Measuring a plurality of analytes using an azinium label

Abstract

Described herein are methods for detecting one or more analytes in a sample using different chemiluminescent labels that have distinct chemiluminescence in the wavelength and kinetic domains. Also described are solid supports, reagents, and compounds for use in these methods. Generally, the methods involve detection via nearly simultaneous detection of multiple analytes in a sample conjugated to different azinium labels.

Description

[0001] Cross-reference to related applications This application claims priority and benefit to U.S. Application No. 63 / 595,386, filed November 2, 2023, the entire contents of which are hereby incorporated by reference. Technical Field

[0002] This disclosure relates to a method for detecting or measuring multiple analytes in a sample by measuring multiple chemiluminescence signals at different emission wavelengths and time frames. The multiple chemiluminescence signals are generated by multiple acridine-based labels, each acridine-based label having a distinguishable emission maximum and / or emission rate (kinetics) and being labeled to a binding compound specific to one of the multiple analytes present in the sample. The invention also relates to stable acridine-based labels capable of emitting light at a slow emission rate (kinetics). The structural features of stable acridine-based compounds required for low-speed light emission are disclosed herein. Background Technology

[0003] Chemiluminescence is a commonly used photogenerating technique designed for the measurement and quantification of analytes in samples. Acridine-labeled compounds (such as acridine esters (AEs) and acridine sulfonamides) are frequently used in these assays. Since the development of stable dimethyl acridine esters (DMAEs) in the 1980s, many useful acridine compounds capable of undergoing chemiluminescence induced by triggering compounds have been developed. Acridines with improved properties, such as higher light output, lower nonspecific binding, and faster light emission kinetics, have been developed, leading to the development of many automated immunoassay systems.

[0004] While these instruments offer high measurement throughput, further expansion of the measurement menu can place significant strain on the system and measurement protocols. For example, a large number of tests require more reagent bottles, calibrators, and controls to be placed on the instrument, which in turn necessitates a larger instrument footprint and more complex system requirements.

[0005] Multiple attempts have been made to use different acridine esters for assays; however, these attempts have been hampered by the problem of implementing the detection of multiple analytes and are generally limited to the simultaneous detection of only two analytes. For example, each of U.S. Patents 5,395,752, 5,702,887 and 5,879,894, which are hereby incorporated in their entirety by reference, describes exemplary long-emitting acridine esters (LEAEs) containing a benzo[b]acridinium ring. Compared to acridine esters that emit light, for example, at λmax 422–428 nm (e.g., DMAE-NHS(1)), benzo[b]acridinium esters (LEAEs) emit light at λmax 508–550 nm, such as LEAE-NHS(2) at λmax 528 nm. Both compounds have the following structures: Similarly, the use of two markers is provided hereby by reference to U.S. Patent 5,656,207, which is incorporated herein by reference in its entirety. However, for example, compounds 3 and 4. Compound 3, however, is an unstable acridine ester that rapidly hydrolyzes to an inactive form in the aqueous medium in which most immunoassays are performed. Using such a marker in commercial products is very difficult (if not impossible), especially in any kind of multianalyte measurement system.

[0006] U.S. Patent 5,879,894 (which is hereby incorporated herein by reference in its entirety) describes acridinium compounds with different structures, including 3-carboxybutadienyl-AE (compound 7) and compound 6. The emission maximum of 3-carboxybutadienyl-AE (compound 7) was determined to be at λmax 464 nm. Compound 7 differs from compound 6 in that compound 7 has a methyl group on the phenyl group of the ester and CF3COO as a counterion. - Compound 6 has an R group at the phenol group and CH3SO4 as a counterion. - Substituents at the R position and counterions do not alter the emission wavelength of acridine onium esters. It is reasonable to assume that structure 6 will have the same emission wavelength profile as compound 7. The small difference of only ~30 nm at the emission maxima between 6 and 5 makes separation of the two wavelengths extremely difficult, thus making it practically impossible to measure the two signals by emission wavelength for multiplexing.

[0007] U.S. Patent 6,165,800 discloses a group of acridinium compounds called energy transfer conjugates (ETCs), in which the AE moiety is covalently linked to a fluorophore. In a reaction leading to chemiluminescence, the excited-state energy contained in the acridinone formed by the reaction with hydrogen peroxide in an alkaline solution is transferred to the covalently linked fluorophore, causing the ETC to emit light at the wavelength of the fluorophore rather than the wavelength of the acridinium ester. By selecting molecules from a variety of fluorophores with different emission wavelengths, the ETCs exhibit light emission in the range of λmax 550 to 718 nm. Compounds 8 and 9 are two examples of ETCs. The authors report three distinguishable color light emission spectra from a mixture of three ETCs and provide an example of dual-analyte determination by simultaneously measuring light at different wavelengths of two acridinium esters.

[0008] Rhodamine-2-AM-DMAE-CO2H (8) Light emission λmax: 620nm CNF-2-AM-DMAE-CO2H (9) Light emission λmax: 718nm There is a persistent need for chemiluminescent compounds that provide high sensitivity and stability for analyte detection. Summary of the Invention

[0009] In accordance with the foregoing and other purposes, this disclosure includes methods for detecting analytes in a sample by using a chemiluminescent label having distinguishable chemiluminescence. Acridinium is also provided for use in chemiluminescent assays, generally possessing stability suitable for these assays. In some embodiments, the sample is blood, saliva, or serum. In some embodiments, the sample is derived from a biological sample, such as a diluted biological sample (e.g., mixed with saline).

[0010] Methods for detecting or quantifying multiple analytes in a sample (e.g., biological samples such as blood, saliva, serum, or samples derived from biological samples such as diluted biological samples) may include: (a) Provide a first set of chemiluminescent markers and a second set of chemiluminescent markers. The first group of chemiluminescent labels includes at least two chemiluminescent labels with separated emission spectra in the wavelength domain, and The second group of chemiluminescent labels comprises at least two chemiluminescent labels with different emission rates; wherein the first and second groups can overlap (e.g., chemiluminescent labels in the first group can also be in the second group). Each chemiluminescent label in the first and second groups can form a binding complex with at least one of a variety of analytes; (c) Mix the first group and the second group with the sample; (e) Prepare a mixture of the first group, the second group and the sample to measure the chemiluminescence of the chemiluminescent markers from the first group and the second group; (f) Triggering chemiluminescence from the first and second groups of chemiluminescent markers after the preparation of the mixture (e.g., by adding one or more triggering compositions that trigger the chemiluminescence of acridine-onium markers); (f) Measuring chemiluminescence in the wavelength domain (e.g., measuring light intensity as a function of wavelength) and the time domain (e.g., measuring light intensity as a function of time from the triggering step); (g) The presence of the at least one analyte or its concentration is detected by comparing the amount of emitted light with a standard dose response curve that correlates the amount of emitted light with a known concentration of at least one of a plurality of analytes.

[0011] This method can detect multiple analytes in a sample. In some embodiments, two chemiluminescent labels in a first group are capable of forming binding complexes with different analytes in the sample. In various embodiments, two chemiluminescent labels in a second group are capable of forming binding complexes with different analytes in the sample. In some embodiments, different acridinium labels are conjugated to the same analyte or its binding partner. In some embodiments, a solid support is conjugated to a first binding partner, such as an antibody (e.g., via biotin / streptavidin). The sample can be mixed with one or more chemiluminescent labels of this disclosure, which are independently conjugated to one or more binding partners of one or more analytes of interest. The sample can also be mixed with one or more conjugates (or intermediate conjugates) that also bind to one or more analytes of interest and to the first binding partner. In this form, the binding complex will be formed by direct binding to a solid phase of an intermediate conjugate, which also binds to the analyte. The analyte will also bind to the chemiluminescent label (via the analyte and the intermediate conjugate). Therefore, by providing one or more immunoassay reagents containing multiple chemiluminescent markers and / or one or more intermediate conjugates, the detection of multiple analytes can be achieved through a single binding complex.

[0012] The first and second groups of acridine-based markers may overlap (e.g., the chemiluminescent acridine-based portion of one group may also exist in the other group). For example, in some embodiments, the acridine-based markers are members of both the first and second groups. In some embodiments, the first acridine-based marker may have a different chemiluminescence wavelength than the second chemiluminescent marker (e.g., measured by the difference in λmax), such that the first and second acridine-based markers form a first group of wavelength-separated acridine-based markers. This first chemiluminescent marker may also be kinetically separated from the third chemiluminescent marker by a third acridine-based marker (e.g., measured by the difference in %RLU measured over an indicated time period), such that the first and third acridine-based markers form a second group of kinetically separated acridine-based markers. In some embodiments, each of the two chemiluminescent markers in the first group may be a corresponding chemiluminescent marker with a different emission rate (e.g., two different groups of chemiluminescent markers separated by emission rates are formed by four different chemiluminescent markers). In some implementations, the four chemiluminescent labels are able to form binding complexes with different analytes in the sample.

[0013] Various assay methods are available, providing the ability to measure many analytes nearly simultaneously (e.g., by collecting chemiluminescence from a single chemiluminescent event, such as by initiation with the addition of one or more triggers). In various embodiments, the first set of chemiluminescent labels comprises at least three chemiluminescent labels having separate emission spectra in the wavelength domain. In some aspects, the three chemiluminescent labels in the first set may be able to form binding complexes with three different analytes in the sample. Each of the three chemiluminescent labels in the first set may have a corresponding chemiluminescent label with different emission kinetics (e.g., three different second set of chemiluminescent labels formed by six different chemiluminescent labels, separated in emission rate), and each of the six chemiluminescent labels may be able to form binding complexes with six different analytes in the sample.

[0014] Each group and each chemiluminescent marker within a group can be mixed with the biological sample in any order. For example, in some embodiments, one or more chemiluminescent markers are mixed with the sample individually. In some embodiments, a first group of chemiluminescent markers is mixed with the sample, for example by adding a composition containing the chemiluminescent markers from the first group. In some embodiments, a second group of chemiluminescent markers is mixed with the sample, for example by adding a composition containing the chemiluminescent markers from the second group. In some embodiments, both the first and second groups of chemiluminescent markers are mixed with the sample, for example by adding a composition containing both the first and second groups of chemiluminescent markers.

[0015] After adding two sets of chemiluminescent labels, a sample is typically prepared to induce chemiluminescence in a manner that allows for the measurement and / or quantification of the analyte's concentration. For example, the preparation steps may include: (e1) A solid support having molecules immobilized thereon, the molecules being capable of forming binding complexes with the at least one analyte and with chemiluminescent markers in the first and / or second groups of chemiluminescent markers; and (e2) Separate the solid support from the mixture.

[0016] In some embodiments, the solid support may comprise at least two molecules (e.g., two, three, four, five, six, seven, eight, nine, or ten), each molecule capable of forming a binding complex with a different analyte and capable of forming a binding complex with at least two different chemiluminescent labels from the first and / or second groups of chemiluminescent labels. In some embodiments, the solid support may comprise molecules capable of forming binding complexes with an analyte or its binding partner, wherein, collectively, the molecules can bind to each chemiluminescent label from the first and / or second groups.

[0017] Typically, chemiluminescent labels have the structure of formula (I): Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: "j" and "k" are independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3 or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N)–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R NEach time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates). For example, chemiluminescent labels can independently have the structure of formula (Ia): Where Ω represents O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L – In some embodiments, the chemiluminescent label independently has the structure of formula (Ib) or (Ic): R4-R7 are independently hydrogen, electron-donating groups, or C. 1-35 Alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and "Y" either does not exist (i.e., it is a key) or is -L C –, –L1–, –R L –, or –R L –L1–. In some embodiments, at least one (e.g., one, two, three, four, five, six, each) of the first group is a zwitterionic acridinenet (e.g., N-sulfopropyl zwitterionic acridinenet, a compound having the structure of formula I, Ia, Ib, or Ic, wherein R1 is selected from –X b , –R L –X b , or –L C –X b For example, –L1–X b R1 is selected from –SO3 - , –R L – SO3 - For example –(CH2) 1-5 -SO3 - , or –L C – SO3- For example –L1– SO3 - In some embodiments, at least one (e.g., one, two, three, four, five, six, each) of the second group is a zwitterionium (e.g., N-sulfopropyl zwitterionium, a compound having the structure of formula I, Ia, Ib, or Ic, wherein R1 is selected from –X). b , –R L –X b , or –L C –X b For example, –L1–X b R1 is selected from –SO3 - , –R L – SO3 - For example –(CH2) 1-5 – SO3 - , or –L C – SO3 - For example –L1– SO3 - In some embodiments, at least one (e.g., one, two, three, four, five, six, each) of the first and / or second groups is an acridineonium salt (e.g., acridineonium carboxylates, such as halocarboxylates, haloalkylcarboxylates, fluoroalkylcarboxylates, acridineonium sulfonates, such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, acridineonium halide salts, such as acridineonium chloride salts, compounds having the structure of formula I, Ia, Ib, or Ic, wherein R1 is selected from –R, –L). C –R,–Z, –R L –Z, –L C –Z,–L1–Z, –R L –L C –R L –Z, –R L –L1–R L –Z, where the counter ion is such as R-COO - R-SO3 - Cl - F - ).

[0018] Measurable differences in chemiluminescence between chemiluminescent labels (e.g., in the wavelength domain and / or in the chemiluminescence rate domain) can be achieved by using acridinium labels with relevant conjugations to elicit such differences. For example, in some embodiments, at least one of R4-R7 (e.g., R4, R5, R7, R7) is an electron-donating group (e.g., and, compared to other identical labels without electron-donating groups, forms chemiluminescent labels with different emission rates). In some embodiments, one chemiluminescent label from the first group has the structure of formula (IIIa): And another chemiluminescent label in the first group has the structure of formula (IIIb): Wherein A1 and A2 are independently analytes or conjugates of analytes, and optionally, A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “j” is 1, 2, 3 or 4; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, -R, electron-donating groups, and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer (IA) such as a fluorophore (e.g., rhodamine); and at least one R2 group is not hydrogen (e.g., at least one R2 is an electron-donating group such as -OG, and at least one R2 group contains a link to a developer). L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(RN )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates). For example, the chemiluminescent label of formula (IIIb) can have the structure of formula (IIIb1): In some embodiments, at least one R3 is not hydrogen (e.g., an electron-donating group such as an alkoxy group). In some embodiments, at least one R3 and / or at least one R2 group is an electron-donating group (e.g., an alkoxy group). In various embodiments, the first group of chemiluminescent labels also comprises compounds having the structure of formula (IIIc): Where IA is the imaging agent (e.g., a fluorophore, such as Texas Red, Rhodamine, or phenol-modified Rhodamine); and A3 is an analyte or a binding partner of an analyte that is different from A1 and A2. In some embodiments, the chemiluminescent label of formula (IIIc) has the structure of formula (IIIc1) or (IIIc2): .

[0019] For example, chemiluminescent labels can have the following structures: .

[0020] In some implementations, the chemiluminescent label of formula (IIIb) can have the structure of formula (IIIb3): Where IA is the developing agent (e.g., a fluorophore, such as rhodamine or phenol-modified rhodamine). For example, a chemiluminescent label of formula (IIIb3) can have the structure of formula (IIIb4) or (IIIb5): .

[0021] For example, chemiluminescent labels can have the following structures: In some implementations, one of the chemiluminescent markers in the first group has the structure of formula (IVa): Furthermore, another chemiluminescent label in the first group has the structure of formula (IVc): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine).

[0022] L C It is divalent C 1-35Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: ; “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: ; "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O)1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0023] The group of acridine-onium labels exhibiting differential chemiluminescence in the wavelength domain can involve these specific compound variations that generate the desired differential chemiluminescence signal. In various embodiments, the differences between the compounds of formulas (IIIa), (IIIb), and (IIIc) are attributed to different A1, A2, and A3 groups and / or conjugations in the acridine-onium ring system (e.g., R1, Ω, L, Y, Y”, IA are the same in formulas (IIIa), (IIIb), and (IIIc)). In some embodiments, the differences between the chemiluminescent labels of formulas (IVa) and (IVb) lie in the conjugation in the acridine-onium ring system (e.g., R1, Ω, L, Y, Y”, IA are the same in formulas (IVa) and different A1 and A2 groups. In various embodiments, the chemiluminescent label of formula (I) (e.g., (Ia), (Ib), (Ic)), (II), (IIIa), (IIIb) (e.g., formula (IIIb1), (IIIb2), (IIIb3), (IIIb4)), and (IV) (e.g., (IVa), (IVb)) is a salt (e.g., a carboxylate, such as a halocarboxylate, a haloalkylcarboxylate, a fluorocarboxylate, a fluoroalkylcarboxylate, F3CCOO). - Salt, where R1 in formula (IIIb2) is an alkyl group, and the counter ion is a carboxylate, a halocarboxylate, a haloalkylcarboxylate, a fluorocarboxylate, a fluoroalkylcarboxylate, or F3CCOO. - ).

[0024] This disclosure is based in part on the identification of stable, slow-emission acridine-2-ylene (AE2-ylene) markers. These stable, slow-emission AE2-ylene (AE2-ylene) markers can be identified by alkyl conjugations (e.g., lower alkyl groups, such as C1 and C2) placed in the AE2-2-ylene system (e.g., at positions 1 and 3 of the AE2-2-ylene system). 1-4 Alkyl groups are formed.

[0025] In some embodiments, at least one chemiluminescent label (e.g., the chemiluminescent label in the second group) has the structure of formula (II): Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: "j" and "k" are independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3 or 4; R1 is hydrogen, –R, –X b–R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: ; “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: ; "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O)1-2 –, –C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –NH–S(O) 1- 2–, –N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–(CH2) 1- 4–, –(CH2) 1-4 –O–, –S–(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –N(R N )–(CH2) 1- 4–, –(CH2) 1-4 –N(R N )–, –(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates). R 2a and R 2b It can be an alkyl group independently (e.g., lower alkyl groups, such as C as methyl, ethyl, propyl, butyl). 1-4 alkyl).

[0026] Stable, slow-emission acridine-onium labels, particularly in their phenyl ester form, can also exhibit slow emission kinetics, where the electron-donating group is positioned on the phenyl ester, for example, at the 4-position. In some embodiments, a chemiluminescent label from the second group has the structure of formula (Va): Furthermore, another chemiluminescent label in the second group has the structure of formula (Vb): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L–L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). R 4a R 5a R 6a and R 7a Independently hydrogen or C 1-35 alkyl; R 4b R 5b R 6b and R 7b Independently hydrogen, C 1-35 Alkyl groups or electron-donating groups (e.g., alkoxy groups); L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1-‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates). In some embodiments, one of the chemiluminescent markers in the second group has the structure of formula (Vc): Furthermore, another chemiluminescent label in the second group has the structure of formula (Vd): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). R 4a R 5a R 6a and R 7a Independently hydrogen or C 1-35 Alkyl groups or electron-donating groups (e.g., alkoxy groups); R 4b R 5b R 6b and R 7b Independently hydrogen, C 1-35 Alkyl groups or electron-donating groups (e.g., alkoxy groups); L C It has an electron-withdrawing linker (e.g., a carboxyl group) relative to the phenyl group; L Cb It is an electron-donating linker relative to the phenyl group (e.g., alkyl, alkoxy, alkylamino); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2–, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0027] Various assay methods can be utilized using the different groups of acridine-based markers described herein. For example, at least two chemiluminescent acridine-based markers with wavelength-separated chemiluminescence (e.g., acridine-based markers in the first group) can have similar chemiluminescence emission rates (e.g., the percentage of light measured at time points such as 1 second, 2 seconds, 4 seconds, or 6 seconds after triggering, relative to the total emission, is within 10%, 5%, or 1%). In some embodiments, at least two chemiluminescent acridine-based markers with time-domain-separated chemiluminescence (e.g., acridine-based markers in the second group) can have similar chemiluminescence wavelengths (e.g., within 10%, 5%, or 1%). max ).

[0028] Chemiluminescent labels are typically conjugated with a binding partner of one of the analytes of interest. Chemiluminescent labels can be formed from chemiluminescent compounds or salts having reactive functional groups for conjugation with the binding partner. For example, chemiluminescent labels may be selected from DMAE-Bz, 3-MeO-DMAE-Bz, DIPAE-Bz, ABAC, LEAE-Bz, DIP-LEAE-Bz, 2-MeO-LEAE-Bz, 3-EtO-LEAE-Bz, 3-QAE-LEAE-Bz, 2-QAE-LEAE-NHS, LEAC-Bz, NSP-LEAE-Bz, 2-MeO-NSE-LEAE-NHS, 2-Meo-LEAE-Imidate, 3-carboxybutadienyl-AE, p-carboxyethyl-AE, rhodamine-2-AM-DMAE-Bz, and rhodamine. Rhodamine-2-AM-DMAE-CO2H, Texas Red-2-AM-DMAE-CO2H, CNF-2-AM-DMAE-CO2H, Texas Red-3-AM-DMAE-CO2H, Rhodamine-3-AM-DMAE-β-alanine, Texas Red-3-AM-DMAE-β-alanine, Texas Red-ED-NCM-DMPAE, Texas Red-ED-NSP-DMPAE, Rhodamine-2-AM-DMAE-HD-theophylline, Texas Red-3-APO-DMAE-Bz, Texas Red-3-ABO-DMAE-Bz, DMAE-Bz, and 2-MeO-LEAE-Bz.

[0029] Compounds having the structure of formula (V) are also provided. Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, “k” can be 0 (e.g., all R3 groups are hydrogen), 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O)1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0030] Compounds for forming these conjugated markers are also provided. These compounds may have the structure of formula (VI): RFG is a reactive functional group used for conjugation to the analyte or its binding partner. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, “k” can be 0 (e.g., all R3 groups are hydrogen), 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C–Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(RN )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0031] For some assays, including positive and / or negative controls or calibrators in the test may be advantageous. Similarly, for some assay combinations, measuring the analytes in the same reaction vessel may be advantageous. For example, if an algorithm (like a ratio) is needed, such as for measuring placental growth factor (PLGF) and soluble FMS-like tyrosine kinase-1 (sFlt-1) to aid in the diagnosis or prognosis of preeclampsia, the variability of results can be reduced because the same sample is aliquoted and the processing error is identical for each analyte. Therefore, it is desirable to develop an assay method in which multiple analytes present in a sample can be detected or measured in a single test reaction. On the other hand, there are many diagnostic assays that are frequently tested as a group, such as thyroid assays, enhanced liver fibrosis (ELF™) assays, fertility hormone assays, cancer marker screening assays, and HIV antibody and antigen assays.

[0032] In another aspect of the invention, a reagent for detecting multiple analytes is provided, comprising a detectable conjugate bound to a chemiluminescent acridinium, wherein different detectable conjugates are bound to different chemiluminescent acridiniums having chemiluminescence separated in the wavelength and / or time domains (e.g., fast reaction kinetics, such as 100% chemiluminescence emission within 1, 2, or 4 seconds of triggering; slow reaction kinetics, such that 100% chemiluminescence emission does not occur until 1, 2, 4, or 6 seconds later). The detectable conjugate may contain one or more (e.g., one or two) zwitterionic functional groups. The reagent may contain each detectable conjugate at a concentration of 10 to 30 ng / mL. Reagents of this disclosure comprise compositions comprising the indicated components, and optionally excipients, carriers, or solvents. Reagents of this disclosure may include surfactants.

[0033] Compositions used in the methods of this disclosure, such as immunoassay reagents, may comprise at least three chemiluminescent acridiniums, wherein each chemiluminescent acridinium is conjugated to a different analyte or a different binding partner of an analyte, and the chemiluminescence of at least two chemiluminescent acridiniums is separated in the wavelength time domain, and the chemiluminescence of at least two chemiluminescent acridiniums from different groups is separated in the time domain. In some embodiments, the composition may comprise at least four chemiluminescent acridiniums conjugated to different analytes or binding partners of analytes, wherein the chemiluminescence of three chemiluminescent acridiniums is separated in the wavelength domain. In various embodiments, at least two chemiluminescent acridiniums with wavelength-separated chemiluminescence have similar chemiluminescence emission rates. In some embodiments, at least two chemiluminescent acridiniums with time-separated chemiluminescence have similar chemiluminescence wavelengths. In some embodiments, the composition comprises a compound having the structure of formula (V).

[0034] These and other aspects of the invention will be better understood by referring to the following detailed description, including the appended claims. Attached Figure Description

[0035] Figure 1 A [3x2] illustration of chemiluminescence spectra measured from samples of six different acridine-tagged compounds that were obtained almost simultaneously (e.g., as if from a single triggering event).

[0036] Figure 2A -J provides exemplary acridine trioxide (ATC) selections for the determination formats described herein. Figure 2A -H provides an example [2x2] measurement, and Figure 2I -J provides an example [3x2] measurement.

[0037] Figure 3A-J provides exemplary acridine trioxide (ATC) selections for the determination formats described herein. Figure 3A -H provides an example [2x2] measurement, and Figure 3I -J provides an example [3x2] measurement.

[0038] Figure 4 The results of comparative chemiluminescence kinetic measurements of fast and slow acridine ring-tagged acridine rings are shown.

[0039] Figure 5 The results show the results of comparative chemiluminescence kinetic measurements of fast and slow acridine-tagged compounds, if they are phenyl esters of acridine compounds, produced by an electron-donating group set at the 4-position. Detailed Implementation

[0040] For convenience, certain terms used in the specification (including embodiments and appended claims) are collected herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0041] Unless otherwise expressly defined, the following terms and phrases are intended to have the following meanings throughout this disclosure: Unless otherwise stated, all percentages given herein refer to the weight percentage of a particular component relative to the entire composition including the carrier (e.g., a percentage may refer to the total percentage of chemiluminescence, by which it implies the amount of chemiluminescence measured from the composition after triggering). It will be understood that the sum of all weight % of the individual components in the composition will not exceed 100%.

[0042] As used herein, the terms “a” or “an” refer to one or more. As used herein, the term “consistently of” is intended to limit the invention to the specified materials or steps and those that do not substantially affect the essential and novel features of the claimed invention, as understood from reading this specification. References to “comprising” include both “consistently of” and “comprises from”.

[0043] Unless otherwise specified, the following definitions of various groups or substituents shall be used. The specific and general values, as well as the ranges, listed below for groups and substituents are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within the scope of the definitions of groups and substituents. Unless otherwise specified, alkyl, alkenyl, alkynyl, alkoxy, etc., represent straight-chain, branched, and cyclic groups, and any combination thereof.

[0044] The term hydrocarbon can refer to a radical or group containing carbon and hydrogen atoms, which can be attached at the indicated positions (e.g., R, R', R'', R'', R''', R'''). N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7). Examples of hydrocarbon groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, and any combination thereof (e.g., alkyl-aryl-alkyl). As used herein, unless otherwise stated, hydrocarbons can be monovalent or polyvalent (e.g., divalent, trivalent) hydrocarbon groups. Form - (CH2) n A group consisting of - (including a methylene group, i.e., -CH2-) is considered an alkyl group if it does not have unsaturated bonds between carbon atoms. Unless otherwise stated, all hydrocarbon groups (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl) may have 1-35 carbon atoms. In other embodiments, the hydrocarbon will have 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. The hydrocarbon may have 2 to 70 atoms, 4 to 40 atoms, or 4 to 20 atoms.

[0045] The substituted hydrocarbon may have one or more hydrocarbon groups as substituents, the substituted hydrocarbon group may contain one or more heteroatoms. Any hydrocarbon substituents disclosed herein (e.g., R, R' ... N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2cR3, R4, R5, R6, R7 may optionally include 1-20 (e.g., 1-10, 1-5) heteroatoms. Examples of substituted hydrocarbon groups include, but are not limited to, heterocycles, such as heteroaryl groups. Unless otherwise stated, a hydrocarbon substituted with one or more heteroatoms will contain 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will contain 1-12, 1-8, 1-6, 1-4, 1-3, or 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorus, halogens (e.g., F, Cl, Br, I), boron, or silicon. In some embodiments, the heteroatom will be selected from oxygen, nitrogen, sulfur, phosphorus, and halogens (e.g., F, Cl, Br, I). In some embodiments, the heteroatom may be selected from O, N, or S. In some embodiments, the heteroatom or group may replace carbon. In some embodiments, the heteroatom or group may replace hydrogen. In some embodiments, the substituted hydrocarbon may contain one or more heteroatoms (e.g., inserted between two carbon atoms, as in "oxa") in the main chain or chain of the molecule. In some embodiments, the substituted hydrocarbon may contain one or more heteroatoms (e.g., covalently bonded to carbon atoms in the chain or main chain, as in "oxo") attached to the main chain or chain side of the molecule.

[0046] When the indicated group is replaced by the indicated substituent, the specified group can be replaced by one or more of any or all named substituents. For example, in the case of a group (e.g., an alkyl or heteroaryl group) being replaced by an unsubstituted C1-C... 20 In the case of alkyl or unsubstituted 2 to 20 heteroalkyl groups, the group may contain one or more unsubstituted C1-C groups. 20 Alkyl and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups. Furthermore, in cases where partially substituted by R substituents, the group may be referred to as "R-substituted". When partially R-substituted, the portion is substituted by at least one R substituent, and each R substituent is optionally different. If the indicated group is used multiple times in a chemical genus (e.g., R group), it will be understood that each group is chosen independently each time it appears.

[0047] Unless otherwise stated, any compound disclosed herein having one or more chiral centers may be in the form of a racemic mixture relative to each chiral center, or may exist as a pure or substantially pure (e.g., greater than 98% ee) R or S enantiomer relative to each chiral center, or may exist as a mixture of R and S enantiomers relative to each chiral center, wherein said mixture contains an enantiomer excess of one or the other configuration, for example, an enantiomer excess (R or S) greater than 60% or greater than 70%, or greater than 80% or greater than 90%, or greater than 95%, or greater than 98%, or greater than 99% enantiomer excess. In some embodiments, any chiral center may be in an "S" or "R" configuration.

[0048] It will be understood that the description of the compounds in this article is limited by the principles of chemical bonding. Therefore, where a group can be substituted by one or more of a plurality of substituents, such substitution is chosen to conform to the principles of chemical bonding, such as with respect to valence, and to give a compound that is not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms aligned with the four valence electrons of carbon.

[0049] Substituent (group) prefixes may be derived from the parent hydride by: (i) replacing "ane" in the parent hydride with the suffix "yl", "diyl", "triyl", or "tetrayl"; or (ii) replacing "e" in the parent hydride with the suffix "yl", "diyl", "triyl", or "tetrayl" (here, the number of atoms(one or more) with free valences is assigned as low as possible, consistent with any given number of the parent hydride). Recognized abbreviated names, such as adamantyl, naphthyl, anthraceneyl, phenanthryl, furyl, pyridyl, isoquinolinyl, quinolinyl, and piperidinyl, as well as common names, such as vinyl, allyl, phenyl, and thiophene, are also used throughout this document.

[0050] Alkyl groups typically refer to saturated hydrocarbon chains, which can be straight or branched chains containing the indicated number of carbon atoms. For example, C1-C6 alkyl indicates that the group may have 1 to 6 carbon atoms. Any atom may optionally be substituted, for example, by one or more substituents. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Any alkyl group mentioned herein (e.g., R, R' ... N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R2c The alkyl groups (R1, R2, R3, R4, R5, R6, R7) may have 1-35 carbon atoms. In other embodiments, the alkyl groups will have 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. The alkyl groups may be lower alkyl groups (e.g., C1-C4 alkyl groups).

[0051] A haloalkyl group is typically an alkyl group in which at least one hydrogen atom is substituted with a halogen. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) is substituted with a halogen. In these embodiments, the hydrogen atoms may each be substituted with the same halogen (e.g., fluorine), or the hydrogen atoms may be substituted with a combination of different halogens (e.g., fluorine and chlorine). The haloalkyl group may include an alkyl moiety in which all hydrogen atoms have been substituted with a halogen (sometimes referred to herein as a perhaloalkyl group, such as a perfluoroalkyl group, e.g., trifluoromethyl). The haloalkyl group may optionally be substituted.

[0052] Typically, alkoxy groups have the formula -O (alkyl). Alkoxy groups can be, for example, methoxy (-OCH3), ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or hexoxy. Similarly, the term "thioalkoxy" refers to a group of the formula -S (alkyl). Finally, the terms "haloalkoxy" and "halothioalkoxy" refer to -O (haloalkyl) and -S (haloalkyl), respectively. The term "sulfhydryl" refers to -SH. As used herein, the term "hydroxyl," used alone or in combination with other terms, refers to a group of the formula -OH. Any alkoxy, thioalkoxy, or haloalkoxy groups mentioned herein (e.g., R, R', R"), N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, and R7 may have 1-35 carbon atoms. In other embodiments, the alkoxy, thioalkoxy, or haloalkoxy group will have 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. The alkoxy group may be a lower alkoxy group (e.g., C1-C4 alkoxy).

[0053] An aralkyl group generally refers to a group in which the alkyl hydrogen atom is replaced by an aryl group. One of the carbon atoms of the alkyl moiety serves as the linking point between the aralkyl group and the other moiety. Any ring or chain atom may optionally be replaced by, for example, one or more substituents. Non-limiting examples of aralkyl groups include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.

[0054] The term "alkenyl" can refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom may optionally be substituted, for example, by one or more substituents. Alkenyl groups may include, for example, vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double-bonded carbons may optionally be the linking point of the alkenyl substituent. Any alkenyl group mentioned herein (e.g., R, R' ... N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7) may have 1-35 carbon atoms. In other embodiments, the alkenyl group will have 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.

[0055] The term alkynyl can refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alynyl groups (e.g., R, R' ... N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7) may optionally be substituted by, for example, one or more substituents. The alkynyl group may include, for example, ethynyl, propynyl, and 3-hexynyl. One of the carbon atoms in the triple bond may optionally be the linking point of the alkynyl substituent.

[0056] The term heterocyclic group generally refers to a fully saturated, partially saturated, or aromatic monocyclic, bicyclic, tricyclic, or other polycyclic cyclic system having one or more independently selected from O and N (it should be understood that one or two additional groups (e.g., R) may be present). N(To complete the nitrogen oxidation state and / or form a salt) or S constituent heteroatom ring atoms. The heteroatom or ring carbon can be the connection point between the heterocyclic substituent and another part. Any atom can optionally be replaced by, for example, one or more substituents (e.g., heteroatom or substituent group X). The heterocyclic group can include, for example, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidinyl), piperazineyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolylalkyl. As an example, the phrase "a heterocycle containing 5-6 ring atoms, wherein 1-2 ring atoms are independently selected from N, NH, N (C1-C6 alkyl), NC (O) (C1-C6 alkyl), O, and S; and wherein said heterocyclic ring is optionally replaced by 1-3 independently selected R" will include (but is not limited to) tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidinyl), piperazineyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolylalkyl.

[0057] The term heterocyclic alkenyl generally refers to a partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group having one or more (e.g., 1-4) heteroatom ring atoms independently selected from O, N (it should be understood that one or two additional groups may be present to complete the nitrogen oxidation state and / or form a salt), or S. The cyclic carbon (e.g., saturated or unsaturated) or heteroatom can be the connecting point for the heterocyclic alkenyl substituent. Any atom may optionally be substituted by, for example, one or more substituents. Heterocyclic alkenyl groups may include, for example, dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydro-pyrimidinyl, and 5,6-dihydro-2H-[1,3]oxazinyl.

[0058] The cycloalkyl group can be a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom can optionally be substituted, for example, by one or more substituents. The cyclic carbon serves as the linking point between the cycloalkyl group and another moiety. The cycloalkyl moiety can include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicyclic [2.2.1]heptyl).

[0059] The cycloalkenyl group can be a partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. The cyclic carbon (e.g., saturated or unsaturated) is the linking point of the cycloalkenyl substituent. Any atom can optionally be substituted, for example, by one or more substituents. The cycloalkenyl moiety can include, for example, cyclohexenyl, cyclohexadienyl, or norbornyl.

[0060] Aryl groups are typically aromatic monocyclic, bicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (> three fused rings) hydrocarbon ring systems. One or more ring atoms may optionally be substituted, for example, by one or more substituents. Aryl moieties include, for example, phenyl and naphthyl groups.

[0061] A heteroaryl group is typically an aromatic monocyclic, bicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (> three fused rings) hydrocarbon group having one or more heteroatomic ring atoms in the ring, said heteroatomic ring atoms being independently selected from O, N (understood to be present with one or two additional groups to complete the nitrification and / or form a salt), or S. One or more ring atoms may optionally be substituted, for example, by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrole, 3H-indolyl, 4H-quinazinyl, acridineyl, benzo[b]thiopheneyl, benzo[b]thiazolyl, β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cenolinyl, dibenzo[b,d]furanyl, furazanyl, furanyl, imidazolyl, imidizolyl, indazole, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, isothiazolyl, and others. Isoxazolyl, naphridinyl, oxazolyl, perimidinyl, phenanthidyl, phenarsazinyl, phenarsazinyl, phenazinyl, phenthiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purine, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrroleyl, quinazolinyl, quinolinyl, quinoxolinyl, thiadiazolyl, thiaanthryl, thiazolyl, thiophenyl, triazolyl, and xanthenyl.

[0062] Generally, when the limitation of a particular variable includes hydrogen and non-hydrogen (halogen, alkyl, aryl) possibilities, the term "one or more substituents other than hydrogen" is collectively referred to as the non-hydrogen possibilities of that particular variable, unless otherwise specified.

[0063] In general, any range of limits (endpoints) described herein is within the scope of this invention and should be understood as the disclosed embodiments. Furthermore, any half-integer values ​​within that range are also considered. For example, the range 0 to 4 explicitly discloses 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and any subset thereof (e.g., from 1 to 2.5).

[0064] The term "substituent" can refer to a group (e.g., alkyl, haloalkyl, cycloalkyl, heterocyclic, heterocyclic alkenyl, cycloalkenyl, aryl, or heteroaryl) that "substitutes" on a hydrocarbon, typically replacing one or more hydrogen atoms at any atom of the group. In one aspect, groups (e.g., R, R' ... N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2cThe substituents (one or more) on R1, R2, R3, R4, R5, R6, R7 are independently any single atom defined for that substituent or any combination of two or more of the permitted atoms or groups of atoms. In another aspect, the substituent itself can be substituted by any of the aforementioned substituents. In some embodiments, the indicated substituent is not further substituted. Furthermore, as used herein, the phrase "optionally substituted" means unsubstituted (e.g., substituted with H) or substituted. It is understood that substitution at a given atom is limited by valence. Common substituents include halogens (e.g., F), C... 1-12 Straight-chain or branched alkyl groups, C 2-12 alkenyl, C 2-12 alkynyl group, C 3-12 cycloalkyl, C 6-12 Aryl, C 3-12 heteroaryl, C 3-12 Heterocyclic group, C 1-12 Alkylsulfonyl, nitro, cyano, –COOR, –C(O)NRR', –OR, –SR, –NRR', and oxo, for example, partially (e.g., trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furanyl, triazolyl, piperazine, pyrazolyl, imidazolyl, etc.) monosubstituted, disubstituted, or trisubstituted, each optionally containing one or more heteroatoms, such as halogen, N, O, S, and P. R and R' are independently hydrogen, C 1-12 Alkyl, C 1-12 Haloalkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 3-12 cycloalkyl, C 4-24 cycloalkylalkyl, C 6-12 Aryl, C 7-24 Aryl alkyl, C 3-12 Heterocyclic group, C 3-24 Heterocyclic alkyl, C 3-12 heteroaryl or C 4-24 Heteroarylalkyl. Unless otherwise stated, all groups described herein optionally contain one or more common substituents to the extent permitted by valence. The term “substituted” generally means that a hydrogen and / or carbon atom has been removed and replaced by a substituent (e.g., a common substituent). When a substituent (group) prefix name (e.g., alkyl) is used without the modifiers “optionally substituted” or “substituted”, it should be understood to mean that the particular substituent is unsubstituted. However, the use of “haloalkyl” without the modifiers “optionally substituted” or “substituted” should still be understood to mean an alkyl group in which at least one hydrogen atom is replaced by a halogen and any other relevant substituent as required. Any hydrocarbon described herein may be considered optionally substituted.

[0065] When a portion of the compounds disclosed herein is described as an analyte or its binding partner, it will be understood that a covalent link is formed with the analyte or its binding partner (e.g., using a reactive functional group that forms a covalent link), for example by replacing the hydrogen on the unconjugated analyte or its binding partner with a covalent bond linked to the indicated portion. The covalent link on the analyte or its binding partner can be formed, for example, at a group on the analyte, its binding partner, or an analyte derivative containing a group for forming the link. This group can be, for example, an amine group, a thiohydroxy group, a carboxyl group, a maleimide group, or a carbohydrate group. For example, if the covalent link is formed by a primary amine of the analyte or its binding partner, the compound can have the following structure: The unconjugated analyte or conjugated partner A has the structure A'–NH2.

[0066] In some embodiments, any hydrocarbon or substituted hydrocarbon disclosed herein (e.g., R, R', R'', R'', R''', R''') N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7 can be substituted by one or more (e.g., 1-6, 1-4, 1-3, one, two, or three) substituents X, wherein X is independently selected each time it appears from one or more (e.g., 1-20) heteroatoms or one or more (e.g., 1-10) heteroatom-containing groups, or X is independently selected each time it appears from –F, –Cl, –Br, –I, –OH, –OR*, –NH2, –NHR*, –N(R*)2, –N(R*)3 + , –N(R*)–OH, –N(→O)(R*)2, –O–N(R*)2, –N(R*)–O–R*, –N(R*)–N(R*)2, –C=N–R*, –N=C(R*)2, –C=N–N(R*)2, –C(=NR*)( –N(R*)2), –C(H)(=N–OH), –SH, –SR*, –CN, –NC, –CHF2, –CCl3, –CF2Cl, –CFCl2, –C(=O)–R*, –CHO, –CO2H, –C(O)CH3, –CO2 -, –CO2R*, –C(=O)–S–R*, –O–(C=O)–H, –O–(C=O)–R*, –S–C(=O)–R*, –(C=O)–NH2, –C(=O)–N( R*)2, –C(=O)–NHNH2, –O–C(=O)–NHNH2, –C(=S)–NH2, –(C=S)–N(R*)2, –N(R*)–CHO, –N(R*) –C(=O)–R*, –C(=NR)–OR*, –O–C(=NR*)–R*, –SCN, –NCS, –NSO, –SSR*, –N(R*)–C(=O)–N(R*) 2. –CH3, –CH2–CH3, –CH2–CH2–CH3, –C(H)(CH2)2, –C(CH3)3, –N(R*)–C(=S)–N(R*)2, –S(=O) 1-2 –R*, –O–S(=O)2–R*, –S(=O)2–OR*, –N(R*)–S(=O)2–R*, –S(=O)2–N(R*)2, –O–SO3, –O–S(=O)2–OR*, –O–S(=O)–OR*, –O–S(=O)–R*, –S (=O)–OR*, –S(=O)–R*, –NO, –NO2, –NO3, –O–NO, –O–NO2, –N3, –N2–R*, –N(C2H4), –Si(R*)3, –CF3, –O–CF3, –O–CHF2, –O–CH3, –O–(CH2) 1-6 CH3, –OC(H)(CH2)2–OC(CH3)3, –PR*2, –O–P(=O)(OR*)2 or –P(=O)(OR*)2; wherein, independently, R* can be H or C in each occurrence. 1-10 Or C 1-8 Or C 1-6 Or C 1-4Hydrocarbons, including but not limited to alkyl, alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), and aryl-alkyl (e.g., tolyl). In some embodiments, X may comprise a C1-C8, C1-C6, or C2-C4 perfluoroalkyl group. In some embodiments, X may be a C1-C8, C2-C6, or C3-C5 heterocyclic ring (e.g., a heteroaryl group). The term "halo" or "halogen" refers to any group of fluorine, chlorine, bromine, or iodine. In some embodiments, X is independently selected each time it appears from –OH, –SH, –NH2, –N(R*)2, –C(O)OR*, –C(O)NR*R*, –C(O)NR*R*, –C(O)OH, –C(O)NH2, F, or –Cl. In some embodiments, X is F. R and R* may be independently saturated or unsaturated alkyl groups (e.g., C1-C8 alkyl groups) each time they appear. In some embodiments, R and R* are independently selected from hydrogen, methyl, ethyl, propyl, or isopropyl. In some embodiments, R and R* are independently selected from hydrogen, methoxy, ethoxy, propoxy, or isopropoxy. In some embodiments, X is –CF3 or –O–CF3.

[0067] L C It can have the following structure: –(X1) 0-1 –(R L ) 0-5 –(X2) 0-1 –(R L ) 0-5 –(X3) 0-1 –(R L ) 0-5 –(X4) 0-1 –(R L ) 0-5 – Where X1 is selected from =N–, –O–, –S– or –NR. N –; X2–X4 are independently selected from –O–, –S–, and –NR. N –、–C(O)–、–NR N –C(O)–、–C(O)–NR N –、–O–C(O)– or –C(O)–O–、–S–C(O)–、 or –C(O)–S–; and R L Each time it appears, it is independently selected from -CH2-, -(CH2CH2O)-, or -(OCH2CH2)-. In various embodiments, L C The chain between A and Ψ (or between A and Z) contains at least one atom (or at least two atoms).

[0068] Anionic groups, such as X a and X b It can, for example, be independently a carboxylate ion (–C(O)O) each time it appears. - ), sulfonate (–S) ), sulfate (–OS) ), phosphate (–OP(O)(OR) P )O - ) or oxygen ions (–O - ), and R P It is hydrogen or C 1-12 Hydrocarbons, optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents). For example, R1 may contain (or) –R L –S In some embodiments, R1 comprises (or) a sulfopropyl group. In some embodiments, R1 is –S(O)2–NH–Z or –(CH2). 1-3 –S(O)2–NH–Z. In various embodiments, R2 and R3 are independently hydrogen, alkyl, or alkoxy (e.g., lower alkoxy groups such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy) each time they appear. In some embodiments, R2 and R3 are each hydrogen. In other embodiments, one of R2 or R3 is hydrogen, and the other of R2 or R3 is an alkoxy group (e.g., lower alkoxy groups such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, X a Sulfonate (–S) ), m is 1, R L It is propyl, and both n and p are 3. For example, Z L It can have the following structure: .

[0069] Compounds can be used to detect the presence of substances (e.g., analytes, such as biomolecules) in a sample. In some embodiments, the analyte is a thyroid hormone (e.g., thyroid-stimulating hormone, and, for example, A is its binding partner, such as anti-thyroid-stimulating hormone monoclonal antibody (AntiTSH-mAb)), androgens, steroid hormones (e.g., androstenedione, testosterone), troponin, thyroglobulin, anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine-binding globulin (TBG), neurofilament light chains (e.g., serum neurofilament light chains), vitamins (e.g., vitamin D, such as 25-hydroxyvitamin D), or viral (e.g., hepatitis) antibodies.

[0070] Compounds for forming conjugates are also provided. For example, such compounds (e.g., compounds for conjugating with an analyte or a binding pair of an analyte (e.g., a peptide, protein, or a macromolecule including an antibody)) may have the structure of formula (VI): RFG is a reactive functional group used for conjugation to the analyte or its binding partner. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, “k” can be 0 (e.g., all R3 groups are hydrogen), 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1-‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates). For example, reactive functional groups (RFGs) can be selected from: –Cl, –Br, –I, or –COOH.

[0071] In some embodiments, the compounds disclosed herein may be zwitterionic and comprise one or more zwitterionic groups. For example, the R1 group of the positively charged nitrogen atom attached to acridine nitrogen may optionally be substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F), and thus may combine with the positively charged acridine nitrogen atom to form a zwitterionic group. For example, the sulfopropyl or sulfobutyl group attached to acridine nitrogen may form a zwitterionic pair. The R1 group may also be neutral (e.g., a lower alkyl group, such as methyl) or zwitterionic itself (e.g., R1 is –Z, –R). L –Z, –L8–Z, or –R L –L8–R M –Z). In some implementations, R1 has the following structure: .

[0072] When the acridineonium label is charged (e.g., R1 has a net neutral charge), the compound can be in its salt form and optionally include a counterion to balance the positively charged nitrogen of the acridineonium nucleus. The counterion can be selected from CH3SO4. - FSO3 - CF3SO4 - C4F9SO4 - CH3C6H4SO3 - halogens (e.g., Cl-) - F - ,Br - ), CF3COO - CH3COO - Or NO3 - In some embodiments, R1 is methyl, ethyl, propyl, or isopropyl. In some embodiments, the acridine compound can be zwitterionic by covalently linking to an anion. For example, R1 may contain –R L –X a And –X a Sulfonate (–S) In some implementations, R1 is –R L –X a And –X a Sulfonate (–S) In some implementations, R1 is –R L –X or –L8–Z. In some implementations, L8 is –S(O)2–NH– or –(CH2). 1-3 –S(O)2–NH–. R1 can contain a sulfopropyl group (–(CH2)3–S In the specific implementation scheme, R1 is sulfopropyl.

[0073] Substituents on chemiluminescent acridine esters can be modified to alter the rate and yield of light emission, thereby reducing nonspecific binding, increasing stability, or increasing hydrophilicity. Typically, these modifications will have minimal interference with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed in U.S. Patent No. 7,309,615, which is hereby incorporated by reference, by describing acridine ester compounds with high quantum yields, for example, containing an electron-donating group such as an alkoxy group (OR*) at C2 and / or C7, wherein R* is a sulfopropyl moiety or an ethylene glycol moiety (e.g., -O(CH2CH2O)). 0-5 CH3) or a combination thereof. In some embodiments, R2 (e.g., R 2a R 2b R 2c R3 and / or R3 can be hydrogen, an electron-donating group such as an alkoxy group (e.g., OR, e.g., -O(CH2CH2O)) each time it appears. 0-5 CH3 and / or OR*). Natrajan et al. also described hydrophilic, high-quantum-yield, chemiluminescent acridine esters in International Publication No. WO2015 / 006174 (which is hereby incorporated herein by reference in its entirety), which also possess certain electron-donating functional groups at the C2 and / or C7 positions. These electron-donating groups (-OG) can have the following structures: Among them, R9-R 14 Each time it appears, the methyl group or the group –(CH2CH2O) is chosen independently. a CH3, where a is an integer from 1 to 5.

[0074] Ψ can include two side-attached methyl groups on the phenolic ester to stabilize the bond, as described by Law et al. Journalof Bioluminescence and Chemiluminescence The information disclosed in 4:88-89 (1989) is hereby incorporated herein by reference in its entirety. The group of chemiluminescent labels can be modified in a manner that achieves appropriate separation of chemiluminescence wavelengths or emission. In some embodiments, Ψ (in the group of one or more chemiluminescent labels) has the following structure: .

[0075] G can be independently selected each time it appears, for example, from hydrogen, alkyl (e.g., C1-C4 alkyl), or -(CH2CH2O). 1-10 -OCH3, such as -(CH2CH2O)2-OCH3 or -(CH2CH2O)5-OCH3, and branched groups having the following structures: Among them, R9-R 14 Each time it appears, it is independently selected from a methyl group or the -(CH2CH2O) group. a CH3, where a is an integer from 1 to 5.

[0076] In some embodiments, A, L, and Ψ are each covalently linked. The covalent link between A and Ψ can be formed by a reactive functional group for forming a covalent link with a peptide, protein, or macromolecule, wherein the functional group includes an electrophilic group, a nucleophilic group, or a photoreactive group. The reactive functional group can be an amine reactive group, a thiol reactive group, a carboxyl reactive group, a maleimide reactive group, or a carbohydrate reactive group. In some embodiments, the reactive functional group can therefore react with the functional group (e.g., a primary amine) of the analyte or binding partner. The reactive functional group can comprise (or) isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, acetaldehyde, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imine esters, carbodiimides, acid anhydrides, fluorophenyl esters, or combinations thereof. In various embodiments, the reactive functional group is therefore labeled with the analyte or binding partner by acylation or alkylation. For example, this connection can be formed from reactive groups selected from the following: .

[0077] In some embodiments, the compound comprises a linker group having the structure -NH-C(O)- or -C(O)-NH-. In a preferred embodiment, the compound or a portion thereof (e.g., L...) C Ψ) contains at least one -NH-C(O)- or -C(O)-NH- linker group.

[0078] Covalent connections between A and Ψ (e.g., L) or between RFG and Ψ (e.g., L) may contain (or be) divalent C. 1-20Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br). In some embodiments, L comprises a zwitterion linker. L may have the structure –L C –(Z L ) z – where z is 0 or 1. L C It can have the following structure –(X1) 0-1 –(R L ) 0-5 –(X2) 0-1 –(R L ) 0-5 –(X3) 0-1 –(R L ) 0-5 –(X4) 0-1 –(R L ) 0-5 – X1 is selected from –O–, –S–, and –NR. N –、–C(O)–、–NR N –C(O)–、–C(O)–NR N –、–O–C(O)– or –C(O)–O–、–S–C(O)– or –C(O)–S–、=N–、–O–、 or –S–; X2–X4 are independently selected from –O–, –S–, and –NR. N –、–C(O)–、–NR N –C(O)–、–C(O)–NR N –、–O–C(O)–、or –C(O)–O–、–S–C(O)–、or –C(O)–S–;and R L Each occurrence is independently selected from ––CH2–, –(CH2CH2O)–, or –(OCH2CH2)–; where, for example, the condition is L C Between A and Ψ (or A and Z) L The chain (between) contains at least one atom (or at least two atoms).

[0079] In some embodiments, L and / or Ψ comprise -C(O)-NH-. In some embodiments, L C It has the following structure: .

[0080] The detectable marker may comprise a dimethyl acridine onium ester (DMAE) moiety and a zwitterion linker, which may contain a zwitterion linker or a polyethylene glycol-derived linker to improve the properties of the compound. When Ψ comprises a zwitterion linker, a polyethylene glycol-derived linker, or a dimethyl phenyl ester, properties such as nonspecific binding, hydrophilicity, or compound stability may be improved. In some embodiments, Z L It has the following structure: .

[0081] In several embodiments, R' is hydrogen or a lower alkyl group (e.g., methyl, ethyl, propyl).

[0082] Exemplary compounds for forming conjugates are disclosed in Table 1. In some embodiments, detectable conjugates are formed by reacting a compound (e.g., a compound of formula (V), a compound from Table 1, or a compound from Table 1 with a different reactive functional group (RFG) or -L-RFG replacing, for example, a benzyl ester or an N-hydroxysuccinimide (NHS) ester) with an analyte, its binding partner, or a derivative thereof capable of reacting with the reactive functional group. Suitable groups of acridine ring labels can be prepared, for example, by alkylating the 1,3 positions of the acridine ring system, attaching a fluorophore to the 2 or 3 position of the acridine ring system, setting an electron-donating group (e.g., -OG) on the acridine ring system (e.g., at the 2 and / or 6 positions), and attaching an electron-donating group (e.g., -OG, -O-, -(CH2)). 0-5 CH3, -NH-) are set on the phenyl ester (e.g., at the 2 and / or 6 positions, such as with -OG, and at the 4 position, such as with a divalent electron-donating group (e.g., -O-, -(CH2)) conjugated with the linker). 0-5 CH3, -NH-)), converting the acridine ring system into a four-membered acridine ring system, or a combination thereof. The compound may be an acridine ester (“AE”). The compound name may include “Z” which may refer to a zwitterionic linker, “CMO” which may refer to a carboxymethyl oxime linker, “CME” which may refer to a carboxymethyl ether linker, “CETE” which may refer to a carboxyethyl sulfide, “ZAE” which may refer to a zwitterionic acridine ester (typically N-sulfopropyl (“NSP”) dimethyl acridine ester (“NSP-DMAE”) in the illustrated examples), and may refer to the acridine core with an isopropoxy functional group attached thereto and the complete zwitterionic group (containing N) attached to the positive N of the acridine ring. + and X - "ISODIZAE" (both)

[0083] In some implementations, the detectable conjugate may have one or more of the following structures: Where z is 0 or 1 independently each time it appears; y is independently 0, 1, 2, 3, 4, or 5 each time it appears; and A' is an analyte or its binding partner that is conjugated to a primary amine of the unconjugated analyte or its binding partner A; Where X1 is selected from –O–, –S–, –NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)– or –C(O)–O–, –S–C(O)– or –C(O)–S–, =N–, –O–, or –S–; X2-X4 are independently selected from -O-, -S-, and –NR. N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; and R L It is selected independently from -(CH2) each time it appears. 1-5 -、-(CH2CH2O) 1-5 -or-(OCH2CH2) 1-5 -

[0084] In the wavelength domain, signals from different emission amplification sources (AEs) with distinguishable emission wavelengths can be measured individually using appropriate selection of detectors and optical filters. In the time-domain detection domain, signals from different AEs with distinguishable emission dynamics can be measured at different time ranges. In both the wavelength and time domains of the combined measurement, the detection or measurement method of the present invention consists of "two-dimensional detection" or 2D detection.

[0085] like Figure 1 As shown, a group of six acridine esters can be grouped into [3x2] mixtures. The number "3" in the bracketed designation indicates three AEs with distinguishable emission maxima that can be measured in the wavelength domain within each wavelength-separated group. The number "2" indicates two AEs with the same emission maxima but different emission kinetics (slow and fast), which can be measured in the time domain within each time-domain-separated group. Figure 1 The diagram illustrates two groups of wavelength-separated acridine-onium markers (Group 1 includes AE1, AE2, and AE3, and Group 2 includes AE3, AE4, and AE5). As can be seen, each group has similar emission dynamics (e.g., slow emission) but is separated in the wavelength domain, exhibiting minimum peak separation, such as 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 100 nm (e.g., 30 nm to 400 nm, 40 nm to 400 nm, 50 nm to 400 nm, 60 nm to 400 nm, 70 nm to 400 nm, 80 nm to 400 nm, 100 nm to 400 nm). Furthermore, three groups of acridine-onium markers with different emission separation but similar wavelengths (e.g., λmax differences less than 30 nm) are used (AE1 and AE4; AE2 and AE5; AE3 and AE6). By selecting acridine-2-tagged markers in this manner, all signals in a reaction can be measured individually in a two-dimensional detection scheme (the two dimensions being wavelength and emission velocity), such as the six signals shown here. As can be seen, acridine-2-tagged markers that are members of one group (e.g., a group of wavelength-separated acridine-2-tagged markers) can also be members of another group (e.g., a group of emission-separated acridine-2-tagged markers). In some embodiments, the system of the present invention utilizes an [m x n] detection scheme, where m and n are each independently integers greater than 2 (e.g., 2, 3, 4, 5), such as [2x2], [3x2], [4x2], [2x3], and [3x3] detection forms. In some embodiments, one or more wavelength-separated acridine-2-tagged markers are not members of a kinetic separation group. In some embodiments, one or more dynamic separation markers are not members of a wavelength separation group.

[0086] Figure 2A-J provides exemplary selections of acridinium-tagged compounds for detecting multiple analytes, presented in matrix form. These figures illustrate relative variations in the selection of acridinium-tagged compounds. Typically, compounds in the same horizontal row have different emission maxima (e.g., λmax1-λmax2 > 30 nm), but compounds in the same vertical row have different reaction kinetics (e.g., fast kinetics, slow kinetics) and different reaction kinetics (e.g., fast kinetics and slow kinetics). In some embodiments, compounds in each vertical row have different reaction kinetics (e.g., fast kinetics and slow kinetics) and similar emission maxima (e.g., λmax1-λmax2 < 30 nm or λmax1-λmax2 < 20 nm or λmax1-λmax2 < 10 nm). These compounds can be used to detect various analytes (e.g., A1, A2, A3, A4, A5, A6) or their binding pairs. In these figures, R1, G1, G2, L, L... C IA and RFG are as described herein. In a particular embodiment, R1 is NSP or methyl (and the compound is a salt).

[0087] exist Figure 2E-2J The present invention provides compounds having reactive functional groups for forming conjugates with analytes or their binding partners (e.g., A, A1, A2, A3, A4, A5, A6). When used for assay, these acridine-labeled compounds are typically conjugated with appropriate conjugates for detection. In some embodiments, each variable (e.g., R1, G1, G2, L, L...) is... C R1 is the same in the various acridine-labeled compounds used in the assay. In some embodiments, particularly those involving imaging agents (IA) such as rhodamine and Texas red, R1 can vary in the acridine-labeled compounds. For example, an acridine-labeled compound without an imaging agent can have zwitterionic acridine (e.g., R1 is –X). a Or –R L –X a (e.g., NSP), and acridine-onium labels with developers are in salt form (e.g., R1 is an alkyl group, such as C1-C4 alkyl). Figure 3A -J provides specific acridine-onium markers that can be conjugated with analytes or their binding partners (e.g., A1, A2, A3, A4, A5, A6) in various [2x2] and [3x2] analytical formats. In various embodiments, A1-A6 are each different analytes or their binding partners.

[0088] Acridine-onium labels can also be characterized by the wavelength of their chemiluminescent emission. For example, some acridine-onium labels may have a maximum emission wavelength (λmax) of 430 nm to 460 nm, 460 nm to 490 nm, 490 nm to 520 nm, 520 nm to 550 nm, 550 nm to 580 nm, 580 nm to 610 nm, 610 nm to 640 nm, 640 nm to 670 nm, 670 nm to 700 nm, 700 nm to 730 nm, 730 nm to 760 nm, 760 nm to 790 nm, 790 nm to 820 nm, or 820 nm to 850 nm. For example, in some embodiments, a compound in the wavelength separation group has a λmax of 400 nm to 500 nm (e.g., Figure 2A Compounds in the first column of -J and 3A-J), and another compound in the wavelength separation group has a λmax of 500-600 nm (e.g., Figure 2A Compounds in the second or third column of -J and 3A-J). In some embodiments, a compound in the wavelength separation group has a λmax of 400 nm to 500 nm (e.g., Figure 2A Compounds in the first column of -J and 3A-J), and another compound in the wavelength separation group has a λmax of 600-700 nm (e.g., Figure 2A Compounds in the second or third column of -J and 3A-J). In some embodiments, a compound in the wavelength separation group has a λmax of 400 nm to 500 nm (e.g., Figure 2A Compounds in the first column of -J and 3A-J), and another compound in the wavelength separation group has a λmax of 700-800 nm (e.g., Figure 2A Compounds in the second or third column of -J and 3A-J). In some embodiments, a compound in the wavelength separation group has a λmax of 500 nm to 600 nm (e.g., Figure 2A Compounds in the first column of -J and 3A-J), and another compound in the wavelength separation set has a λmax of 600-700 nm (e.g., Figure 2A Compounds in the second or third column of -J and 3A-J). In some embodiments, a compound in the wavelength separation group has a λmax of 500 nm to 600 nm (e.g., Figure 2A Compounds in the first column of -J and 3A-J), and another compound in the wavelength separation group has a λmax of 700-800 nm (e.g., Figure 2A Compounds in the second or third column of -J and 3A-J). In some embodiments, a compound in the wavelength separation group has a λmax of 600 nm to 700 nm (e.g., Figure 2ACompounds in the first column of -J and 3A-J), and another compound in the wavelength separation group has a λmax of 700-800 nm (e.g., Figure 2A (Compounds in the second or third column of -J and 3A-J).

[0089] Chemiluminescence from multiple acridine esters with different emission wavelengths can be measured using multiple photomultiplier tubes (PMTs), each equipped with an optical filter that allows light from the acridine ester of interest to pass through while blocking unwanted light from other acridine esters. In some embodiments, a single PMT can be used in conjunction with a filter wheel mounted in front of the PMT's detection window. The filter wheel can be equipped with multiple filters corresponding to, for example, the number of acridine esters used for chemiluminescence, where each filter allows light from the acridine ester of interest to pass through while blocking unwanted light from other acridine esters. Another alternative detector is a charge-coupled device (CCD), where pixels can be grouped into segments of a two-dimensional detector, each segment corresponding to the number of acridine esters to be detected. Chemiluminescence can pass through a grating, allowing wavelength separation to occur along the detector (e.g., a CCD detector), and the image can be analyzed accordingly. Each group segment of the pixel can have a specific optical filter that allows light from the acrylonitrile ester of interest to pass through while blocking unwanted light from other emission sources (AEs). Other types of detection systems can be used for the detection or measurement of light from AEs with different emission distributions.

[0090] The presence of overlapping signals due to the emission wavelengths of acridine esters can be minimized by appropriately selecting optical filters (such as long-pass and short-pass filters for detecting two AEs and band-pass filters for detecting three or more AEs). The residual overlapping signal after filtering can be deconvolved by an algorithm or artificial intelligence (AI) that measures the emission wavelength distribution of individual AEs at multiple different wavelengths. For example, the emission wavelength distribution of acridine ester markers with similar emission kinetics can be adapted to the sum of typical chemiluminescence distributions (e.g., normal, Gaussian, Poisson, or combinations thereof) to identify the peaks and widths contributed by each individual acridine ester marker. The signal of the acridine ester marker can be determined by subtracting the overlapping signal due to stray light from the unwanted signal, which may include noise and signals from other acridine ester markers present in the system. Differentiation can be further improved by utilizing different emission kinetics and measuring the emission wavelength distribution of individual AEs at multiple wavelengths at different time points. Algorithms or AIs trained on datasets of acridine-onium markers from various groups can further improve the differentiation of overlapping signals through multi-wavelength detection and the application of different dynamic variations. The multi-analyte detection schemes disclosed herein offer increased sensitivity and a wider range of analyte measurements.

[0091] An important consideration is that acridine-tagged markers possess sufficiently dissociated emission kinetics. Individual light measurements can be performed at different time points and durations following chemiluminescence triggering to provide distinct measurements between kinetically separated groups. This can be accomplished by turning detectors on and off at specific times, or by collecting light at small intervals (binning) and appropriately grouping and binning it over suitable time widths to allow for precise light measurements over specific time ranges. For example, in a group of emission-separated acridine-based markers, where most of the emission of one acridine-based marker occurs in one time domain (e.g., fast emission, such as greater than 90%, greater than 95%, greater than 98%, greater than 100% occurring within three seconds of triggering, or within two seconds of triggering, or within one second of triggering), and most of the emission of another acridine-based marker (e.g., slow emission, such as greater than 90%, greater than 95%, greater than 98%, greater than 100% occurring within 120 seconds of triggering, or within 60 seconds of triggering, or within 15 seconds of triggering, or within 10 seconds of triggering), bins can be selected to collect light from one marker in some bins and collect light from another marker in other bins. The measured intensity of each binned bin (or appropriately grouped bins) can be analyzed individually to quantify the corresponding analyte.

[0092] Faster acridine-based labels typically complete emission within 5 seconds of chemiluminescence triggering. In some embodiments, the fast acridine-based labels of this disclosure can exhibit faster photoemission kinetics compared to other acridine-based compounds, e.g., emitting at least 90% of their light within 2 seconds, measured over 5 seconds. Slower acridine-based labels can complete emission within 120 seconds of chemiluminescence triggering (and have a negligible light contribution during the triggering period of the faster labels). For example, slower acridine-based labels can complete less than 30%, less than 20%, or less than 10% of their emission within 5 seconds or 2 seconds, and emit at least 90% of their light as measured over 120 seconds, 60 seconds, or 30 seconds.

[0093] For example, the acridine-onium marker of U.S. Patent No. 8,119,422 (which is hereby incorporated herein by reference in its entirety) can be used as a material for forming a group of acridine-onium markers for wavelength separation and emission separation. Table 2 provides the percentage of relative optical units (RLUs) of various acridine-onium markers measured at 0.5 s, 1.0 s, 2.0 s, 5.0 s, and 10 s, similar to those described in the examples.

[0094] The structures of the compounds in Table 2 are as follows: It will be understood that any inconsistencies between the structures and compounds in U.S. Patent No. 8,119,422, both compounds, will be considered acceptable under this disclosure. The use of these acridine-onium labels coupled with the wavelength and kinetic differences provided herein allows for the creation of various assays capable of detecting multiple analytes. For example, each of these acridine-onium labels can be used in… Figure 2A -J and Figure 3A The assay forms described in -J. These compounds can be integrated into various assay forms of this disclosure by utilizing the differences between acridine ring conjugations and phenyl ester conjugations, as well as the related conjugations described herein (e.g., dialkyl substitution of the acridine ring (e.g., 1,3-methyl substitution of the acridine ring), and the arrangement of electron-donating groups at the 4' position of the phenyl group).

[0095] If an acridine ester label has relatively close emission kinetics that do not allow for clear or “sufficient” separation of chemiluminescent signals, then superposition of signals from different acridine esters can exist over a time span. In this case, the overlapping signals can be deconvolved using algorithms or artificial intelligence (AI) that measure the emission kinetics distribution of individual AEs at multiple different time points and obtain the signal of the acridine ester of interest by subtracting the overlapping signal due to stray light from the unwanted signal. Differentiation can be further improved by utilizing different emission wavelengths and measuring the emission kinetics of individual AEs at multiple time points at different wavelengths. Algorithms or AI, combined with different emission wavelengths or spectral variations and kinetic measurements (measured at different time points), can further improve the differentiation of overlapping signals.

[0096] The compounds disclosed herein can be characterized by their stability. Acridine-labeled compounds used in the assays disclosed herein, such as fast acridine-labeled compounds and / or slow acridine-labeled compounds, can be characterized as stable. For example, a compound can be considered stable if it exhibits minimal loss of chemiluminescent activity, measured by the loss of relative light units (“RLUs”), when typically stored in aqueous solutions in a pH range of 6–9. Compounds exhibiting increased instability compared to other compounds may have a greater loss of chemiluminescent activity. For example, compounds of this disclosure (e.g., compounds having the structure of formulas (I)–(VI)) can be characterized as exhibiting increased stability for more than 33 days at pH 6 and / or 7 and / or 8 at 4 °C (common reagent storage temperature) and / or 37 °C (acceleration temperature). These compounds may exhibit increased stability compared to other identical compounds that do not have fused heterocycles conjugated with an acridine system. In some embodiments, the compound may be characterized in that the change in chemiluminescent activity after storage at 37°C and pH 7 and / or pH 8 for 33 days is less than (or from 1% to) 40% (e.g., less than 30%, less than 20%, from 10% to 40%, from 10% to 30%, from 10% to 20%).

[0097] This compound can be prepared from commercially available starting materials, compounds known in the literature, or readily prepared intermediates using standard synthetic methods (other than those provided herein). Standard synthetic methods and procedures for the preparation of organic molecules and the transformation and manipulation of functional groups are readily available from relevant scientific literature or from standard textbooks in the field. It will be understood that other method conditions may also be used when typical or preferred method conditions (e.g., reaction temperature, time, molar ratio of reactants, solvent, pressure) are given, unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through conventional optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and sequence of the presented synthetic steps can be modified to optimize the formation of the compounds described herein.

[0098] The synthetic chemical transformations (including protecting group methods) used to synthesize the compounds described herein are known in the art and include, for example, those described below: RC Larock, Comprehensive Organic Transformations Second edition, Wiley-VCH Publishing (1999); PGM Wuts and TW Greene, Protective Groups in Organic Synthesis Fourth edition, John Wiley and Sons (2007); L. Fieser and M. Fieser, Fieser and Fieser ' s Reagents for Organic Synthesis John Wiley and Sons (1994); and L. Paquette, editor, Encyclopedia of Reagents for Organic Synthesis John Wiley and Sons (1995) and their subsequent editions, each hereby incorporated in its entirety by reference.

[0099] The methods described herein can be monitored using any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) Infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-Vis), or mass spectrometry (MS), or by chromatography (e.g., high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC)).

[0100] The preparation of compounds can involve the protection and deprotection of various chemical groups. Those skilled in the art can readily determine the requirements for protection and deprotection, as well as the selection of appropriate protecting groups. The chemical properties of the protecting groups can be determined, for example, by Greene et al. Protective Groups in Organic Synthesis The reference is found in the 2nd edition, Wiley & Sons, 1991, and is incorporated herein by reference in its entirety.

[0101] The reactions described herein can be carried out in suitable solvents, which can be easily selected by those skilled in the art of organic synthesis. Suitable solvents, at the temperatures in which the reaction proceeds (i.e., the temperature range from the solvent's freezing point to its boiling point), are substantially unreactive with the starting materials (reactants), intermediates, or products. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the specific reaction step, a solvent suitable for that particular reaction step can be selected.

[0102] The resolution of racemic mixtures of compounds can be performed by any of the numerous methods known in the art. For example, the absolute configuration of stereoisomers can be determined by 1D and 2D NMR techniques (e.g., COSY, NOESY, HMBC, and HSQC). Specific implementations of these NMR techniques can be found in Hauptmann, H et al. Bioconjugate Chem . 11(2000):239-252 or Bowler, J. Steroids The information was found in 54 / 1(1989):71-99, each of which is hereby incorporated herein by reference in its entirety. Another exemplary method involves preparing Mosher esters or amide derivatives of the respective alcohols or amines, respectively. Then, by protonation and / or... 19 F NMR spectroscopy determines the absolute configuration of the ester or amide. An exemplary method includes fractional recrystallization using a "chiral resolving acid" (which is an optically active salt forming an organic acid). Suitable resolving agents for fractional recrystallization are, for example, optically active acids such as D- and L-type tartaric acid, diacetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be performed by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Those skilled in the art can determine suitable elution solvent compositions.

[0103] Typically, zwitterionic acridine esters (“ZAEs”) containing reactive functional groups for forming covalent linkages, such as those described in U.S. Patent Nos. 6,664,043, 7,309,615, 9,575,062, or 9,487,480 to Natrajan et al., each hereby incorporated in its entirety (and particularly with respect to the zwitterionic acridine esters and their synthesis thereof) can be used to synthesize the compounds disclosed herein. For example, zwitterionic acridine ester starting materials may include an N-sulfopropyl (“NSP”) group in the zwitterionic moiety and / or include a charged nitrogen atom attached to a charged acridine core (“DIZAE”) and / or include a sterically stabilized dimethyl acridine ester (“DMAE”) and / or include an isopropoxy-functionalized acridine core (“ISO”) and / or include a zwitterionic (“Z”) and / or hexaethylene glycol derivative (“HEG”) and / or glutarate derivative (e.g., –C(O)–(CH2)3–C(O)–) ​​linking moiety between the acridine ester and the reactive functional group. The reactive functional group may be NH2 or N-hydroxysuccinimide ester (“NHS”). For example, a compound (e.g., a compound for conjugating an analyte or a binding pair of an analyte (e.g., a peptide, protein, or a macromolecule including an antibody)) may have the structure of formula (IV): RFG is a reactive functional group used for conjugation with analytes or their binding partners. L does not exist (i.e., it is a bond) or a linker, and Ψ is a chemiluminescent acridine. The chemiluminescent conjugates or compounds used to form the conjugates can also be synthesized using acridine sulfonamide reactants. For example, the acridine sulfonamide disclosed in U.S. Patent No. 5,543,524 to Mattingly et al. (incorporated herein by reference in its entirety) is a useful starting material for the preparation of the chemiluminescent compounds disclosed herein.

[0104] This assay can be, for example, a competitive immunoassay, which typically involves the detection of macromolecules (also known as macromolecular analytes) using binding molecules (e.g., antibodies). Antibodies are immobilized or attached to a solid phase, such as particles, beads, membranes, microtiter plates, or any other solid surface.

[0105] In examples of competitive heterogeneous assays, a support to which an antibody for the analyte (e.g., 3C3, 3H10, 4G8 bovine monoclonal antibody) is bound is contacted with a medium containing a sample suspected of containing the analyte and the chemiluminescent conjugate (or “labeled analog”) described herein. The analyte from the sample competes with the labeled analog for binding to the analyte antibody. After separation of the support and medium, the labeling activity of the support or medium is determined using conventional techniques, and is correlated with the amount of analyte in the sample. In variations of the above competitive heterogeneous assays, the support contains an analyte analog that competes with the analyte in the sample for binding to the antibody reagent, according to the principles described herein. The labeled analyte analog may be covalently linked to a chemiluminescent or fluorescent molecule commonly referred to as a label or tracer.

[0106] When a solid phase containing immobilized antibodies is mixed with a sample containing both analytes and labeled analytes, binding complexes typically form between the analytes or labeled analytes. Because the solid phase is involved, this type of assay is often referred to as a heterogeneous assay. The chemiluminescent signal associated with the binding complex can then be measured, and the presence or absence of the analyte in the sample can be inferred. Typically, the binding complex is separated from the remainder of the binding reaction components (e.g., excess labeled analyte) before a signal is generated. For example, if the binding complex is associated with magnetic beads, a magnet can be used to separate the binding complex associated with the beads from the bulk solution.

[0107] In an example employing a sandwich assay using two antibodies (or fragments thereof), a solid support having a first immobilized antibody or fragment thereof for the analyte is mixed with a sample containing the analyte and a labeled conjugate containing a second antibody or fragment thereof. A binding complex is formed between the solid particles and the labeled conjugate via the analyte in the sample. Different chemiluminescent moieties may conjugate to the same binding pair of the analyte. In some embodiments, different chemiluminescent moieties conjugate to different binding pairs of the analyte. In some embodiments, different chemiluminescent moieties conjugate to different binding pairs of different analytes (all of which can be detected on the same solid support). The signal associated with the binding complex can be measured, and the presence or absence or amount of the analyte can be inferred. Typically, the binding complex is separated from the remaining binding reaction components, such as excess labeled analyte, before the signal is generated. For example, if the binding complex is associated with magnetic beads, a magnet can be used to separate the binding complex associated with the beads from the bulk solution. In some embodiments, the first immobilized antibody is a biotinylated mouse monoclonal antibody bound to optionally paramagnetic particles coated (e.g., streptavidin-coated). In some implementations, the second antibody is a mouse monoclonal antibody fragment labeled with acridine (e.g., acridine ester).

[0108] By using a series of "standards"—analytes at known concentrations—dose-response curves can be generated for known labeled analytes. These dose-response curves can be identified individually for any acridineonium label or based on a combination of acridineonium labels used in the assay. Therefore, the dose-response curve correlates a certain amount of measured signal with a specific concentration of the analyte. In competitive assays, if chemiluminescence from the binding complex is measured, the signal intensity decreases as the analyte concentration increases. The concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated by the unknown sample containing the macromolecular analyte with the dose-response curve.

[0109] Methods for attaching binding molecules (e.g., antibodies) to a solid phase typically involve mixing necessary components to induce attachment. For example, antibodies can be covalently attached to particles containing amines on their surface by using cross-linking molecules (e.g., glutaraldehyde). Attachment can also be non-covalent and can involve simply adsorbing the binding molecule onto the surface of a solid phase (e.g., polystyrene beads and microtiter plates). Labeling of binding molecules (e.g., antibodies and other binding proteins) is also well known in the art and is commonly referred to as a conjugation reaction, with labeled antibodies often referred to as conjugates. Typically, the amine-reactive portion on the label reacts with the amine on the antibody to form an amide link. Other links between the antibody and the label (e.g., thioethers, esters, carbamates, etc.) can also be used.

[0110] In another aspect of the invention, a reagent for detecting an analyte can be provided, the reagent comprising a chemiluminescent acridine compound that binds to the analyte or its binding pair. The reagent may comprise 0.1 to 100 ng / mL of the chemiluminescent acridine compound, or 1 to 50 ng / mL of the chemiluminescent acridine compound, or 5 to 30 ng / mL of the chemiluminescent acridine compound. In some embodiments, the compound is provided in a reagent that further comprises a buffer.

[0111] Typically, determinations used for the detection or quantification of analytes in a sample include: (a) Provide a first set of chemiluminescent markers and a second set of chemiluminescent markers. The first group of chemiluminescent labels includes at least two chemiluminescent labels with separated emission spectra in the wavelength domain, and The second group of chemiluminescent labels comprises at least two chemiluminescent labels with different emission rates; wherein the first and second groups can overlap (e.g., chemiluminescent labels in the first group can also be in the second group). Each chemiluminescent label in the first and second groups can form a binding complex with at least one of a variety of analytes; (c) Mix the first group and the second group with the sample; (e) Prepare a mixture of the first group, the second group and the sample to measure the chemiluminescence of the chemiluminescent markers from the first group and the second group; (f) Triggering chemiluminescence from the first and second groups of chemiluminescent markers after the preparation of the mixture (e.g., by adding one or more triggering compositions that trigger the chemiluminescence of acridine-onium markers); (f) Measuring chemiluminescence in the wavelength domain (e.g., measuring light intensity as a function of wavelength) and the time domain (e.g., measuring light intensity as a function of time from the triggering step); (g) The presence of the at least one analyte or its concentration is detected by comparing the amount of emitted light with a standard dose response curve that correlates the amount of emitted light with a known concentration of at least one of a plurality of analytes.

[0112] In some embodiments, the sample is derived from a mammal (e.g., a human). In some embodiments, the sample contains saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0113] In some assays, the sample to be analyzed is pretreated to release the analyte from endogenous binding substances (e.g., plasma or serum proteins bound to the analyte). Release of the analyte from endogenous binding substances can be achieved, for example, by adding a digestive agent or a releasing agent, or a combination of digestive and releasing agents used sequentially. A digestive agent is a substance that breaks down endogenous binding substances so that they can no longer bind to the analyte.

[0114] Conditions for measuring a portion of a sample according to the principles described herein may include measurement in an aqueous buffer medium of moderate pH, which generally provides optimal assay sensitivity. The aqueous medium may be water alone or may include 0.1 to 40% by volume a co-solvent. The pH of the medium may range from 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8. Typically, the pH of the solution will be a trade-off between optimal binding of any particular binding pair member, optimal pH of the other reagents being measured (e.g., members of the signal generation system), etc. A variety of buffers can be used to achieve the desired pH and maintain it during the assay. Illustrative buffers include, for example, borates, phosphates, carbonates, TRIS, barbiturates, PIPES, HEPES, MES, ACES, MOPS, and BICINE.

[0115] Various auxiliary materials can be used in the assay method. For example, in addition to buffers, the composition, reagents, or reaction media may contain stabilizers for the media and reagents used. In some embodiments, the media may contain proteins (e.g., albumin), organic solvents (e.g., formamide), quaternary ammonium salts, polyanionic compounds (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), and combinations thereof.

[0116] The chemiluminescence of analogues can be triggered by adding a chemiluminescence triggering agent. The chemiluminescence triggering agent can be acidic or basic. Multiple chemiluminescence triggering agents can be added sequentially. For example, an acidic solution can be added first, followed by a basic solution. In some embodiments, the chemiluminescence triggering agent comprises hydrogen peroxide, hydrogen peroxide salts, nitric acid, nitrates, sodium hydroxide, ammonium salts, or combinations thereof.

[0117] The kits according to this disclosure may relate to immunoassay reagent compositions comprising one or more AEs conjugated to a binding partner (e.g., an antibody (or a fragment thereof)). The kit may include auxiliary components such as buffers, blocking reagents, ions (e.g., divalent or monovalent cations), calibration proteins, secondary antibodies, detection reagents (e.g., detection dyes), and any other suitable compounds or liquids necessary for the performance of analyte detection. Additionally, the kit may include instruction sheets and / or provide (or access) information regarding the relevance of the obtained results and / or additional tests (which may be applicable to additional risk assessments). In some embodiments, the kit also includes a solid-phase reagent. In some embodiments, the kit also includes a chemiluminescent triggering reagent. Example

[0118] The following examples illustrate the synthesis of a representative number of compounds, the characterization of relevant parameters in assay development, and the use of these compounds in the measurement of samples in heterogeneous competitive assays. Therefore, the examples are intended to be illustrative but not limiting of this disclosure. Additional compounds not specifically illustrated may be synthesized using conventional methods in conjunction with the methods described herein.

[0119] Example 1: Synthesis of stable slow-emission acridine ester 1,3-dimethyl-DMAE-Bz(10) Synthesis of B: 4-Hydroxybenzoic acid (A, 7.8 g) was dissolved in 50 mL of methanol, and insoluble substances were filtered off. A solution of 2.3 g of potassium hydroxide in 15 mL of water was added dropwise to the solution until the pH reached 7.6. The solution was stirred at room temperature for 1 hour and then evaporated to dryness under vacuum using a rotary evaporator to obtain product B (9.35 g).

[0120] Synthesis of C A suspension of compound B (9.35 g) and dibenzo-18-crown-6 (1.975 g) in 210 mL of a mixed acetonitrile / DMF (2:1) was stirred at 80–90 °C for 30 min, followed by the addition of benzyl bromide (6.1 mL). The mixture was stirred at 80–90 °C for 3 h, then cooled to room temperature and stored in a refrigerator over the weekend. The resulting mixture was filtered. The filtrate was concentrated under reduced pressure to a brown oily substance. The crude substance was diluted with chloroform and applied to a rapid silica gel column packed with silica gel (600 mL) in hexane. The column was eluted with 5%, 10%, and 15% ethyl acetate / hexane. Fractions containing the desired product were combined and evaporated to dryness under reduced pressure using a rotary evaporator to yield 7.12 g of compound C.

[0121] Synthesis of F A solution of indigo (D, 5 g) in anhydrous N,N-dimethylformamide (DMF, 150 mL) was treated with sodium hydride (60% suspension, 1.632 g) for approximately 20 minutes until no gas was released. 5-Bromo-xylene (E, 9.24 mL) was added to the mixture, followed by cuprous iodide (I) (12.9 g). The mixture was heated at 150 °C for 18 hours with stirring. The mixture was cooled to room temperature, diluted with approximately 750 mL of chloroform, and filtered to remove the off-white precipitate (CuI). The filtrate was evaporated under reduced pressure using a rotary evaporator to give crude substance F, as a brown material (15 g).

[0122] G synthesis The mixture of compound F in 100 mL of 10% sodium hydroxide aqueous solution was heated at 120 °C under reflux for 16 hours. The mixture was cooled to room temperature and filtered. The filter cake was washed with ~50 mL of water. The combined filtrate and washing solution were acidified to pH 2-3 with concentrated hydrochloric acid in an ice-water bath. The resulting precipitate was collected and washed with water (2 x 50 mL). The material was air-dried overnight to give 4.1 g of compound G.

[0123] Synthesis of H The mixture of G (110 mg) in pyridine (5 mL) was treated with toluenesulfonyl chloride (146 mg) at room temperature for 5 minutes, followed by the addition of C (98 mg). The solution was heated at 60 °C for 24 hours and cooled to room temperature. Stirring was continued at room temperature for 2 days. The resulting mixture was evaporated to dryness under reduced pressure using a rotary evaporator. The solid was dissolved in chloroform (50 mL), and the chloroform layer was washed with 1 N sodium hydroxide solution (2 × 20 mL), brine (1 × 20 mL), and dried over sodium sulfate. The solvent was removed to obtain the crude product, which was purified by passing it through two 2 mm thick (20 × 20 cm) preparative silica gel plates and eluting with ethyl acetate / hexane (1:1). The major band was collected and extracted with diethyl ether (200 mL). The diethyl ether was removed under reduced pressure using a rotary evaporator to give 29 mg of H.

[0124] Synthesis of 1,3-dimethyl-DMAE-Bz (10, batch number #QJ4-30-3) A solution of H (23 mg) in 2 mL of dichloromethane was stirred with methyl fluorosulfonate (38 μL) under nitrogen atmosphere at room temperature for two days. The reaction was stopped by removing the solvent by purging nitrogen. The crude product was purified by preparative HPLC (solvent A: 0.5% TFA / water, solvent B: 0.5% acetonitrile, flow rate: 16 mL / min, detection: 260 nm) to obtain 20 mg of 1,3-dimethyl-DMAE-Bz (10, batch number #QJ4-30-3).

[0125] Example 2: Synthesis of stable slow-emission acridine ester p-CE-DMPAE (12, batch number #WJW4-188A) J synthesis A solution of 2,6-dimethylphenol (I, 10 g) and acrylonitrile (8.08 mL) was cooled to 15 °C in a water bath, and aluminum chloride (10.91 g) was slowly added over 15 minutes. After addition, the mixture was heated at 100 °C with stirring for 45 minutes. The resulting solid was added to 200 g of crushed ice along with a small amount of methanol. The precipitate was collected and recrystallized from methanol to give 4.944 g J.

[0126] Synthesis of K A solution of potassium hydroxide (23 g) in 35 mL of water was added to a round-bottom flask containing 3.6 J, followed by 50 mL of ethanol. The mixture was stirred overnight at 110 °C. The mixture was cooled to room temperature and evaporated under reduced pressure using a rotary evaporator to remove most of the ethanol, then diluted with 300 mL of water. The solution was acidified to pH 4 with 50% sulfuric acid. The resulting precipitate was filtered, washed with water (2 x 100 mL), and dried under vacuum as a white solid to produce 3.07 g K.

[0127] Synthesis of L Potassium hydroxide (0.848 g) in 7 mL of water was added to a solution of 2.69 g K in 25 mL of methanol to make it slightly alkaline. The solution was stirred at room temperature for 1 hour, and then evaporated under reduced pressure using a rotary evaporator to obtain solid material K, which was used in the next reaction.

[0128] Synthesis of M Before adding benzyl bromide (2.6 g), a suspension of K (all materials obtained in the above steps) and dibenzo-8-crown-6 (511 mg) in a mixed solvent of acetonitrile (30 mL) and DMF (15 mL) was stirred at 80 °C for 15 min. The suspension was stirred at 80 °C for 4 h and then overnight at room temperature. The resulting mixture was filtered and the solid was washed with ethyl acetate. The solid was evaporated under reduced pressure using a rotary evaporator to give an oil. This oil was purified by rapid silica gel column chromatography, eluting with 15% ethyl acetate / hexane to give 2.11 g of product M as an oil.

[0129] O synthesis A suspension of acridine-9-carboxylic acid hydrochloride (N, 1.67 g) in 10 mL of thionyl chloride was stirred at 95 °C for 3 hours. It was then cooled and concentrated to a small volume by purge with nitrogen. The suspension was quenched with 200 mL of anhydrous diethyl ether, and the precipitate was filtered off, washed with more diethyl ether, and then dried under high vacuum to give 1.96 g of O as a yellow solid.

[0130] Synthesis of P DMAP (188 mg) was added to a solution of M (2.01 g) in pyridine (25 mL). The solution was stirred at 0 °C for 5 minutes, and then O (1.96 g) was added. The suspension was stirred at 100 °C for 3 hours and then allowed to stand overnight at room temperature. The solution was evaporated to dryness under reduced pressure by co-evaporation with toluene. The solution was purified by rapid silica gel column chromatography, eluting with 20% ethyl acetate in hexane to yield 1.84 g of product P.

[0131] Synthesis of Q A solution of P (969 mg) and methyl fluorosulfonate (1.56 mL) in 25 mL of dichloromethane was stirred overnight at room temperature under nitrogen. The reaction was stopped by purging the solvent with a nitrogen stream. The resulting solid was dissolved in dichloromethane, and the solution was added to 450 mL of anhydrous diethyl ether. The yellow precipitate was redissolved in dichloromethane (75 mL), and the solution was added to 350 mL of anhydrous diethyl ether to give 715 mg of product Q.

[0132] Synthesis of p-CE-DMPAE (12, batch number #WJW4-188A) A solution of Q (572.9 mg) in 3 mL of acetic acid with 30% hydrogen bromide was stirred at 100 °C for 2 hours under nitrogen. The solution was purged with nitrogen to a small volume and then poured into anhydrous diethyl ether. The resulting precipitate was filtered, collected, and washed with diethyl ether (6 × 10 mL). The yellow material was dried under high vacuum to give 373 mg p-CE-DMPAE (11, batch number #WJW4-188A).

[0133] Example 3: Measurement of Chemiluminescence Wavelength and Kinetics General procedure for optical emission dynamics measurements: All optical measurements are performed on MLA1. TM The procedure was performed on [a specific surface area]. To measure the RLU from acridine ester, a 1 mg / mL acridine ester DMF solution was sequentially diluted 10 [units]. 6 It contains up to 10 mM phosphate, 150 mM NaCl, pH 8, and also 0.05% BSA and 0.1% sodium azide. Photometric measurements were performed using a 25 μL sample, initiated in the instrument by adding 0.350 mL of Reagent 1 (containing 0.5% hydrogen peroxide in 0.1 N nitric acid), followed by 0.35 mL of Reagent 2 (containing a surfactant in 0.25 N NaOH). A very short delay of 0.1 seconds was used between the addition of Reagent 1 and Reagent 2.

[0134] 1,3-Acridine-onium ring conjugation alters chemiluminescence kinetics Kinetic measurements of 1,3-dimethyl-DMAE-Bz 10 and DME-Bz 11 were compared. Measurement times varied from 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 to 10.0 seconds. The structures used for 1,3-dimethyl-DMAE-Bz 10 and DMAE-Bz 11 are as follows: The amount of light emitted at each measurement time was reported by the instrument as RLU, and then converted to a percentage by assigning a 100% value to the RLU recorded for acridine ester at 10 s. Table 3 and Figure 4 The percentage of light from 1,3-dimethyl-DMAE-Bz and DMAE-Bz measured over 10 seconds under standard flash conditions is shown.

[0135] exist Figure 4 In the diagram, for each, 1,3-dimethyl-DMAE-Bz is shown on the left (black) and DMAE-Bz is shown on the right (white). It can be seen that the 1,3-conjugation of acridine-onium labels results in slower emission kinetics compared to acridine-onium labels without conjugation at these positions.

[0136] The combination of electron-donating groups at the 4-position of phenyl esters Kinetic measurements were also performed on p-CE-DMPAE 12 and DMAE-Bz 11. Measurement times varied from 2.0 s, 5.0 s, 10.0 s, 25.0 s to 60.0 s. The structures of p-CE-DMPAE 12 and DMAE-Bz 11 are as follows: The amount of light emitted at each measurement time was reported by the instrument as RLU, and then converted to a percentage by assigning a 100% value to the RLU recorded at 60.0 seconds for acridine ester. Table 4 and Figure 5 The percentage of light measured over 10 seconds for 1,3-dimethyl-DMAE-Bz and DMAE-Bz under standard flash conditions is shown.

[0137] p-CE-DMPAE(12) takes about 60 seconds to emit all the light, while DMAE-Bz(11) takes only 2 seconds to emit all the light (98% light emission).

[0138] Using these compounds, the possible sequences for detecting a single signal are shown in Table 5 below. The first time range is from 0 to 2 seconds of light collection, during which 8% of the signal from p-CE-DMPAE (12) and 98% of the signal from DMAE-Bz (11) can be obtained, respectively. Due to the significant difference in emission rates between the two compounds, light collection in time range 2 can begin at any time after 2 seconds without delay. The significantly slower emission of light from DMAE-Bz (11) provides the option to begin measuring light at a much later time (e.g., after 10 seconds). This provides the option to use a third AE in the time domain.

[0139] Table 5 Measurement range 1 Delay (no light collected) Measurement range 2 2 seconds 0 2 until 60 seconds CE-DMPAE (12) ~8% signal NA Up to ~92% signal strength DMAE-Bz (11) ~98% signal NA ~0% signal

[0140] Non-restrictive illustrative implementation scheme The following are non-limiting illustrative embodiments, each of which should be considered part of the disclosure of this application. These embodiments can be applied to any of the embodiments described herein.

[0141] Illustrative Embodiment 1. A method for detecting or quantifying multiple analytes in a sample (e.g., a biological sample, such as blood, saliva, serum, or a sample derived from a biological sample, such as a diluted biological sample), comprising: (a) Provide a first set of chemiluminescent markers and a second set of chemiluminescent markers. The first group of chemiluminescent labels includes at least two chemiluminescent labels with separated emission spectra in the wavelength domain, and The second group of chemiluminescent labels comprises at least two chemiluminescent labels with different emission rates; wherein the first and second groups can overlap (e.g., chemiluminescent labels in the first group can also be in the second group). Each chemiluminescent label in the first and second groups can form a binding complex with at least one of a variety of analytes; (c) Mix the first group and the second group with the sample; (e) Prepare a mixture of the first group, the second group and the sample to measure the chemiluminescence of the chemiluminescent markers from the first group and the second group; (f) Triggering chemiluminescence from the first and second groups of chemiluminescent markers after the preparation of the mixture (e.g., by adding one or more triggering compositions that trigger the chemiluminescence of acridine-onium markers); (f) Measuring chemiluminescence in the wavelength domain (e.g., measuring light intensity as a function of wavelength) and the time domain (e.g., measuring light intensity as a function of time from the triggering step); (g) The presence of the at least one analyte or its concentration is detected by comparing the amount of emitted light with a standard dose response curve that correlates the amount of emitted light with a known concentration of at least one of a plurality of analytes.

[0142] Illustrative Implementation Scheme 2. The method according to Illustrative Implementation Scheme 1, wherein the two chemiluminescent labels in the first group are capable of forming binding complexes with different analytes in the sample.

[0143] Illustrative Implementation Scheme 3. The method according to Illustrative Implementation Scheme 1 or 2, wherein the two chemiluminescent labels in the second group are capable of forming binding complexes with different analytes in the sample.

[0144] Illustrative Embodiment 4. The method according to any one of Illustrative Embodiments 1-3, wherein each of the two chemiluminescent markers in the first group has a corresponding chemiluminescent marker with a different emission rate (e.g., so as to form two different second groups of chemiluminescent markers separated by emission rates from four different chemiluminescent markers), and the four chemiluminescent markers are capable of forming binding complexes with different analytes in the sample.

[0145] Illustrative Embodiment 5. The method according to any one of Illustrative Embodiments 1-4, wherein the first group of chemiluminescent labels comprises at least three chemiluminescent labels having emission spectra separated in the wavelength domain.

[0146] Illustrative Implementation Scheme 6. The method according to Illustrative Implementation Scheme 5, wherein the three chemiluminescent labels in the first group are capable of forming binding complexes with three different analytes in the sample.

[0147] Illustrative Embodiment 7. The method according to Illustrative Embodiment 5, wherein each of the three chemiluminescent markers in the first group has a corresponding chemiluminescent marker with a different emission rate (e.g., to form three different second groups of chemiluminescent markers separated by emission rates from six different chemiluminescent markers), and each of the six chemiluminescent markers is capable of forming a binding complex with six different analytes in the sample.

[0148] Illustrative Embodiment 8. The method according to any one of Illustrative Embodiments 1-7, wherein the preparation step comprises: (e1) A solid support having molecules immobilized thereon, the molecules being capable of forming binding complexes with the at least one analyte and with chemiluminescent markers in the first and / or second groups of chemiluminescent markers; and (e2) Separate the solid support from the mixture.

[0149] Illustrative Embodiment 9. The method according to Illustrative Embodiment 8, wherein the measurement step includes adding one or more chemiluminescent triggering reagents to the separated solid support and / or the separated mixture.

[0150] Illustrative Embodiment 10. The method according to Illustrative Embodiment 8 or 9, wherein the solid support comprises at least two types of molecules (e.g., two, three, four, five, six, seven, eight, nine, or ten), each molecule being capable of forming a binding complex with a different analyte and capable of forming a binding complex with at least two different chemiluminescent labels from the first and / or second group of chemiluminescent labels.

[0151] Illustrative Embodiment 11. The method according to any one of illustrative embodiments 1-10, wherein the chemiluminescent label independently has the structure of formula (I): Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: "j" and "k" are independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3 or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2–, –C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –NH–S(O) 1- –, –N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–(CH2) 1- –, –(CH2) 1-4 –O–, –S–(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –N(R N )–(CH2) 1- –, –(CH2) 1-4 –N(R N )–, –(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –(CH2CH2O) 1-10 –; RL It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0152] Illustrative Embodiment 12. The method according to Illustrative Embodiment 11 or 12, wherein the chemiluminescent label independently has the structure of formula (Ia): Where Ω represents O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –

[0153] Illustrative Embodiment 13. The method according to Illustrative Embodiment 11, wherein at least one (e.g., one, two, three, four, five, six) of the chemiluminescent labels has a structure of formula (Ib) or (Ic): R4-R7 are independently hydrogen, electron-donating groups, or C. 1-35 Alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and "Y" either does not exist (i.e., it is a key) or is -L C –, –L1–, –R L –, or –R L –L1–.

[0154] Illustrative Embodiment 14. The method according to Illustrative Embodiment 13, wherein at least one of R4-R7 (e.g., R4, R5, R7, R7) is an electron-donating group (e.g., forming a chemiluminescent label with a different emission rate compared to another identical label without an electron-donating group).

[0155] Illustrative Embodiment 15. The method according to any one of illustrative embodiments 1-14, wherein each of the two chemiluminescent markers in the first group is conjugated to a binding coupler of one of the analytes, and the chemiluminescent marker is formed from a chemiluminescent compound or salt having reactive functional groups for conjugation to the binding coupler.

[0156] Illustrative Embodiment 16. The method according to Illustrative Embodiment 15, wherein the chemiluminescent compound or salt is selected from DMAE-Bz, 3-MeO-DMAE-Bz, DIPAE-Bz, ABAC, LEAE-Bz, DIP-LEAE-Bz, 2-MeO-LEAE-Bz, 3-EtO-LEAE-Bz, 3-QAE-LEAE-Bz, 2-QAE-LEAE-NHS, LEAC-Bz, NSP-LEAE-Bz, 2-MeO-NSE-LEAE-NHS, 2-Meo-LEAE-iminocyanate, 3-carboxybutadienyl-AE, p-carboxyethyl-AE, rhodamine-2-AM-DMAE- Bz, Rhodamine-2-AM-DMAE-CO2H, Texas Red-2-AM-DMAE-CO2H, CNF-2-AM-DMAE-CO2H, Texas Red-3-AM-DMAE-CO2H, Rhodamine-3-AM-DMAE-β-alanine, Texas Red-3-AM-DMAE-β-alanine, Texas Red-ED-NCM-DMPAE, Texas Red-ED-NSP-DMPAE, Rhodamine-2-AM-DMAE-HD-theophylline, Texas Red-3-APO-DMAE-Bz, Texas Red-3-ABO-DMAE-Bz, DMAE-Bz, and 2-MeO-LEAE-Bz.

[0157] Illustrative Embodiment 17. The method according to any one of Illustrative Embodiments 1-16, wherein at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label from the first group is a zwitterionium (e.g., N-sulfopropyl zwitterionium, a compound having the structure of formula I, Ia, Ib, or Ic, wherein R1 is selected from –X). b , –R L –X b , or –L C –X b For example, –L1–X b R1 is selected from –SO3 - , –R L – SO3 - For example –(CH2) 1-5 – SO3 - , or –L C – SO3 - For example –L1– SO3 - ).

[0158] Illustrative Embodiment 18. The method according to any one of illustrative embodiments 1-17, wherein at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label is a zwitterionium (e.g., N-sulfopropyl zwitterionium, a compound having the structure of formula I, Ia, Ib or Ic, wherein R1 is selected from –X b , –R L –X b , or –L C –X b For example, –L1–X b R1 is selected from –SO3 - , –R L – SO3 - For example –(CH2) 1-5 – SO3 - , or –L C – SO3 - For example –L1– SO3 - ).

[0159] Illustrative Embodiment 19. The method according to any one of Illustrative Embodiments 1-18, wherein at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label is an acridineonium salt (e.g., acridineonium carboxylate, such as fluoroalkyl carboxylate, acridineonium sulfonate, such as fluoroalkyl sulfonate, acridineonium halide salt, such as acridineonium chloride salt, a compound having the structure of formula I, Ia, Ib or Ic, wherein R1 is selected from –R, –L C–R, –Z, –R L –Z, –L C –Z,–L1–Z, –R L –L C –R L –Z, –R L –L1–R L –Z, where the counter ion is such as R-COO - R-SO3 - Cl - ).

[0160] Illustrative Embodiment 20. The method according to any one of Illustrative Embodiments 1-19, wherein at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label of the first group is an acridineonium salt (e.g., acridineonium carboxylates, such as halocarboxylates, haloalkylcarboxylates, fluoroalkylcarboxylates, acridineonium sulfonates, such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, acridineonium halide salts, such as acridineonium chloride salts, compounds having the structure of formula I, Ia, Ib, or Ic, wherein R1 is selected from –R, –L). C –R, –Z, –R L –Z, –L C –Z,–L1–Z, –R L –L C –R L –Z, –R L –L1–R L –Z, where the counter ion is such as R-COO - R-SO3 - Cl - F - ).

[0161] Illustrative Embodiment 21. The method according to any one of illustrative embodiments 1-20, wherein at least one chemiluminescent label (e.g., the chemiluminescent label in the second group) has the structure of formula (II): Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: "j" and "k" are independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3 or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N)–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –‍O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10–, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates) Illustrative Implementation Scheme 22. The method according to Illustrative Implementation Scheme 21, wherein R 2a and R 2b Independently alkyl (e.g., lower alkyl, such as C10) 1-4 Alkyl groups, such as methyl, ethyl, propyl, and butyl.

[0162] Illustrative Embodiment 23. The method according to any one of embodiments 1-22, wherein one of the chemiluminescent markers in the first group has the structure of formula (IIIa): And another chemiluminescent label in the first group has the structure of formula (IIIb): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –RL –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “j” is 1, 2, 3 or 4; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups, and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer (IA) such as a fluorophore (e.g., rhodamine); and at least one R2 group is not hydrogen; L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4–, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0163] Illustrative Embodiment 24. The method according to Illustrative Embodiment 23, wherein the chemiluminescent label of formula (IIIb) has the structure of formula (IIIb1): .

[0164] Illustrative embodiment 25. The method according to illustrative embodiment 23 or 24, wherein at least one R3 is not hydrogen (e.g., an electron-donating group such as an alkoxy group).

[0165] Illustrative Embodiment 26. The method according to Illustrative Embodiment 23 or 24, wherein at least one R3 and / or at least one R2 group is an electron-donating group (e.g., alkoxy).

[0166] Illustrative Embodiment 27. The method according to any one of Illustrative Embodiments 23-26, wherein the first group of chemiluminescent labels further comprises a compound having the structure of formula (IIIc): Where IA is the imaging agent (e.g., a fluorophore, such as rhodamine or phenol-modified rhodamine); and A3 is an analyte or a combination partner of analytes that is different from A1 and A2.

[0167] Illustrative Embodiment 28. The method according to Illustrative Embodiment 27, wherein the chemiluminescent label of formula (IIIc) has the structure of formula (IIIc1) or (IIIc2): .

[0168] Illustrative Embodiment 29. The method according to Illustrative Embodiment 23, wherein the chemiluminescent label of formula (IIIb) has the structure of formula (IIIb3): IA is the developing agent (e.g., a fluorophore, such as rhodamine or phenol-modified rhodamine).

[0169] Illustrative Embodiment 30. The method according to Illustrative Embodiment 29, wherein the chemiluminescent label of formula (IIIb3) has the structure of formula (IIIb4) or (IIIb5): .

[0170] Illustrative Embodiment 31. The method according to any one of Illustrative Embodiments 23-30, wherein the difference between the compounds of formula (IIIa), formula (IIIb) and formula (IIIc) is different A1, A2 and A3 groups, and in the acridine ring system concatenation (e.g. R1, Ω, L, Y, Y”, IA in formulas (IIIa), (IIIb) and (IIIc) are the same).

[0171] Illustrative Embodiment 32. The method according to any one of Illustrative Embodiments 23-31, wherein the chemiluminescent label of formula (IIIb) (e.g., formula (IIIb1), formula (IIIb2), formula (IIIb3), formula (IIIb4)) is a salt (e.g., a carboxylate, such as a halocarboxylate, haloalkylcarboxylate, fluorocarboxylate, fluoroalkylcarboxylate, F3CCOO). -The salt, R1 in formula (IIIb2) is an alkyl group, and the counter ion is a carboxylate, a halocarboxylate, a haloalkylcarboxylate, a fluorocarboxylate, a fluoroalkylcarboxylate, or F3CCOO. - ).

[0172] Illustrative Embodiment 33. The method according to any one of illustrative embodiments 1-31, wherein one of the chemiluminescent markers in the first group has the structure of formula (IVa): Furthermore, another chemiluminescent label in the first group has the structure of formula (IVb): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine).

[0173] L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N)–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 A divalent hydrocarbon group (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, or combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C.1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0174] Illustrative Embodiment 34. The method according to Illustrative Embodiment 33, wherein the difference between the chemiluminescent labels of formula (IVa) and formula (IVv) lies in the conjugation of the acridine ring system (e.g., R1, Ω, L, Y, Y”, IA are the same in formulas (IVa) and (IVb) and the different A1 and A2 groups.

[0175] Illustrative Embodiment 35. The method according to any one of illustrative embodiments 1-34, wherein one of the chemiluminescent markers in the second group has a structure of formula (Va): Furthermore, another chemiluminescent label in the second group has the structure of formula (Vb): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or Y does not exist when Ω is 0; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –RL –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). R 4a R 5a R 6a and R 7a Independently hydrogen or C 1-35 alkyl; R 4b R 5b R 6b and R 7b Independently hydrogen, C 1-35 Alkyl groups or electron-donating groups (e.g., alkoxy groups); L C It is divalent C 1-35 Alkyl, alkenyl, ynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3–, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0176] Illustrative Embodiment 36. The method according to any one of illustrative embodiments 1-35, wherein one of the chemiluminescent markers in the second group has a structure of formula (Vc): Furthermore, another chemiluminescent label in the second group has the structure of formula (Vd): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –,–L1–R L –L1, and –R L –L1–R L –; “k” can be 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –X b–R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). R 4a R 5a R 6a and R 7a Independently hydrogen or C 1-35 Alkyl groups or electron-donating groups (e.g., alkoxy groups); R 4b R 5b R 6b and R 7b Independently hydrogen, C 1-35 Alkyl groups or electron-donating groups (e.g., alkoxy groups); L C It has an electron-withdrawing linker (e.g., a carboxyl group) relative to the phenyl group; L Cb It is an electron-donating linker relative to the phenyl group (e.g., alkyl, alkoxy, alkylamino); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xa and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4–N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 A divalent hydrocarbon group (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, or combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0177] Illustrative Embodiment 37. The method according to any one of Illustrative Embodiments 1-36, wherein the at least two chemiluminescent acridinium having wavelength-separated chemiluminescence have similar chemiluminescence emission rates (e.g., the percentage of light measured at time points such as 1 second, 2 seconds, 4 seconds, or 6 seconds after triggering is within 10%, 5%, or 1%, compared to the total emission).

[0178] Illustrative Embodiment 38. The method according to any one of Illustrative Embodiments 1-37, wherein the at least two chemiluminescent acridinium having time-domain separated chemiluminescence have similar chemiluminescence wavelengths (e.g., within 10%, 5%, or 1%). max ).

[0179] Illustrative Embodiment 39. The method according to any one of Illustrative Embodiments 1-38, wherein the first set of chemiluminescent markers is provided as a composition to be mixed with the sample.

[0180] Illustrative Embodiment 40. The method according to any one of Illustrative Embodiments 1-39, wherein the second set of chemiluminescent markers is provided as a composition to be mixed with the sample.

[0181] Illustrative Embodiment 41. The method according to any one of Illustrative Embodiments 1-40, wherein the first group and the second group of chemiluminescent markers are provided as a composition to be mixed with the sample.

[0182] Illustrative Implementation Scheme 42. A compound having the structure of formula (V) Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, “k” can be 0 (e.g., all R3 groups are hydrogen), 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1 to 20 heteroatoms, having 1 to 20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O)1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0183] Illustrative Scheme 43. A compound having the structure of formula (VI) RFG is a reactive functional group used for conjugation to the analyte or its binding partner. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, “k” can be 0 (e.g., all R3 groups are hydrogen), 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z (e.g., –L1–Z), or –R L –L C –R L –Z (e.g., –R) L –L1–R L –Z); R 2a and R 2b Independently selected from optionally substituted alkyl groups (e.g., having 1-20 heteroatoms and 1-20 substituents); R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups may together form a fused heterocyclic group (e.g., a 5-7 member fused heterocyclic group), and wherein R3 may contain a link to a developer such as a fluorophore (e.g., rhodamine). L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl or arylalkyl groups, optionally substituted (e.g., having 1 to 20 heteroatoms); Z L It is a zwitterionic linker group with the following structure: “m” can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 (i.e., they are keys) to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are independently 0 or 1; “r” is an integer from 0 to 10 independently (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2)1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, ynyl, arylalkyl, and combinations thereof), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents); R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms and 1-20 substituents); R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C. 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0184] Illustrative Embodiment 44. A composition comprising at least three chemiluminescent acridines, wherein each chemiluminescent acridine is conjugated to a different analyte or a different binding pair of the analyte, and the chemiluminescence of at least two chemiluminescent acridines is separated in the wavelength time domain, and the chemiluminescence of different groups of at least two chemiluminescent acridines is separated in the time domain.

[0185] Illustrative Embodiment 45. The composition according to Illustrative Embodiment 44, wherein the composition comprises at least four chemiluminescent acridines conjugated to different analytes or binding partners of analytes, wherein the chemiluminescence of three of the chemiluminescent acridines is separated in the wavelength domain.

[0186] Illustrative Embodiment 46. The composition according to Illustrative Embodiment 44 or 45, wherein the at least two chemiluminescent acridinium having wavelength-separated chemiluminescence have similar chemiluminescence emission rates.

[0187] Illustrative Embodiment 47. The composition according to any one of Illustrative Embodiments 44-46, wherein the at least two chemiluminescent acridiniums having time-domain separation have similar chemiluminescence wavelengths.

[0188] Illustrative Embodiment 47. The composition according to any one of Illustrative Embodiments 44-47, wherein the chemiluminescent acridine trioxide (e.g., the at least three chemiluminescent acridine trioxides, the at least four chemiluminescent acridine trioxides) is independently selected from compounds having the structure of formula (I) (e.g., (Ia), (Ib), (Ic)), (II), (IIIa), (IIIb) (e.g., formula (IIIb1), formula (IIIb2), formula (IIIb3), formula (IIIb4)), (IV) (e.g., (IVa), (IVb)).

[0189] Illustrative Embodiment 48. A kit comprising a composition according to any one of Illustrative Embodiments 44-48 and at least one of a triggering reagent and / or a solid-phase reagent.

[0190] All references cited herein (including patent applications and publications) are incorporated herein by reference for all purposes, to the extent that each individual publication or patent or patent application is expressly and individually indicated as being incorporated herein by reference in its entirety for all purposes. As will be apparent to those skilled in the art, many modifications and variations of the invention may be made without departing from the spirit and scope of the invention. The embodiments described herein are provided by way of example only, and the invention is limited only by the terms of the appended claims and the full scope of their equivalents.

Claims

1. A method for detecting or quantifying multiple analytes in a sample, comprising: (a) Provide a first set of chemiluminescent markers and a second set of chemiluminescent markers. The first group of chemiluminescent labels includes at least two chemiluminescent labels with separated emission spectra in the wavelength domain, and The second group of chemiluminescent labels contains at least two chemiluminescent labels with different emission rates; The first and second groups can overlap. Each chemiluminescent label in the first and second groups can form a binding complex with at least one of a variety of analytes; (c) Mix the first group and the second group with the sample; (e) Prepare a mixture of the first group, the second group and the sample to measure the chemiluminescence of the chemiluminescent markers from the first group and the second group; (f) Trigger chemiluminescence from the chemiluminescent markers of the first and second groups after the preparation of the mixture; (f) Measurement of chemiluminescence in the wavelength and time domains; (g) The presence of the at least one analyte or its concentration is detected by comparing the amount of emitted light with a standard dose response curve that correlates the amount of emitted light with a known concentration of at least one of a plurality of analytes.

2. The method according to claim 1, wherein the two chemiluminescent markers in the first group are capable of forming binding complexes with different analytes in the sample and / or the two chemiluminescent markers in the second group are capable of forming binding complexes with different analytes in the sample.

3. The method according to any one of claims 1-2, wherein each of the two chemiluminescent markers in the first group is a corresponding chemiluminescent marker with a different emission rate, and the four chemiluminescent markers are capable of forming binding complexes with different analytes in the sample.

4. The method according to any one of claims 1-3, wherein the first group of chemiluminescent markers comprises at least three chemiluminescent markers having emission spectra separated in the wavelength domain.

5. The method according to any one of claims 1-4, wherein the chemiluminescent label independently has the structure of formula (I): Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: "j" and "k" are independently 0, 1, 2, 3 or 4; R1 is hydrogen, –R, –X b , –R L –X b , –L C –R, –L C –X b , –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group, and wherein R2 or R3 may contain a link to a developer such as a fluorophore; L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

6. The method according to any one of claims 1-5, wherein each of the two chemiluminescent markers in the first group is conjugated to a binding partner of one of the analytes, and the chemiluminescent marker is formed from a chemiluminescent compound or salt having a reactive functional group for conjugation to the binding partner.

7. The method according to claim 6, wherein the chemiluminescent compound or salt is independently selected from DMAE-Bz, 3-MeO-DMAE-Bz, DIPAE-Bz, ABAC, LEAE-Bz, DIP-LEAE-Bz, 2-MeO-LEAE-Bz, 3-EtO-LEAE-Bz, 3-QAE-LEAE-Bz, 2-QAE-LEAE-NHS, LEAC-Bz, NSP-LEAE-Bz, 2-MeO-NSE-LEAE-NHS, 2-Meo-LEAE-iminocyanate, 3-carboxybutadienyl-AE, p-carboxyethyl-AE, rhodamine-2-AM-DMAE-Bz, etc. Rhodamine-2-AM-DMAE-CO2H, Texas Red-2-AM-DMAE-CO2H, CNF-2-AM-DMAE-CO2H, Texas Red-3-AM-DMAE-CO2H, Rhodamine-3-AM-DMAE-β-alanine, Texas Red-3-AM-DMAE-β-alanine, Texas Red-ED-NCM-DMPAE, Texas Red-ED-NSP-DMPAE, Rhodamine-2-AM-DMAE-HD-theophylline, Texas Red-3-APO-DMAE-Bz, Texas Red-3-ABO-DMAE-Bz, DMAE-Bz, and 2-MeO-LEAE-Bz.

8. The method according to any one of claims 1-7, wherein at least one chemiluminescent label has the structure of formula (II): Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: "j" and "k" are independently 0, 1, 2, 3 or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R 2a and R 2b Independently selected from optionally substituted alkyl groups; R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups can form a fused heterocyclic group together, and wherein R3 may contain a link to a developer such as a fluorophore; L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

9. The method according to any one of claims 1-8, wherein one of the chemiluminescent markers in the first group has the structure of formula (IIIa): And another chemiluminescent label in the first group has the structure of formula (IIIb): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist, or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –, –L1–R L –L1, and –R L –L1–R L –; "j" can be 1, 2, 3, or 4; "k" can be 0, 1, 2, 3 or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group, and wherein R2 or R3 may contain a link to a developer (IA) such as a fluorophore; and at least one R2 group is not hydrogen; L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

10. The method according to any one of claims 1-9, wherein one of the chemiluminescent markers in the first group has the structure of formula (IVa): Furthermore, another chemiluminescent label in the first group has the structure of formula (IVb): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist (i.e., it is a key), or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –, –L1–R L –L1, and –R L –L1–R L –; "k" can be 0, 1, 2, 3 or 4; R1 is hydrogen, –R, –X b –R L –X b –L C –R, –L C –X b (For example, –L1–X) b ), –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 or R3 groups may together form a fused cyclic group, and wherein R2 or R3 may contain a link to a developer such as a fluorophore; L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

11. The method according to any one of claims 1-10, wherein one of the chemiluminescent markers in the second group has a structure of formula (Vc): Furthermore, another chemiluminescent label in the second group has the structure of formula (Vd): Wherein A1 and A2 are independently analytes or binding partners of analytes, and A1 is different from A2; Ω can be O or N; Y is selected from -R or –R L –Z, or if Ω is 0, then Y does not exist; and Y' either does not exist, or it is selected from –L1–, –R L –, –R L –L1–, –L1–L1–, –‍L1–‍R L –, –L1–R L –L1, and –R L –L1–R L –; "k" can be 0, 1, 2, 3 or 4; R1 is hydrogen, –R, –X b , –R L –X b , –L C –R, –L C –X b , –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R2 and R3 are independently selected from hydrogen, –R, electron-donating groups and -Z each time they appear; wherein two adjacent R2 or R3 groups may together form a fused cyclic group, and wherein R2 or R3 may contain a link to a developer such as a fluorophore; R 4a R 5a R 6a and R 7a Independently hydrogen or C 1-35 Alkyl groups or electron-donating groups; R 4b R 5b R 6b and R 7b Independently hydrogen, C 1-35 Alkyl groups or electron-donating groups; L C It has an electron-withdrawing linker relative to the phenyl group; L Cb It is an electron-donating linker relative to the phenyl group; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 (i.e., it is a key) or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

12. The method according to any one of claims 1-11, wherein the at least two chemiluminescent acridinium having wavelength-separated chemiluminescence have similar chemiluminescence emission rates and / or The at least two chemiluminescent acridine trioxides with time-domain separation have similar chemiluminescence wavelengths.

13. The method according to any one of claims 1-12, wherein the first set of chemiluminescent markers is provided as a composition to be mixed with the sample and / or The second set of chemiluminescent markers is provided as a composition to be mixed with the sample.

14. Compounds having the structure of formula (V) Where A is the analyte or its binding pair. L is absent (i.e., it is a bond) or is a linker that optionally contains the groups LC or ZL. "k" can be 0, 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b , –R L –X b , –L C –R, –L C –X b , –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R 2a and R 2b Independently selected from optionally substituted alkyl groups; R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups can form a fused heterocyclic group together, and wherein R3 may contain a link to a developer such as a fluorophore; L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

15. Compounds having the structure of formula (VI) RFG is a reactive functional group used for conjugation to the analyte or its binding partner. L is absent or optionally contains the group L. C or Z L The connector, "k" can be 0, 1, 2, 3 or 4 independently; R1 is hydrogen, –R, –X b , –R L –X b , –L C –R, –L C –X b , –Z, –R L –Z, –L C –Z, or –R L –L C –R L –Z; R 2a and R 2b Independently selected from optionally substituted alkyl groups; R 2c It can be hydrogen, –R, an electron-donating group, or -Z; R3 is independently selected from hydrogen, –R, electron-donating groups and -Z each time it appears; wherein two adjacent R2 groups can form a fused heterocyclic group together, and wherein R3 may contain a link to a developer such as a fluorophore; L C It is divalent C 1-35 Alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups, optionally substituted; Z L It is a zwitterionic linker group with the following structure: "m" can be 0 or 1; "n" and "p" are independent integers from 0 to 10 each time they appear; Z is a zwitterionic group, and each time it appears, it independently has the following structure: "q" and "l" are 0 or 1 independently; "r" can be an integer from 0 to 10 independently; X a and X b It is an anionic group each time it appears; L1 occurs independently of –O–, –S–, –NH–, –N(R) each time it appears. N )–, –(CH2) 1-10 –, –S(=O) 1-2 –, –‍C=C–,–C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –‍C(O)–‍O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –‍C(O)–‍N(R N )–‍(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–, –(CH2) 1-3 –N(R N )–C(O)–, –‍NH–‍S(O) 1- ‍2–‍, –N(R N )–S(O) 1-2 –, –‍S(O) 1-2 –N(R N )–, –S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –‍, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –‍(CH2) 1-3 –S(O) 1-2 –N(R N )–, –(CH2) 1-3 –S(O) 1-2 –NH–, –O–‍(CH2) 1- ‍4–, –(CH2) 1-4 –O–, –‍S–‍(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –, –‍N(R N )–‍(CH2) 1- ‍4–, –(CH2) 1-4 –N(R N )–, –‍(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 –, or –‍(CH2CH2O) 1-10 –; R L It is C independently each time it appears. 1-20 Divalent hydrocarbon groups, optionally having one or more substitution sites; R is independently either hydrogen or C each time it appears. 1-35 Hydrocarbon group, optionally having one or more substitution sites; R' and R” are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups; and R' is hydrogen or C. 1-10 Alkyl; or Its salt.

16. A composition comprising at least three chemiluminescent acridines, wherein each chemiluminescent acridine is conjugated to a different analyte or a different binding pair of the analyte, and the chemiluminescence of at least two chemiluminescent acridines is separated in the wavelength time domain, and the chemiluminescence of different groups of at least two chemiluminescent acridines is separated in the time domain.

17. The composition of claim 16, wherein the composition comprises at least four chemiluminescent acridine ions conjugated to different analytes or binding partners of analytes, wherein the chemiluminescence of three of the chemiluminescent acridine ions is separated in the wavelength domain.

18. The composition according to claim 16 or 17, wherein the at least two chemiluminescent acridine trioxides having wavelength-separated chemiluminescence have similar chemiluminescence emission rates.

19. The composition according to any one of claims 16-18, wherein the at least two chemiluminescent acridine trioxides having time-domain separation have similar chemiluminescence wavelengths.

20. A kit comprising the composition according to any one of claims 16-19 and at least one of a triggering reagent and / or a solid-phase reagent.

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