【Chemistry 4】 [In the formula, Each R is independently C １ ~C ４ Alkyl, C １ ~C ４ Alkoxy, C １ ~C ４ Haloalkyl, C １ ~C ４ selected from the group consisting of haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl; m is 0, 1, 2, 3, 4 or 5; n is 1, 2, 3, 4, 5 or 6.], or a salt thereof.

12. m is 1 and R is C １ ~C ４ 12. The compound of claim 11, or a salt thereof, wherein:

13. 13. The compound according to claim 11 or 12, wherein m is 1 and R is methyl, or a salt thereof.

14. The compound according to any one of claims 11 to 13, wherein n is 3, or a salt thereof.

15. Formula (Ib) 【Chemistry 5】 The compound according to any one of claims 11 to 14, or a salt thereof, having the formula:

16. 16. The compound according to any one of claims 1 to 15, or a salt thereof, wherein the fluorophore is selected from fluoresceins, rhodamines, boron-dipyrromethenes, cyanines, oxazines, thiazines, coumarins, naphthalimides, rhodols, naphthalenes, squaraines, porphyrins, flavins, and lanthanide dyes.

17. The fluorophore is 【Chemistry 6】 【change】 The compound according to any one of claims 1 to 16, or a salt thereof, selected from the group consisting of

18. A conjugate of formula (II) or a salt thereof 【Chemistry 7】 [In the formula, X is -NH- or a diamine linker; Y is selected from nitrogen, oxygen, and sulfur; When Y is nitrogen, R １ is -SO ２ -A, where A is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocyclylalkyl; When Y is oxygen or sulfur, R １ does not exist, Q is -SO ２ - or -CO-, L １ is selected from alkylene and heteroalkylene; L ３ is the linker, R ａ , R ｂ , R ｃ , R ｄ , R ｅ , R ｆ , R ｇ , and R ｈ are hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ Alkoxy, C １ ~C ４ Haloalkyl, C １ ~C ４ independently selected from haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl; A binding member is a molecule capable of binding to a target analyte; Each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, alkylene, and heteroalkylene is independently optionally substituted with 1, 2, 3, 4, or 5 substituents.

19. X, 【Chemistry 8】 20. The conjugate of claim 18, or a salt thereof, wherein the diamine linker is selected from the group consisting of:

20. X, 【Chemistry 9】 20. The conjugate of claim 18 or 19,

21. The conjugate according to any one of claims 18 to 20, or a salt thereof, wherein Y is nitrogen.

22. A is unsubstituted or C １ ~C ４ Alkyl, C １ ~C ４ Alkoxy, C １ ~C ４ Haloalkyl, C １ ~C ４ 22. The conjugate of claim 21, or a salt thereof, which is aryl substituted with 1, 2, 3, 4, or 5 substituents selected from haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl.

23. Q is -SO ２ The compound according to any one of claims 18 to 22, or a salt thereof, wherein

24. L １ But, C １ ~C ４ The conjugate of any one of claims 18 to 23, wherein -alkylene.

25. The compound has the formula (IIa) 【Chemistry 10】 [In the formula, R is C １ ~C ４ Alkyl, C １ ~C ４ Alkoxy, C １ ~C ４ Haloalkyl, C １ ~C ４ selected from the group consisting of haloalkoxy, halo, hydroxy, cyano, nitro, amino, carboxy, sulfonyl, phosphoryl, and selenyl; m is 0, 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, 5, or 6.]. The conjugate according to any one of claims 18 to 24, or a salt thereof.

26. m is 1 and R is C １ ~C ４ 26. The conjugate of claim 25, or a salt thereof, wherein:

27. 27. The conjugate of claim 25 or 26, or a salt thereof, wherein m is 1 and R is methyl.

28. The conjugate according to any one of claims 25 to 27, or a salt thereof, wherein n is 3.

29. 29. The conjugate according to any one of claims 18 to 28, or a salt thereof, wherein the linker is selected from alkylene and heteroalkylene linkers.

30. 30. The conjugate of any one of claims 18 to 29, wherein the linker comprises a moiety E that is the product of a reaction between two reactive groups.

31. 31. The conjugate of claim 30, wherein E is selected from the group consisting of amides, esters, carbamates, and triazoles.

32. 32. The conjugate according to any one of claims 18 to 31, or a salt thereof, wherein the fluorophore is selected from fluoresceins, rhodamines, boron-dipyrromethenes, cyanines, oxazines, thiazines, coumarins, naphthalimides, rhodols, naphthalenes, squaraines, porphyrins, flavins, and lanthanide dyes.

33. The fluorophore is 【Chemistry 11】 【change】 【change】 The conjugate according to any one of claims 18 to 32, selected from:

34. 34. The conjugate according to any one of claims 18 to 33, or a salt thereof, wherein the binding member is selected from a protein, a peptide, a small molecule, a nucleic acid, a carbohydrate, and a dendrimer or dendritic structure.

35. 35. The conjugate of claim 34, wherein the binding member is a protein, the protein being selected from an antibody, an antigen, a receptor, an enzyme, and a glycoprotein.

36. 36. The conjugate of claim 35, or a salt thereof, wherein the protein is selected from Immunoglobulin G, Immunoglobulin M, an HIV antibody, an HIV antigen, an HCV antibody, an HCV antigen, a p24 antigen, a troponin, and a brain natriuretic peptide.

37. 35. The conjugate of claim 34, wherein the binding member is a small molecule, the small molecule being selected from an enzyme substrate, an enzyme inhibitor, a steroid, a retinoid, a lipid, a vitamin, a nutrient, a nutrient metabolite, a pharmaceutical drug or a drug of abuse.

38. 38. The conjugate of any one of claims 18 to 37, or a salt thereof, wherein the binding member is attached to the remainder of the conjugate of formula (II) via an amino acid residue selected from lysine, cysteine, aspartic acid, and glutamic acid.

39. The conjugate of any one of claims 18 to 38, further comprising an additional binding member covalently linked to the conjugate.

40. 1. A method for detecting an analyte of interest in a biological sample, comprising: a) contacting a biological sample with at least one specific binding member that binds to an analyte of interest to form at least one complex, said specific binding member comprising a conjugate according to any one of claims 18 to 39; and b) detecting the presence or absence of a signal from the specific binding member, the detection of the signal indicating the presence of the analyte in the sample and the absence of the signal indicating the absence of the analyte in the sample. A method comprising:

41. a) contacting a biological sample with at least one first specific binding member and at least one second specific binding member, wherein each of the at least one first specific binding member and the at least one second specific binding member specifically binds to an analyte of interest, thereby generating one or more first complexes comprising first specific binding member-analyte-second specific binding member, wherein the second specific binding member comprises a conjugate according to any one of claims 18 to 39; b) detecting the presence or absence of a signal from a second specific binding member, wherein detection of said signal indicates the presence of said analyte in said sample and the absence of said signal indicates the absence of said analyte in said sample.

41. The method of claim 40, comprising:

42. 1. A method for detecting two or more analytes of interest in a biological sample, comprising: a) simultaneously or sequentially contacting a biological sample with (i) at least one first specific binding member that binds to a first analyte of interest to form at least one first complex; and (ii) at least one second specific binding member that binds to a second analyte of interest to form at least one second complex, wherein each of the first and second specific binding members comprises a conjugate according to any one of claims 18 to 39, and the fluorophores of the conjugates in each of the first and second specific binding members are different; and b) detecting the presence or absence of a signal from each of the first and second specific binding members, where (i) detection of a signal from the first specific binding member indicates the presence of a first analyte in the sample and the absence of a signal from the first specific binding member indicates the absence of the first analyte in the sample, and (ii) detection of a signal from the second specific binding member indicates the presence of the second analyte in the sample and the absence of a signal from the second specific binding member indicates the absence of the second analyte in the sample. A method comprising:

43. 1. A method for detecting two or more analytes of interest in a biological sample, comprising: a) contacting a biological sample with at least one first specific binding member and at least one second specific binding member, wherein the at least one first specific binding member and the at least one second specific binding member each specifically bind to a first analyte of interest, thereby generating one or more first complexes comprising the first specific binding member-first analyte-second specific binding member, wherein the second specific binding member comprises a conjugate according to any one of claims 18 to 39; and b) simultaneously or sequentially contacting the biological sample with at least one third specific binding member and at least one fourth specific binding member, wherein the at least one third specific binding member and the at least one fourth specific binding member each specifically bind to a second analyte of interest, thereby generating one or more second complexes comprising the third specific binding member-second analyte-fourth specific binding member, the fourth specific binding member comprising a conjugate according to any one of claims 16 to 34, wherein the fluorophores in the conjugate for each of the second and fourth specific binding members are different; and c) detecting the presence or absence of a signal from each of the second and fourth specific binding members, where (i) detection of a signal from the second specific binding member indicates the presence of the first analyte in the sample and the absence of a signal from the second specific binding member indicates the absence of the first analyte in the sample, and (ii) detection of a signal from the fourth specific binding member indicates the presence of the second analyte in the sample and the absence of a signal from the fourth specific binding member indicates the absence of the second analyte in the sample. A method comprising:

44. contacting the biological sample simultaneously or sequentially with at least one fifth specific binding member and at least one sixth specific binding member, wherein the at least one fifth specific binding member and the at least one sixth specific binding member each specifically bind to a third analyte of interest, thereby generating one or more third complexes comprising the fifth specific binding member-third analyte-sixth specific binding member, the sixth specific binding member comprising a conjugate according to any one of claims 18 to 39, wherein the fluorophores of the conjugates for each of the second, fourth and sixth specific binding members are different; 44. The method of claim 43, further comprising detecting the presence or absence of a signal from each of the second, fourth and sixth specific binding members, wherein: (i) detection of a signal from the second specific binding member indicates the presence of a first analyte in the sample and the absence of a signal from the second specific binding member indicates the absence of the first analyte in the sample; (ii) detection of a signal from the fourth specific binding member indicates the presence of a second analyte in the sample and the absence of a signal from the fourth specific binding member indicates the presence of the second analyte in the sample; and (iii) detection of a signal from the sixth specific binding member indicates the presence of a third analyte in the sample and the absence of a signal from the sixth specific binding member indicates the presence of the third analyte in the sample.

45. The method according to any one of claims 40 to 44, wherein the biological sample is whole blood, serum, urine, cerebrospinal fluid, amniotic fluid, saliva or plasma.

46. 42. The method of claim 41 , wherein the first specific binding member and / or the second specific binding member are immobilized on a solid support.

47. 43. The method of claim 42, wherein the first specific binding member, the second specific binding member, the third specific binding member, and / or the fourth specific binding member are immobilized on a solid support.

48. 45. The method of claim 44, wherein the first specific binding member, the second specific binding member, the third specific binding member, the fourth specific binding member, the fifth specific binding member and / or the sixth specific binding member are immobilized on a solid support.

49. The method of any one of claims 40 to 48, which is carried out using a clinical chemistry assay, an immunoassay or a single molecule detection assay.

50. 50. The method of any one of claims 40 to 49, further comprising the step of adding hydrogen peroxide to the biological sample prior to the detecting step.

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