WO2025096599A1

WO2025096599A1 - Measurement of multiple analytes using acridinium labels

Info

Publication number: WO2025096599A1
Application number: PCT/US2024/053642
Authority: WO
Inventors: Qingping Jiang; Frank Vitzthum; William Bedzyk
Original assignee: Siemens Healthcare Diagnostics Inc
Current assignee: Siemens Healthcare Diagnostics Inc
Priority date: 2023-11-02
Filing date: 2024-10-30
Publication date: 2025-05-08
Anticipated expiration: 2026-05-02

Abstract

Methods of detecting one or more analytes in a sample are described herein using different chemiluminescent labels having differentiated chemiluminescence in the wavelength and kinetic domain. Solid supports, reagents, and compounds for use in these methods are also described. Typically, the methods involve the detection of nearly simultaneous detection of multiple analytes in a sample via conjugation to different acridinium labels.

Description

MEASUREMENT OF MULTIPLE ANALYTES USING ACRIDINIUM LABELS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of US App No 63/595,386, filed November 2, 2023. the entire contents of which are hereby incorporated by reference in their entirety.

FIELD OF DISCLOSURE

[0002] The present disclosure relates to an assay method of detection or measurement of multiple analytes in a sample by measurement of multiple chemiluminescent signals at different emission wavelengths and time frames, multiple chemiluminescent signals resulting from multiple acridinium labels, each having distinguishable emission maximum and/or emission speed (kinetics) from others and labeled to a binder specific to one of the multiple analytes present in the sample. The present invention also relates to stable acridinium labels capable of emitting a light at a slow emission speed (kinetics). Structural features of stable acridinium compounds necessary' for emitting a light at a slow speed are disclosed herein.

BACKGROUND

[0003] Chemiluminescence is an often-utilized light generation technique in assays designed to measure and quantify analytes in a sample. Acridinium labels, such as acridinium ester (AE) and acridinium sulfonamides are often used in these assays. Since the inception of stable dimethyl acridinium ester (DMAE) in 1980’s, there have been a number of useful acridinium compounds developed which are able to undergo chemiluminescence induced by a triggering compound. Acridiniums have been developed with improved properties such as higher light output, lower non-specific binding and faster light emission kinetics, resulting in the development of many automated immunoassay systems.

[0004] Although these instruments are capable of high assay through-put, further assay menu expansion could put significant stress on the systems and measurement protocols. For instance, a larger number of tests requires more reagent bottles, calibrators and controls placed on the instrument, which would in turn require a large footprint of the instrument and complicated system requirements. [0005] Multiple attempts at assay formats using different acridinium esters has been attempted however these attempts have been plagued with problems implementing multiple analyte detection and are often limited to the simultaneous detection of only two analytes. For example, U.S. Patents 5,395,752, 5,702,887, and 5,879,894, each of which are hereby incorporated by reference in their entirety, which describe a exemplify Long Emission Acridinium Esters (LEAE) containing benz[b]acridinium ring. In comparison with acridinium esters such as DMAE-NHS (1) which emits a tight at Xmax 422-428 run, benz[b]acridinium esters (LEAE) emit a tight at Xmax 508-550 nm, e.g., LEAE-NHS (2) at Xmax 528 nm. These two compounds have the structure:

[0006] Similarly, U.S. Patent 5,656,207, which is hereby incorporated by reference in its entirety, provides assays two using two labels. However, such as compound 3 and compound 4. However, compound 3 is an unstable acridinium ester which hydrolyzes rapidly to an inactive form in an aqueous medium where most of immunoassays are performed. Such a label is extremely difficult, if not impossible, for use in a commercial product, particularly in any sort of multi analyte measurement system.

[0007] U.S. Patent 5,879,894, which is hereby incorporated by reference in its entirety describes acridinium compounds with varying structures including 3-cafboxybutadienyl-AE (compound 7) and compound 6. The emission maximum of 3-carboxybutadienyl AE (compound 7) was determined to be at Xmax 464 nm. Compound 7 and compound 6 differ in that compound 7 has a methyl group on the phenyl group of the ester and a CT'iCOO group as a counter ion, and compound 6 has a R group at the phenol group and the CH3SO4 group as a counter ion. It is known to a skilled person in the art that a substitution group at the R position and a counter ion do not change the emission wavelength of an acridinium ester. It is reasonable to assume that structure 6 would have the same emission wavelength profile as compound 7. A small difference by only ~30 nm at emission maxima between 6 and 5 makes the separation of two wavelengths extremely difficult, thus making the measurement of two signals by emission wavelength for multiplexing practically impossible.

[0008] U.S. Patent 6,165,800 discloses a group of acridinium compounds termed as Energy Transfer Conjugate (ETC) where an AE moiety is covalently linked to a fhiorophore. In a reaction leading to chemiluminescence, the excited state energy embedded in the acridone formed from the reaction with hydrogen peroxide in an alkali solution transfers to the covalently linked fluorophore, causing the ETC to emit a light at the wavelength of the fhiorophore instead of the wavelength of the acridinium ester. By selecting a molecule from a variety of fluorophores having different emission wavelengths, the ETCs are shown to emit light in a range of λ_max 550 to 718 nm. Compounds 8 and 9 are two examples of ETCs. The authors report the light emission spectrum of three distinguishable colors from a mixture of three ETCs and provide an example of a dual-analyte assay by simultaneous measurement of lights at different wavelengths of two acridinium esters.

CNF-2-AM-DMAE-CO2H (9) Light Emission λ_max: 718 nm

[0009] There is a continuing need for chemiluminescent compounds that can provide high sensitivity for analyte detection while also being stable.

SUMMARY

[0010] In accordance with the foregoing objectives and others, the present disclosure includes methods for the detection of analytes in a sample through the use of chemiluminescent labels separated that have differentiated chemiluminescence. Acridiniums that can be used in the chemiluminescent assays are also provided, typically having a stability suitable for these assays. In some embodiments, the sample is blood, saliva, or serum. In some embodiments, the sample derived from a biological sample such as a diluted biological sample (e.g., as mixed with saline).

[0011] The methods for the detection or quantification of multiple analytes in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) may comprise: (a) providing a first set of chemiluminescent labels and a second set of chemiluminescent labels, wherein the first set of chemiluminescent labels comprise at least two chemiluminescent labels having emission spectra separated in the wavelength domain, and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first set and the second set may overlap (e.g., a chemiluminescent label in the first set may also be in the second set), wherein each chemiluminescent label in the first and second set is capable of forming a binding complex with at least one of the multiple analytes;

(c) mixing the first set and said second set with said sample;

(e) preparing the mixture of said first set, second set, and sample to measure chemiluminescence from the first set and the second set of chemiluminescent labels;

(f) triggering chemiluminescence from the first set and second set of chemiluminescent labels following preparation of the mixture (e.g., by the addition of one or more triggering compositions which trigger chemiluminescence of the acridinium labels);

(I) measuring the chemiluminescence in the wavelength domain (e.g.. measuring light intensity as a function of wavelength) and in the time domain (e g., measuring light intensity as a function of time from the triggering step);

(g) detecting the presence or calculating the concentration of said at least one analyte by comparing the amount of light emitted with a standard dose response curve which relates the amount of light emitted to a known concentration of the at least one of the multiple analytes.

[0012] The method may detect multiple analytes in a sample. In some embodiments, the two chemiluminescent labels in the first set are capable of forming a binding complex with different analytes in the sample. In various implementations, the two chemiluminescent labels in the second set are capable of forming a binding complex with different analytes in the sample. In some embodiments, different acridinium labels are conjugated to the same analyte or binding partner thereof. In some embodiments, the solid support is conjugated to a first binding partner such as an antibody (e.g., via biotin/streptavidin). The sample may be mixed with one or more of the chemiluminescent labels of the present disclosure independently conjugated to one or more binding partners of one or more analytes of interest. The sample may also be mixed with one or more conjugates (or intermediary conjugates) which also bind to the one or more analy tes of interest and bind to the first binding partner. In this format, the binding complex will be formed by the solid phase binding directly to the intermediary conjugate, which also binds to the analyte. The analyte will also be bound to the chemiluminescent label (via the analyte and the intermediary conjugate). Accordingly, by providing one or more immunoassay reagents comprising multiple chemiluminescent labels and/or one or more intermediary conjugates, detection of multiple analytes may be achieved through a single binding complex.

[0013] The first and second set of acridinium labels may overlap (e.g. , the chemiluminescent acridinium moiety in one set may also be present in another set). For example, in some embodiments, an acridinium label is a member of the first and second set. In some embodiments, a first acridinium label may have a different chemiluminescence wavelength (e.g., as measured by the difference in /.max) than a second chemiluminescent label such that the first and second acridinium label form a first set of w avelength separated acridinium labels. The first chemiluminescent label may also be separated from a third chemiluminescent label in the kinetic domain (e.g., as measured as a difference % RLU measured over an indicated time period) with a third acridinium label such that the first and third acridinium label form the second set of kinetic separated acridinium labels. In some embodiments, each of the two chemiluminescent labels in the first set may have a corresponding chemiluminescent label of different emission speed (e.g.. to form two different second sets of chemiluminescent labels separated in emission speed from four different chemiluminescent labels). In some embodiments, the four chemiluminescent labels are capable of forming a binding complex with different analytes in the sample.

[0014] Various assay formats may be employed which afford the ability to measure many analytes nearly simultaneously (e.g., by collection of the chemiluminescence from a single chemiluminescence event such as one initiated by the addition of one or more triggering agents). In various embodiments, the first set of chemiluminescent labels comprise at least three chemiluminescent labels having emission spectra separated in the wavelength domain. In certain aspects, the three chemiluminescent labels in the first set may be capable of forming a binding complex with three different analytes in the sample. Each of the three chemiluminescent labels in the first set may have a corresponding chemiluminescent label w ith different emission kinetics (e.g., to form three different second sets of chemiluminescent labels separated in emission speed from six different chemiluminescent labels), and each of the six chemiluminescent labels are capable of forming a binding complex with six different analytes in the sample.

[0015] Each set, and each chemiluminescent label within a set, may be mixed with the biological sample in any order. For example, in some embodiments, one or more of the chemiluminescent labels is mixed with the sample individually. In some embodiments, the first set of chemiluminescent labels is mixed with the sample together, such as by the addition of a composition comprising the chemiluminescent labels in the first set. In some embodiments, the second set of chemiluminescent labels is mixed with the sample together, such as by the addition of a composition comprising the chemiluminescent labels in the second set. In some embodiments, the first and second set of chemiluminescent labels is mixed with the sample together, such as by the addition of a composition comprising the chemiluminescent labels in the first set and second set.

[0016] Following addition of the two sets of chemiluminescent labels, the sample is typically- prepared to induce chemiluminesence in a manner that the analytes can be measured and/or their concentration quantified. For example, the preparing step may comprise:

(el) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with said at least one analyte and capable of forming a binding complex with a chemiluminescent label in the first set and/or the second set of chemiluminescent labels; and

(e2) separating said solid support from said mixture.

In some embodiments, the solid support may comprise at least two molecules (e.g.. two, three, four, five, six, seven, eight, nine ten), each capable of forming a binding complex with different analytes and capable of forming a binding complex with at least two different chemiluminescent labels in the first set and/or second set of chemiluminescent labels. In some embodiments, the solid support may comprise molecules capable of forming a binding complex with an analyte or binding partner thereof, wherein, collectively, the molecules can bind with each chemiluminescent label in the first and/or second set. [0017] Typically, the chemiluminescent labels have the structure of formula (T):

wherein A is an analyte or binding partner for an analyte,

L is absent (i.e., it is a bond) or a linker optionally comprising a group L^c or Z^L, and

V is a chemiluminescent acridinium comprising the structure:

“j" and “H” are independently 0 (e.g., all Rz groups are hydrogen, all Rs groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, -R, -X*. -L^c-R -L^c-X^b (e.g., -L_l-X^b), -Z, -R^L-Z, -L^c-Z (e.g., - L_l-Z), or — R^L— L^c— R^L-Z (e.g., -R^L-L_l-R^L-Z);

Rz and Rs are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Rz or Rs groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R₂ or R₃ may comprise a linkage to an imaging agent such as a fhiorophore (e.g., rhodamine);

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);

Z^L is a zwitterionic linker group having the structure:

“m ” is 0 (i.e. it is a bond) or 1;

“n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10;

Z is a zwitterionic group independently at each occurrence has the structure:

“r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

X^a and X^b are independently at each occurrence an anionic group; L_l is independently at each occurrence -O-, -S-, -NH-, -N(R^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 , (CH₂0)_1-10 , (OCH₂CH₂)_1-10 , or -(CH₂CH₂0)_1-10-;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g.. 1-10. 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl (e.g., methyl, ethyl, propyl); and

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). For example, the chemiluminescent labels may independently have the structure of formula (la):

wherein 52 is O or N;

Y is selected from -R or -R^L-Z, or in the case where 52 is O then Y is absent; and

Y’ is either absent (i.e. it is a bond), or is selected from -L_l- -R^L-, -R^L-L_l-, -L1-L1-, -L_l-R^L- -L_l-RL-L_l, and -R^L-L_l-R^L— . In some embodiments, the chemiluminescent labels independently have the structure of formula (lb) or (Ic):

wherein R₄-R₇ are independently hydrogen, an electron donating group, or C1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and Y” is either absent (i.e., it is a bond) or-L^c- -L_l- -R^L-, or-R^L-L_l- In some embodiments, at least one (e g., one, two, three, four, five, six, each) chemiluminescent label in the first set is a zwitterionic acridinium (e.g., N-sulfopropyl zwitterionic acridinium, a compound having the structure of formula I, la, lb, or Ic wherein Ri is selected from -X^b, — R^L-X^b, or — L^c-X^b such as -L_l-X^b; Ri is selected from -SO3", -R^L- SO3" such as -(CH2)_1-5- SO3", or -L^c- SO3" such as -L_l- SO3"). In some embodiments, at least one (e.g., one, two, three, four, five , six, each) chemiluminescent label in the second set is a zwitterionic acridinium (e.g., N-sulfopropyl zwitterionic acridinium, a compound having the structure of formula I, la, lb, or Ic wherein Ri is selected from -X^b, -R^L-X^b, or -I ^C-X^b such as -L_l-X^b, Ri is selected from -SO3", -R^L- SO3" such as (CH ₂)_1-5 SO3", or -L^c- SO3" such as -L_l- SO3"). In some embodiments, at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label in the first and/or second set is an acridinium salt (e.g., acridinium carboxylate salts such as halocarboxylate salts, haloalkyl carboxylate salts, fluoroalkyl carboxylate salts, acridinium sulfonate salts such a halo sulfonate salts, haloalkyl sulfonate salts, fluoroalkyl sulfonate salts, acridinium halide salts such as acridinium chloride salts, a compound having the structure of formula I, la, lb, or Ic wherein Ri is selected from-R, -L^c-R, -Z, -R^L-Z, -L^c-Z,-L_l-Z, -R^L-L^C-R^L-Z, -R^L-L_l-R^L-Z with a negative counterion such as R-COO", R-SO3", Cl", F").

[0018] Measurably differential chemiluminesence between chemiluminesent labels (e.g., in the wavelength domain and/or in the chemiluminesence rate domain) can be achieved through use of acridinium labels with relevant conjugations to induce such a differentian. For example, in some embodiments, at least one of R4-R7 (e.g., R4, IC. R?, R?) are an electron donating group (e.g., and forms a chemiluminescent label with different emission speed as compared to an otherwise identical label without the electron donating group). In some embodiments, one chemiluminescent label in the first set has the structure of formula (Illa):

and another chemiluminescent label in the first set has the structure of formula (IIIb):

wherein A1 and A2 are independently an analyte or binding partner for an analyte and, optionally, A1 is different than A2;

Ω is O or N;

Y is selected from -R or -R^L-Z, or in the case where Ω is O then Y is absent; and

Y’ is either absent (i.e. it is a bond), or is selected from -L_l-, -R^L- -R^L-L_l-, -L1-L1- -L_l-R^L-, -L_l-R^L-L_l, and -R^L-L_l-R^L-;

T is 1, 2, 3, or 4; 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, -R, -X^b, -R^L-X^b. -L^c-R, -L^c-X^b (e.g., -L_l-X^b), -Z, -R^L-Z. -L^c-Z (e.g., - L_l-Z), or -R^L-L^C-R^L-Z (e.g., -R^L-L_l-R^L-Z);

R2 and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent (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, at least one R2 group comprises a linkage to an imaging agent);

L^c is a divalent C1-35 alkyd, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);

Z^L is a zwitterionic linker group having the structure:

“m ” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

“r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10);

X¹ and X^b are independently at each occurrence an anionic group;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g, 1-10, 1-5) points of substitution (e.g, with 1-10 heteroatoms, with 1-10 substituents);

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g, alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g, with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a )_1-10 alkyl;

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g, a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). For example, the chemiluminescent label of formula (IIIb) may have the structure of formula (TUb 1):

In some embodiemnts, at least one R3 is not hydrogen (eg., an electron donating group such as alkoxy). In some embodiments, least one R₃ and/or at least one R₃ group is an electron donating group (e.g., alkoxy). In various implementations, the first set of chemiluminescent labels further comprise a compound having the structure of formula (IIIe):

wherein IA is an imaging agent (e.g., a fluorophore such as Texas Red, rhodamine or phenol modified rhodamine); and

A3 is a different analyte or binding partner for an analyte than A₁ and A₂. In some embodiments, the chemiluminescent label of formula (IIIe) has the structure of formula (Kiel) or (nic2):

For example, the chemiluminescent label may have the structure:

In some embodiments, the chemi luminescent label of formula (IIIb) may have the structure of formula (IIIb3):

wherein IA is an imaging agent (e.g., a fluorophore such as rhodamine or phenol modified rhodamine). For example, the chemiluminescent label of formula (IIbI3) may have the structure of formula (IIMII ) or (IIIb5):

For example, the chemiluminescent label may have the structure:

[0019] In some embodiments, one chemiluminescent label in the first set has the structure of formula (IVa):

and another chemiluminescent label in the first set has the structure of formula (IVc):

wherein A1 and A₂ are independently an analyte or binding partner for an analyte and A1 is different than A₂;

Ω is O orN;

Y is selected from -R or -R^L-Z, or in the case where Ω is O then Y is absent; and Y’ is either absent (i.e. it is a bond), or is selected from -L_l- -R^L-, -R^L-L_l-, -L1-L1-, -L_l— R^L-, — L_l— R^L-L_l, and -R^L-L_l-R^L-;

“k" is 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, -R, -X* R^L X^b, -L^c-R, -L^c-X^b (eg., -Lr-X^b), -Z, -R^L-Z, -L^c-Z (eg., L_l-Z), or — R^L— L^c— R^L-Z (eg., -R^L-L_l-R^L-Z);

Ri is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5- 7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (eg., rhodamine).

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (eg., with 1 to 20 heteroatoms, with 1-20 substituents);

Z^L is a zwitterionic linker group having the structure:

“m” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

“r” is independently an integer from 0 to 10 (eg, from 1 to 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10);

X¹ and X^b are independently at each occurrence an anionic group; L_l is independently at each occurrence -O-, S , -NH-, -N(R^N)-, -(CH2)_1-10-, -S(=O)_1-2-, c c , -C=C-(CH2)_1-3-, c(o) . o c(o) . c(o) (ci b)i -i , (ci b)i .1 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^K)-, -(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)I-₃-S(O)_1-2-N(R^N)-, -(CH₂)I-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- -(CH₂0)_1-10-. -(OCH₂CH₂)_1-10- or -(CH₂CH₂0)_1-10-;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyd, alkenyl, alky nyl, or aralkyl) radical, optionally having one or more (e.g., 1-20. 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alky l (e.g, methyl, ethyl, propyl); and

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g. , a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxy late salt).

[0020] Sets of acridinium labels having differential chemiluminescence in the wavelength domain may involve these specific compound alterations yielding the required differential chemiluminescent signal. In various implementations, the difference between compounds of formula (Illa), formula (Illb), and formula (IIIc) is relegated to the different A₁, A₂, and A3 groups and/or in the conjugation of the acridinium ring system (e.g., R₁, Ω, L, Y, Y”, IA in formulas (Illa), (Illb), and (IIIc) are identical). In some embodiments, the difference between the chemiluminescent labels of formula (IVa) and formula (IVb) is located in the conjugation of the acridinium ring system (e.g., Ri, Ω. L, Y, Y”, IA in formulas (IVa) and (IVb) are identical) and the different A1 and A₂ groups. In various implementations, the chemiluminescent label of formula (I) (e.g., (la). (Ib), (Ic)), (II), (Illa), (Illb) (e.g., formula (IIIbl), formula (IIbI2), formula (IIbI3), formula(IIIb 4)), (IV) (e.g., (IVa), (IVb)) is a salt (e.g., a carboxylate salt such as a halocarboxylate salt, halo alkyl carboxylate salt, fluorocarboxylate salt, fluro alkyl carboxylate salt, F3CCOO" salt, Ri in formula(IIIb 2) is alkyl and the counterion is carboxylate, halocarboxylate, halo alkyl carboxylate, fluorocarboxylate, fluoroalkyl carboxylate, F3CCOO ).

[0021] The present disclosure is partially premised on the identification of stable slow emitting acridinium labels. These stable slow emitting acridinium labels may be formed by alkyl conjugation (e.g., lower alkyl such as (j -i alkyl) placed in the acridinium system (e.g., at the 1 and 3 positions of the acridinium system).

[0022] In some embodiments, at least one chemiluminescent label (e.g., a chemiluminescent label in the second set) has the structure of formula (II):

wherein A is an analyte or binding partner for an analyte,

T is a chemiluminescent acridinium comprising the structure:

“f and “K* are independently 0 (eg., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, -R, -X* 1<^L X^b, -L^c-R, I ^c X^b (e.g., -L_l-X^b), -Z, -R^L-Z, -L^c-Z (e.g.,

La— Z), or — R^L— L^c— R^L-Z (e.g., R' l.i R^L Z):

R2a and R2b are independently selected from alkyl optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);

R2c is hydrogen, -R, an electron donating group, or -Z;

R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 groups may together form a fused heterocyclic group (e.g., 5-7 membered fused heterocyclic group) and wherein R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms);

Z^L is a zwitterionic linker group having the structure:

“/n” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

X¹ and X^b are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g, alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g, with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halo sulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). R2a and R2b may be independently alkyl (e.g., lower alkyl such including C1-4 alkyl as methyl, ethyl, propyl, butyl).

[0023] The stable slow emitting acridinium labels, particularly when in phenyl ester form, may also have slower emission kinetics with electron donating groups placed on the phenyl ester, such as at the 4 position. In some embodiments, one chemiluminescent label in the second set has the structure of formula (Va):

and another chemiluminescent label in the second set has the structure of formula (Vb):

wherein A1 and Aa are independently an analyte or binding partner for an analyte and A1 is different than Aa;

Ω is O orN;

Y’ is either absent (i.e. it is a bond), or is selected from -L_l-, -R^L-, -R^L-L_l-, -L1-L1-, -LL-R^L-, — L_l— R^L-L_l, and -R^L-L_l-R^L-;

Z^L is a zwitterionic linker group having the structure:

“/n” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

X¹ and X^b are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g, alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g, with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g, a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). In some embodiments, one chemiluminescent label in the second set has the structure of formula (Vc):

and another chemiluminescent label in the second set has the structure of formula (Vd):

Ω is O orN;

R₃ and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R₃ or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein Ra or R3 may comprise a linkage to an imaging agent such as a fhiorophore (e.g., rhodamine);

L^c has an electron withdrawing linker (e.g., carboxyl) with respect to the phenyl;

L⁰⁶ is an electron donating linker (e.g., alkyl, alkoxy, alkylamino) with respect to the phenyl;

Z^L is a zwitterionic linker group having the structure:

“m” is 0 (i.e. it is a bond) or 1 ;

Z is a zwitterionic group independently at each occurrence has the structure:

“r’' is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

X^a and X^b are independently at each occurrence an anionic group; L_l is independently at each occurrence -O-, -S-, -NH-, -N(R^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^K)- -(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- -(CH₂0)_1-10-. -(OCH₂CH₂)_1-10- or -(CH₂CH₂0)_1-10-;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alky l, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g.. 1-20. 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents);

R' and R " are independently at each occurrence hydrogen or a C1-10 alkyl;

R' is hydrogen or a C1-10 alkyl; or a salt thereof (e.g. , a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0024] Various assay formats may be utilized through the different sets of acridinium labels described herein. For example, the at least two chemiluminescent acridiniums with wavelength separated chemiluminescence (e.g., acridinium labels in the first set) may have similar chemiluminescence emission rates (e.g., the percentage of light measured at a time point such as 1 second or 2 seconds or 4 seconds or 6 seconds following triggering as compared to total emission is within 10% or within 5% or within 1%). In some embodiments, the at least two chemiluminescent aciidiniums with time domain separated chemiluminescence (e.g., acridinium labels in the second set) may have similar chemiluminescence wavelengths (e.g., λmax within 10% or within 5% or within 1%).

[0025] The chemiluminescent labels are typically conjugated to a binding pair of one of the analytes of interest. The chemiluminescent labels may be formed from a chemiluminescent compound or salt having a reactive functional group for conjugation to the binding pair. For example, the chemiluminescent label 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, 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-b-Alanine,

Texas Red-3 -AM-DMAE-b- 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.

[0026] Compounds are also provided having the structure of formula (V)

wherein A is an analyte or binding partner for an analyte,

L is absent (i.e., it is a bond) or a linker optionally comprising a group L^c or Z^L,

“k” is independently 0 (e.g., all R₃ groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, L_l-Z), or — R^L

R2a and R2b are independently selected from alkyl optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); Rac is hydrogen, -R, an electron donating group, or -Z;

R₃ is independently selected at each occurrence from hydrogen, -R, an electron donating group, and — Z; wherein two vicinal R2 groups may together form a fused heterocyclic group (e.g., 5-7 membered fused heterocyclic group) and wherein R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g, with 1 to 20 heteroatoms);

Z^L is a zwitterionic linker group having the structure:

“m” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

“r” is independently an integer from 0 to 10 (eg., from 1 to 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10);

X¹ and X^b are independently at each occurrence an anionic group;

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0027] Compounds for forming these conjugated labels are also provided. These compounds may have the structure of formula (VI)

wherein RFG is a reactive functional group for conjugating to an analyte or a binding partner for an analyte,

L is absent (i.e., it is a bond) or a linker optionally comprising a group L^c or Z^L, is independently 0 (e.g., all Rs groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, -R, -X*. R¹' X^b _: -L^c-R, I ^c X*¹ (e.g., -L_l-X^b), -Z, -R^L-Z, -L^c-Z (e.g., L_l— Z), or — R^L— L^c— R^L-Z (e.g., -R^L-L_l-R^L-Z); R2a and R2b are independently selected from alkyl optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);

Rzc is hydrogen, -R, an electron donating group, or -Z;

Rs is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Rz groups may together form a fused heterocyclic group (e.g., 5-7 membered fused heterocyclic group) and wherein Rs may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

L^c is a divalent Ci-ss alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms);

Z^L is a zwitterionic linker group having the structure:

“m” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

X¹ and X⁶ are independently at each occurrence an anionic group;

-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(0)_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-, -(CH₂0)_1-10-, -(OCH₂CH₂)_1-10- or -(CH₂CH₂0)_1-10-;

R’ and R” are independently at each occurrence hydrogen or a C1-10 alk d;

R^N is independently at each occurrence from hydrogen or C1-5 alkyd (e.g., methyl, ethyl, propyl); and

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g. , a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalky 1 carboxylate salt, fluoroalky l carboxylate salt).

[0028] For certain assays, there may be an advantage to include positive control and/or negative control, or calibrators into the test. Also, for certain assay combination there may be an advantage to measure the analytes in the same reaction vessel. E.g., if an algorithm like a ratio is required, e.g.. for the measurement of 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 the results may be reduced, because the same sample aliquot is used and the processing errors are the same for each analyte. Therefore, it is desirable to develop an assay method where multiple analytes present in a sample can be detected or measured in one test reaction. On the other hand, there are a number of diagnostic assays that are frequently tested as a group, such as a thyroid test panel, the Enhanced Liver Fibrosis (ELF™) test panel, the fertility hormone test panel, the cancer marker screen panel, and HIV antibody and antigen panels.

[0029] In another aspect of the invention, a reagent is provided for the detection of multiple analytes comprising a detectable conjugate bound to a chemiluminescent acridinium, wherein different detectable conjugates are bound to different chemiluminescent acridiniums which have chemiluminescence separated in the wavelength domain and/or the time domain (e.g., fast reaction kinetics such as 100% of chemiluminescence is emitted within 1 second or 2 seconds or 4 seconds of triggering, slow reaction kinetics such that 100% of chemiluminescence is not emitted until after 1 second or after 2 seconds or after 4 seconds or after 6 seconds). The detectable conjugate may comprise one or more (e.g, one. two) zwitterionic functional groups. The reagent may comprise a concentration of each detectable conjugate of from 10 to 30 ng/mL. Reagents of the present disclosure include compositions comprising the indicated components, and optionally an excipient, carrier, or solvent. The reagents of the present disclosure may include a surfactant.

[0030] Compositions such as immunoassay reagents for use in the methods of the present disclosure may comprise at least three chemiluminescent acridiniums, wherein each chemiluminescent acridinium is conjugated to a different analyte or a different binding pair for an analyte, and the chemiluminescence of at least two chemiluminescent acridiniums are separated in the wavelength time domain and the chemiluminescence of a different set of at least two chemiluminescent acridiniums are separated in the time domain. In some embodiments, the composition may comprise at least four chemiluminescent acridiniums conjugated to a different analyte or binding pair for an analyte, wherein the chemiluminescence of three chemiluminescent acridiniums is separated in the wavelength domain. In various implementations, at least two chemiluminescent acridiniums with wavelength separated chemiluminescence have similar chemiluminescence emission rates. In some embodiments, at least two chemiluminescent acridiniums with time domain separated chemiluminescence have similar chemiluminescence wavelengths. In some embodiments, the composition comprises a compound having the structure of formula (V).

[0031] These and other aspects of the invention will be better understood by reference to the following detailed description including the appended claims. BRIEF DESCRIPTION OF FIGURES

[0032] FIG. 1 provides an illustration of a [3x2] assay chemiluminescence spectra from a sample measuring signal from six different acridinium labels nearly simultaneously (e.g., as from a single triggering event).

[0033] FIGS. 2A-J provide exemplary acridinium selection for use in the assay formats described herein. FIGS. 2A-H provide exemplary [2x2] assays and FIGS. 2I-J provide exemplary [3x2] assays.

[0034] FIGS. 3A-J provide exemplary acridinium selection for use in the assay formats described herein. FIGS. 3A-H provide exemplary [2x2] assays and FIGS. 3I-J provide exemplary [3x2] assays.

[0035] FIG. 4 illustrates the results of comparative chemiluminescence kinetic measurements of fast and slow acridinium labels as produced by alky l conjugation of the acridinium ring (e.g., alkyl conjugation at the 1 and 3 positions).

[0036] FIG. 5 illustrates the results of comparative chemiluminescence kinetic measurements of fast and slow acridinium labels as produced by electron donating groups installed at the 4 position if the phenyl ester of an acridinium compound.

DETAILED DESCRIPTION

[0037] For convenience, certain terms employ ed in the specification, including the examples and appended claims, are collected here. Unless defined otherwise, 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.

[0038] Unless otherwise explicitly defined, the following terms and phrases are intended to have the following meanings throughout this disclosure:

[0039] All percentages given herein refer to the weight percentages of a particular component relative to the entire composition, including the carrier, unless otherwise indicated (e.g., a percentage may relate to the total percentage of chemiluminescence by which is meant the amount of chemiluminescence measured from a composition following triggering). It will be understood that the sum of all weight % of individual components within a composition will not exceed 100%.

[0040] The terms “a” or '‘an,” as used in herein means one or more. As used herein, the term “consisting essentially of’ is intended to limit the invention to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention, as understood from a reading of this specification. Recitations of “comprising” include “consisting essentially” and '‘consisting.”

[0041] The following definitions of various groups or substituents are used, unless otherwise described. Specific and general values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents. Unless otherwise indicated, alky l, alkenyl, alkynyl, alkoxy, and the like denote straight, branched, and cyclic groups, as well as any combination thereof.

[0042] The term hydrocarbon may refer to a radical or group containing carbon and hydrogen atoms which may be bound at an indicated position (e.g., R, R‘, R”, R^N, Y, Y’, Ω. L_l, L^c, R^L, Examples of hydrocarbon radicals include, without

limitation, alkyd, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-ary 1, and any combination thereof (e.g., alkyl-ary 1-alkyl). As used herein, unless otherwise indicated, hydrocarbons may be monovalent or multivalent (e.g. , divalent, trivalent) hydrocarbon radicals. A radical of the form -(CH2)n-, including a methylene radical, i.e., -CH2-, is regarded as an alkyl radical if it does not have unsaturated bonds between carbon atoms. Unless otherwise specified, all hydrocarbon radicals (including substituted and unsubstituted alkyl, alkenyl, alkynyl, ary 1, aryl-alkyl, alkylaryl) may have from 1-35 carbon atoms. In other embodiments, hydrocarbons will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Hydrocarbons may have from 2 to 70 atoms or from 4 to 40 atoms or from 4 to 20 atoms.

[0043] A substituted hydrocarbon may have as a substituent one or more hydrocarbon radicals, substituted hydrocarbon radicals, or may comprise one or more heteroatoms. Any hydrocarbon substituents disclosed herein (e.g, R, R’, R”, R^N, Y, Y’, Ω, L_l, L^c, R^L, R^c, Ri,

may optionally include from 1-20 (e.g, 1-10, 1-5) heteroatoms. Examples of substituted hydrocarbon radicals include, without limitation, heterocycles, such as heteroaryls. Unless otherwise specified, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorous, halogen (e.g., F, Cl, Br, I), boron, or silicon. In some embodiments, heteroatoms will be selected from the group consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen (e.g., F. Cl, Br, I). In certain embodiments, the heteroatoms may be selected from O, N, or S. In some embodiments, a heteroatom or group may substitute a carbon. In some embodiments, a heteroatom or group may substitute a hydrogen. In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms in the backbone or chain of the molecule (e.g.. interposed between two carbon atoms, as in '‘oxa”). In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms pendant from the backbone or chain of the molecule (e.g. , covalently bound to a carbon atom in the chain or backbone, as in “oxo"’).

[0044] When an indicated group is substituted with an indicated substituent, the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl, the group may contain one or more unsubstituted C1-C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. If an indicated group is used multiple times in chemical genus (e.g., R groups), it will be understood that each group is independently selected at each occurrence.

[0045] Unless otherwise specified, any compound disclosed herein which has one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may exist as pure or substantially pure (e.g. , great than 98% ee) R or S’ enantiomers with respect to each chiral center, or may exist as mixtures of R and S enantiomers with respect to each chiral center, wherein the mixture comprises an enantiomeric excess of one or the other configurations, for example an enantiomeric excess (of R or <S) of more than 60% or more than 70% or more than 80% or more than 90%. or more than 95%. or more than 98%. or more than 99% enantiomeric excess. In some embodiments, any chiral center may be in the “S’" or “R^: configurations.

[0046] It will be understood that the description of compounds herein is limited by principles of chemical bonding. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding such as regard to valencies, and to give compounds which are not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon.

[0047] Substituent (radical) prefix names may be derived from the parent hydride by either (i) replacing the “ane” or in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl;” or (ii) replacing the “e” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl,” (here the atom(s) with the free valence, when specified, is (are) given numbers as low as is consistent with any established numbering of the parent hydride). Accepted contracted names, e.g, adamantyl, naphthyl, anthryl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, and trivial names, e.g, vinyl, allyl, phenyl, and thienyl are also used herein throughout.

[0048] Alkyl groups typically refer to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, Ci- Ce alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionally substituted, e.g., by one or more substituents. Examples of alkyl groups include without limitation methyl, ethyl, «-propyl, /.sopropyl. and tert-butyl. Any alkyl group referenced herein (e g, R, R’, R”, R^N, Y, Y’, Ω, L_l, L^c, R^L, R^c, Ri, R₂, R_2a, Ra, R_2c, R3, R₄, Rs, Re, R7) may have from 1-35 carbon atoms. In other embodiments, alkyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five. six. seven, eight, nine, or ten carbon atoms. Alkyl groups may be lower alkyl (e.g., C1-C4 alkyl).

[0049] Haloalkyl groups are typically alkyl groups where at least one hydrogen atom is replaced by halo. In some embodiments, more than one hydrogen atom (e.g, 2. 3, 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, or 14) are replaced by halo. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g, fluoro) or the hydrogen atoms can be replaced by a combination of different halogens (e.g., fluoro and chloro). Haloalkyl may include alky l moieties in which all hydrogens have been replaced by halo (sometimes referred to herein as perhaloalkyl. e.g, perfluoroalkyl, such as trifluoromethyl). Haloalkyl groups may be optionally substituted.

[0050] Typically, alkoxy groups have the formula -O(alkyl). Alkoxy can be, for example, methoxy (-OCHs). ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2- pentoxy, 3-pentoxy, or hexyloxy. Likewise, the term “thioalkoxy’’ refers to a group of 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,” employed alone or in combination with other terms, refers to a group of formula - OH. Any alkoxy, thioalkoxy, or haloalkoxy group referenced herein (e.g, R, R’, R”. R^N. Y,

may have from 1-35 carbon atoms. In other embodiments, alkoxy, thioalkoxy, or haloalkoxy groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Alkoxy groups may be lower alkoxy (e.g., C1-C4 alkoxy).

[0051] Aralkyl groups typically refers to groups where an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbons of the alkyl moiety serves as the point of attachment of the aralkyl group to another moiety. Any ring or chain atom can be optionally substituted, e.g, by one or more substituents. Non-limiting examples of aralkyl include benzy l, 2-phenylethyl, and 3-phenylpropyl groups.

[0052] The term “alkenyl” may 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 can be optionally substituted, e.g., by one or more substituents. Alkenyl groups can include, e.g.. vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent. Any alkenyl group referenced h

may have from 1-35 carbon atoms. In other embodiments, alkenyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six. seven, eight, nine, or ten carbon atoms.

[0053] The term alkynyl may refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups

Re, R?) can be optionally substituted, e.g., by one or more substituents. Alkynyl groups can include, e.g., ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons can optionally be the point of attachment of the alkynyl substituent.

[0054] The term heterocyclyl typically refers to a fully saturated, partially saturated, or aromatic monocyclic, bicyclic, tricyclic, or other polycyclic ring system having one or more constituent heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups (e.g., R^N) may be present to complete the nitrogen valence and/or form a salt), or S. The heteroatom or ring carbon can be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, e.g.. with one or more substituents (e g. heteroatoms or substituent groups X). Heterocyclyl groups can include, e.g., tetrahydro furyl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, the phrase “heterocyclic ring containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms is independently selected from N, NH, O, and S: and

wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected R” would include (but not be limited to) tetrahydro furyl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0055] The term heterocycloalkenyl typically refers to partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups having one or more (e.g., 1-4) heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and/or form a salt), or S. A ring carbon (e g., saturated or unsaturated) or heteroatom can be the point of attachment of the heterocycloalkenyl substituent. Any atom can be optionally substituted, e.g., by one or more substituents. Heterocycloalkenyl groups can include, e.g., dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4.5-dihydro-lH-imidazolyl, 1.2.5.6-tetrahydro- pyrimidinyl, and 5,6-dihydro-2H-[l,3]oxazinyl.

[0056] Cycloalkyl groups may be fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. Any atom can be optionally substituted, e.g., by one or more substituents. A ring carbon serves as the point of attachment of a cycloalkyl group to another moiety. Cycloalkyl moieties can include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbomyl (bicycle[2.2.1]heptyl).

[0057] Cycloalkenyl groups may be partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ring carbon (e.g. , saturated or unsaturated) is the point of attachment of the cycloalkenyl substituent. Any atom can be optionally substituted, e.g, by one or more substituents. Cycloalkenyl moieties can include, e.g., cyclohexenyl, cyclohexadienyl, or norbomenyl.

[0058] Aryl groups are often aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon ring system. One or more ring atoms can be optionally substituted, e.g, by one or more substituents. Aryl moieties include, e g., phenyl and naphthyl.

[0059] Heteroaryl groups typically are aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and/or form a salt), or S in the ring. One or more ring atoms can be optionally substituted, e.g. , by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyL 4H- quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, 0-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazoly l, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl. naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl. thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl. and xanthenyl.

[0060] In general, when a definition for a particular variable includes both hydrogen and non-hydrogen (halo, alkyl, and) possibilities, the term “substituent(s) other than hydrogen” refers collectively to the non-hydrogen possibilities for that particular variable, unless otherwise specified.

[0061] In general, the limits (end points) of any range recited herein are within the scope of the invention and should be understood to be disclosed embodiments. Additionally, any half- integral value within that range is also contemplated. For example, a range of from 0 to 4 expressly discloses 0, 0.5, 1. 1.5, 2, 2.5, 3, 3.5, 4, and any subset within that range (e.g.. from 1 to 2.5).

[0062] The term “substituent” may refer to a group “substituted” on, on a hydrocarbon (e.g. , an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, heteroaryl) group at any atom of that group, typically replacing one or more hydrogen atoms therein. In one aspect, the substituent(s) on a group (e.g., R, R’, R”, R^N, Y, Y’, Ω. L_l, L^c, R^L, are independently any one single, or any

combination of two or more of the permissible atoms or groups of atoms delineated for that substituent. In another aspect, a substituent may itself be substituted with any one of the above substituents. In some embodiments, an indicated substituent is not further substituted. Further, as used herein, the phrase “optionally substituted” means unsubstituted (e.g., substituted with an H) or substituted. It is understood that substitution at a given atom is limited by valency. Common substituents include halo (e.g. F), C1-12 straight chain or branched chain alkyl. C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, C3-12 heteroaryl, C3-12 heterocyclyl, C1-12 alkylsulfonyl, nitro, cyano, -COOR, -C(O)NRR’, -OR, -SR, -NRR’, and oxo, such as mono- or di- or tri-substitutions with moieties such as trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl. imidazoyl. and the like, each optionally containing one or more heteroatoms such as halo, N, O. S, and P. R and R’ are independently hydrogen, C1-12 alkyl, C1-12 haloalkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-24 cycloalkydalkyl, C6-12 aryl, C7-24 aralkyl, C3-12 heterocyclyl, C3-24 heterocyclylalkyl, C3-12 heteroaiyl, or C4-24 heteroarylalkyl. Unless otherwise noted, all groups described herein optionally contain one or more common substituents, to the extent permitted by valency. The term “substituted” typically means that a hydrogen and/or carbon atom is removed and replaced by a substituent (e.g., a common substituent). The use of a substituent (radical) prefix names such as alkyl without the modifier “optionally substituted” or “substituted” is understood to mean that the particular substituent is unsubstituted. However, the use of “haloalkyl” without the modifier “optionally substituted” or “substituted” is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo and any other associated substitutions as necessary'. Any hydrocarbon described herein may be considered optionally substituted. [0063] When a moiety of the compounds of the present disclosure are described as an analyte or a binding partner thereof it will be understood that a covalent linkage is formed with an analyte or binding partner thereof (eg., using the reactive functional groups which form covalent linkages), for example, by replacing a hydrogen on the unconjugated analyte or binding partner thereof with a covalent bond to the indicated moiety. The covalent linkage on the analyte or binding partner thereof may be formed, for example, at a group on the analyte, binding partner thereof, or derivatized version of the analyte containing a group for forming a linkage. The group may be, for example, an amine group, a thiol group, a carboxy group, a maleimidyl group, or a carbohydrate group. For example, if a covalent linkage is formed through a primary amine of the analyte or binding partner thereof, the compound may have the structure:

where the unconjugated analyte or binding partner A has the structure

[0064] In some embodiments, any hydrocarbon or substituted hydrocarbon disclosed herein

aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), aryl-alkyl (e.g., tolyl) In some embodiments, X may comprise a Ci-Cs or Ci-Ce or C2-C4 perfluoroalkyl. In some embodiments, X may be a Ci-Cs or C2-C6 or C3-C5 heterocycle (e.g., heteroaryl radical). The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine. In some embodiments, X is independently selected at each occurrence from -OH, -SH, -NH2, -N(R*)2, -C(O)OR*, - C(O)NR*R*, -C(O)NR*R*. -C(O)OH, -C(0)NH2, F, or -Cl. In some embodiments, X is F. R and R* may be, independently at each occurrence, saturated or unsaturated alkyl (e.g., Ci- Cs alkyl). 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.

[0065] L^c may have the structure:

-(XI)O-I-(R^L)O-5-(X2)O-I- (R^L)O-5- (XS)O-I- (R^L)o-5- (X4)O-I- (R^L)O-5- wherein Xi is selected from =N-, -O-, -S-. or -NR^N-;

X2-X4 are independently 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-; and

R^L is independently selected at each occurrence from -CH2-, -(CH2CH2O)-, or -(OCH2CH2)-. In various embodiments, L^c comprises at least one atom (or at least two atoms) in the chain between A and T (or between A and Z^L).

[0066] Anionic groups, such as X^a and X^b may be, for example, independently at each occurrence carboxylate (-C(O)O’), sulfonate (-SO3 ), sulfate (-OSO3 ), phosphate (- OP(O)(OR^p)O ), or oxide (-O’), and R^p is hydrogen or C1-12 hydrocarbon optionally having one or more (e.g., 1-10, 1-5) points of substitution (e g, with 1-10 heteroatoms, with 1-10 substituents). For example, Ri may comprise (or be) -R^L-SO₃ (e.g., sulfopropyl). In some embodiments, Ri comprises (or is) sulfopropyl. In some embodiments, Ri is -S(O)2-NH-Z or -(CH2)i-3-S(O)2-NH-Z. In various implementations, R2 and R3 are independently at each occurrence hydrogen, alkyl, or alkoxy (e.g, lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). 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 alkoxy (e.g., lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, X¹ is sulfonate (— SO₃ ), m is 1 , R^L is propyl, and n and p are each 3. For example, Z^L may have the structure:

[0067] The compounds may be used to detect for the presence of a material in a sample such as an analyte (e.g., a biomolecule). In some embodiments, the analyte is a thyroid hormone (e.g. , a thyroid stimulating hormone and, for example, A is a binding partner therefor such as an anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), an androgen, a steroid hormone (e.g., androstenedione, testosterone), a troponin, thyroglobulin, anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine- binding globulin (TBG), neurofilament light chain (e.g., serum neurofilament light chain), a vitamin (e.g, vitamin-D such as 25-hydroxy- vitamin D), or an antibody for a virus (e.g., hepatitis).

[0068] Compounds for forming the conjugates are also provided. For example, the compound (e.g., a compound for conjugating with an analyte or binding partner of an analyte such as a peptide, a protein, or a macromolecule including an antibody) may have the structure the structure of formula (VI)

L is absent (i.e., it is a bond) or a linker optionally comprising a group L^c or Z^L, “k” is independently 0 (eg., all R3 groups are hydrogen), 1, 2, 3, or 4;

R2c is hydrogen, -R, an electron donating group, or -Z;

R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and — Z; wherein two vicinal Rs groups may together form a fused heterocyclic group (e.g., 5-7 membered fused heterocyclic group) and wherein R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (eg., with 1 to 20 heteroatoms);

Z^L is a zwitterionic linker group having the structure:

“m” is 0 (i.e. it is a bond) or 1;

“ri” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10;

Z is a zwitterionic group independently at each occurrence has the structure:

X¹ and X^b are independently at each occurrence an anionic group; L

R is independently at each occurence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). For example, the reactive functional group (RFG) may be selected from:

[0069] In some embodiments, the compounds of the present disclosure may be zwitterionic and include one or more zwitterionic groups. For example, the Ri group attached to the positively charged nitrogen of the acridinium may optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F) and therefore may in combination with the positively charged acridinium nitrogen atom, constitute a zwitterionic group. For example, a sulfopropyl or sulfobutyl group attached to the acridinium nitrogen may form a zwitterionic pair. The Ri group may also be neutral (e.g., lower alkyl such as methyl) or by itself be zwitterionic (e.g., Ri is — Z, -R^L-Z, -LS-Z, or -R^L-LB-R^M-Z). In some embodiments, Ri has the structure:

When the acridinium label is charged (e.g., Ri has a net neutral charge), the compound may be in its salt form and optionally include a counterion to balance the positively charged nitrogen

[0070] The substituents on the chemiluminescent acridinium ester may be modified to vary the rate and yield of light emission, to reduce the non-specific binding, increase stability, or increase hydrophilicity. Typically, these modifications will have minimal interference substantially with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed in Natrajan et al. in U.S. Pat No 7,309,615, hereby incorporated by reference herein, which describes high quantum yield acridinium compounds containing electron donating groups such as alkoxy groups (OR*) at, for example, C2 and/or C7, wherein R* is a group comprising a sulfopropyl moiety or ethylene glycol moieties (e.g., - or combinations thereof. In some embodiments,

and/or R3 may be independently at each occurrence hydrogen an electron donating group such as an alkoxy groups Natrajan et al. in

International Pub. No. WO2015/006174, hereby incorporated by reference in its entirety, also describes hydrophilic high quantum yield, chemiluminescent acridinium esters possessing certain eleclron-donaling functional groups at the C2 and/or C7 positions as well. These electron donating groups (-OG) may have the structure:

[0071] ψ may comprise two flanking methyl groups on a phenolic ester to stabilize the bond as disclosed in Law et al. Journal of Bioluminescence and Chemiluminescence 4: 88-89 (1989), hereby incorporated by reference in its entirety. The sets of chemiluminescent labels may be modified in a manner to achieve the proper separation of chemiluminescent wavelength or emission. In some embodiments ψ (in one or more of the sets of chemiluminescent labels) has the structure:

,

[0072] In some embodiments, A, L and ψ are each covalently linked. Portions of the covalent linkage between A and ψ may be formed from a reactive functional group for forming covalent linkages with a peptide, a protein, or a macromolecule, wherein the functional group comprises an electrophilic group, nucleophilic group, or a photoreactive group. The reactive functional group may an amine-reactive group, a thiol-reactive group, a carboxy-reactive group, a maleimidyl-reactive group, or a carbohydrate-reactive group. In some embodiments, the reactive functional group may react with a functional group of the analyte or binding partner therefore such as a primary amine. The reactive functional group may comprise (or be) an isiothiocyanate, isocyantate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, fluorophenyl ester, or combinations thereof. In various implementations, the reactive functional group labels the analyte or binding partner therefor through acylation or alkylation. For example, the linkage may be formed from a reactive group selected from:

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 moiety thereof (e.g., L^c, ψ) comprises at least one — NH-T2(O)— or -X)(O)-NH- linker group.

[0073] The covalent linkage between A and Y (e.g. , L) or RFG and T (e.g. , L) may comprise (or be) a divalent C1-20 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br). In some embodiments L comprises a zwitterionic linker. L may have the structure — L^C-(Z^L)Z-, wherein z is 0 or 1. L^c may have the structure

R^L is independently selected at each occurrence

; with, for example, the proviso that L^c comprises at least one atom (or at least two atoms) in the chain between A and V (or between A and Z^L).

[0074] In some embodiments, L and/or ψ comprises C(O) NI I . In some embodiments, L^c has the structure:

[0075] The detectable label may coirprise a dimethyl acridinium ester (DMAE) moiety and a zwitterionic linker comprising a zwitterionic linker or a polyethylene glycol derived linker to improve properties of the compound. Such properties as non-specific binding, hydrophilicity, or compound stability may be improved when T comprises a zwitterionic linker or a polyethylene glycol derived linker or a dimethyl phenyl ester. In some embodiments, Z^L has the structure:

In several embodiments, R’ is hydrogen or lower alkyl (e.g., methyl, ethyl, propyl).

[0076] Exemplary compounds for forming the conjugates are disclosed in Table 1. In some embodiments, the detectable conjugate is formed by reacting a compound (e.g., a compound of Formula (V), a compound from Table 1 , a compound from Table 1 with a different reactive functional group (RFG) or -L-RFG in place of, for example, the benzyl ester or the N- hydroxysuccinimid (NHS) ester) with an analyte, binding partner thereof, or derivatized version of the foregoing capable of reacting with a reactive functional group). Suitable sets of acridinium labels may be prepared, for example, by alkylating the 1,3 positions of the acridinium ring system, by attaching a fluorophore to the 2 or 3 position of the acridinium ring system, by installing electron donating groups (e.g., -OG) on the acridinium ring system (e.g., at the 2 and/or 6 positions), by installing electron donating groups (e.g., -OG, -O-, -(CH2) 0-5CH3, -NH-) on the phenyl ester (e.g., at the 2 and/or 6 positions such as with -OG, at the 4 position (e g., with a divalent electron donating group conjugated to the linker such as -O-, -(CH2) 0-5CH3, -NH-), by converting the acridinium ring system into a four membered acridinium system, or combinations thereof). The compounds may be an acridinium ester (“AE”). The compounds designations may include “Z” which may refer to a zwitterionic linker, ”CMO" which may refer to a carboxy methyl oxime linker, '’CME" which may refer to a carboxy methyl ether linker, ' CETE which may refer to carboxy ethyl thioether, ”ZAE ‘ which may refer to a zwitterionic acrinidium ester (which is typically an N-sulfopropyl (“NSP”) dimethyl acridinium ester in the examples shown ("NSP-DMAE")). “ISODIZAE” which may refer to an acridinium nucleus with an isopropoxy functional group attached thereto and a full zwitterionic group (comprising both N⁺ and X ) attached to the positive N of the acridinium.

Table 1

[0077] In some embodiments, the detectable conjugate may have the structure of one or more of:

wherein z is independently at each occurrence 0 or 1 ; y is independently at each occurrence 0, 1, 2, 3, 4, or 5; and

A’ is the analyte or binding partner thereof conjugated via a primary amine of an unconjugated analyte or binding partner thereof A; wherein Xi is selected from -O-, -Sr-, -NR^N-, -0(0)-, -NR^N-0(0)-, -C(O)-NR^N-, C(O)-, or C(O) O , -8-0(0)-, or-C(O)-S-, =N-, -O-, or-S-;

X2-X4 are independently selected from -O-, -S-, -NR^N- -0(0)-, -NR^N-C(O)-, -0(0)- NR^N- -0-0(0)-, or -0(0)-0-, -S-O(O)-, or C(O) S ; and R^L is independently selected at each occurrence from -(CH2)_1-5-, -(CH₂CH₂O)i-5-, or - (OCH₂CH2)l-5-.

[0078] In the wavelength domain, signals from different AEs with distinguishable emission wavelength can be measured individually with proper selection of detector and optical filter. In the time detection domain, signals from different AEs with distinguishable emission kinetics can be measured at different time frames. In combining both wavelength and time domains of measurement, the detection or measurement method of the present invention consists of a “Two-Dimensional Detection” or 2D-Detection.

[0079] As represented in FIG. 1. a group of six acridinium esters can be grouped into a [3x2] mixture. Number “3” in this bracket nomenclature indicates that three AEs that have distinguishable emission maxima which can be measured at the wavelength domain in each wavelength separated set. Number “2” indicates two AEs that have the same emission maxima but different emission kinetics (slow and fast) which can be measured at the time domain in each time domain separated set. In FIG. 1, two sets of wavelength separated acridinium labels are shown (set 1 includes AE1, AE2, and AE3 and set 2 includes AE3, AE4, and AE5). As can be seen, each set has similar emission kinetics (e.g., slow' emission), but are separated in the wavelength domain with a minimum separation of peaks, for example, 30 nm or 40 nm or 50 nm or 60 nm or 70 nm or 80 nm or 100 nm (e.g., from 30 nm to 400 nm, from 40 nm to 400 nm, from 50 nm to 400 nm, from 60 nm to 400 nm, from 70 nm to 400 nm, from 80 nm to 400 nm, from 100 nm to 400 nm). Furthermore, three different sets of emission separated but similar wavelength (e.g., a /.max difference of less than 30 nm) acridinium labels are used (AE1 and AE4; AE2 and AE5; AE3 and AE6). By choosing acridinium labels in this manner, all signals in one reaction, such as the six signals shown here, can be individually measured in a tw o-dimensional detection scheme (the two dimensions being w avelength and emission speed). As can be seen, acridinium labels that are the member of one set (e.g., a set of wavelength separated acridinium labels) may also be a member of another set (e.g.. a set of emission separated acridinium labels). In some embodiments, the systems of the present invention utilize [mxn] detection schemes, 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 formats. In some embodiments, one or more wavelength separated acridinium labels is not a member of a kinetic separated set. In some embodiments, one or more kinetic separated label is not a member of a wavelength separated set. [0080] Exemplary selections of acridinium labels for the detection of multiple analytes are provided in a matrix format are provided in FIG. 2A-J. In these figures, relative alterations for the selections of acridinium labels are provided. Typically, compounds in the same horizontal row have different emission maxima (e.g., /.max i - Xmax2 > 30 nm) but similar reaction kinetics (e.g., fast kinetics, slow kinetics) and compounds in the same vertical row have different reaction kinetics (e.g., fast kinetics and slow kinetics). In some embodiments, the compounds in each vertical row have different reaction kinetics (e.g.. fast kinetics and slow kinetics) and similar emission maxima (e.g., λmax 1 - λmax2 < 30 nm or /λmax 1 - λmax2 < 20 nm or λmax 1 - λmax2 < 10 nm). These compounds may be used for the detection of various analytes (e.g., Ai, A2, A3, A4, As, Ae) or binding partners thereof. In these figures, Ri, Gi, G2, L, L^c, IA, RFG, are as described herein. In particular embodiments, Ri is NSP or methyl (and the compound is a salt).

[0081] In FIGS. 2E-2J, compounds with the reactive functional group for forming a conjugation with an analyte or binding partner thereof are

provided. When used in an assay, these acridinium labels are typically conjugated with the appropriate conjugate for detection. In some embodiments, each variable (e.g., Ri, Gi, G2, L, L^c, RFG, IA) is the same in the multiple acridinium labels used in the assay. In some embodiments, particularly the embodiments involving imaging agents (IA) such as rhodamine and Texas Red, Ri may be different in the acridinium labels. For example, acndimum labels without the imaging agent may have zwitterionic acridiniums

such as NSP) and acridinium labels with the imaging agent are in salt form (e.g., Ri is alkyd such as C1-C4 alkyl). Specific acridinium labels which may be conjugated to analytes or binding partners thereof (e.g., Al, A2. A3, A4, A5, A6) in various [2x2] and [3x2] assay formats are provided in FIGS. 3A-J. In various implementations A1-A6 are each different analytes or binding partners thereof.

[0082] The acridinium labels may also be characterized by their wavelengths of chemiluminescent emission. For example, some acridinium labels may have an emission w avelength maxima ( λmax) of from 430 nm to 460 nm or from 460 nm to 490 nm or from 490 nm to 520 nm or from 520 nm to 550 nm or from 550 nm to 580 nm or from 580 nm to 610 nm or from 610 nm to 640 nm or from 640 nm to 670 nm or from 670 nm to 700 nm or from 700 nm to 730 nm or from 730 nm to 760 nm or from 760 nm to 790 nm or from 790 nm to 820 nm or from 820 nm to 850 nm. For example, in some embodiments, one compound in a wavelength separated set has a Xmax of from 400 nm to 500 nm (e.g.. the compounds in the first column of FIGS. 2A-J and 3A-J) and another compound in the wavelength separated set has a Xmax of from 500-600 nm (e.g., the compounds in the second or third column of FIGS. 2A-J and 3A-J). In some embodiments, one compound in a wavelength separated set has a Xmax of from 400 nm to 500 nm (e.g., the compounds in the first column of FIGS. 2A-J and 3 A- J) and another compound in the wavelength separated set has a Xmax of from 600-700 nm (e.g.. the compounds in the second or third column of FIGS. 2A-J and 3A-J). In some embodiments, one compound in a wavelength separated set has a Xmax of from 400 nm to 500 nm (e.g., the compounds in the first column of FIGS. 2A-J and 3A-J) and another compound in the wavelength separated set has a λmax of from 700-800 nm (e.g.. the compounds in the second or third column of FIGS. 2A-J and 3A-J). In some embodiments, one compound in a wavelength separated set has a Xmax of from 500 nm to 600 nm (e.g., the compounds in the first column of FIGS. 2A-J and 3A-J) and another compound in the wavelength separated set has a Xmax of from 600-700 nm (e.g., the compounds in the second or third column of FIGS. 2A-J and 3A-J). In some embodiments, one compound in a wavelength separated set has a Xmax of from 500 nm to 600 nm (e.g., the compounds in the first column of FIGS. 2A-J and 3A-J) and another compound in the wavelength separated set has a Xmax of from 700-800 nm (e.g., the compounds in the second or third column of FIGS. 2A-J and 3A-J). In some embodiments, one compound in a wavelength separated set has a Xmax of from 600 nm to 700 nm (e.g.. the compounds in the first column of FIGS. 2A-J and 3A-J) and another compound in the wavelength separated set has a Xmax of from 700-800 nm (e.g., the compounds in the second or third column of FIGS. 2A-J and 3A-J).

[0083] Chemiluminescence from multiple acridinium labels with different emission wavelengths can be measured by using multiple photomultiplier tubes (PMT), each of the PMT’s equipped with an optical filter that allows the light from an acridinium ester of interest to pass through while blocking the unwanted light from other acridinium labels such as acridinium esters. In some embodiments, a single PMT can be utilized with a filter wheel installed in the front of the PMT’s detection window. The filter wheel may be mounted with multiple filters corresponding to, for example, the number of acridinium labels used for chemiluminescence, wherein each filter may allow the light from an acridinium label of interest to pass through while blocking the unwanted light from other AEs. Yet another alternative detector is a charge-coupled device (CCD), where the pixels can be grouped into several sections of the two dimensional detector, each section corresponding to the number of AEs to be detected. The chemiluminescence may pass through a grating, such that the wavelength separation may occur along the detector (e.g.. CCD detector) and the images can be analyzed accordingly. Each grouped section of the pixels can have a specific optical filter that allows the light from an acridinium ester of interest to pass through while blocking the unwanted light from other AEs. Other ty pes of detection systems are available for detection or measurement of lights of AEs with different emission profiles.

[0084] The presence of overlapping signals due to emission wavelengths of acridinium esters can be minimized by proper selection of optical filters, such as long pass and short pass filters for detection of two AEs and bandpass filters for detection of three or more AEs. The residual overlapping signals after the use of filters can be deconvolved by an algorithm or artificial intelligence (Al) which can measure emission wavelength profiles of individual AEs at multiple different wavelengths. For example, the emission wavelength profile of acridinium labels having similar emission kinetics can be fit to a summation of typical chemiluminescence distributions (e.g., Normal distributions, Gaussian distribution, Poisson distribution, combinations thereof) to identify the peak and width of each individual acridinium label contribution. The signal of an acridinium label may be ascertained by subtracting the overlapping signal due to the stray light from the unwanted signal which could include noise and signal from other acridinium labels present in the system. The differentiation may be further improved by leveraging the different emission kinetics and measuring the emission wavelength profiles of the individual AEs at multiple wavelengths at different points in time. Through algorithms or Al, trained on data sets involving various sets of acridinium labels, the different kinetic changes together with the multiple wavelength detection can further improve the differentiation of overlapping signals. The multiple analyte detection schemes of the present disclosure offer increased sensitivity and a wider variety of multiple analyte measurement.

[0085] One important consideration is for the acridinium labels have emission kinetics of sufficient separation. The measurement of individual lights can be made at a different time point and duration following chemiluminescent triggering to afford different measurement between kinetic separated sets. This can be done by turning on and off the detector at a particular time, or by collecting light in small intervals (binning) and property grouping and binning with an appropriate time width to allow precise measurement of light at a particular time frame. For example, for emission separated acridinium label sets where most emission of one acridinium label occurs in one time domain (e.g., fast emission such as more than 90% more than 95%, more than 98%. more than 100% occurs within three seconds of triggering or within two seconds of triggering or with one second of triggering), and most emission of another acridinium label (e.g., slow- emission such as more than 90% more than 95%, more than 98%, more than 100% occurs within 120 seconds of triggering or within 60 seconds of triggering or within 25 second of triggering or within 15 second of triggering or within 10 seconds of triggering), the bins may be chosen to collect the light from one label in certain bins and to collect the light of the other label in other bins. Each binned measured intensity (or appropriately grouped bins) may individually be analyzed for quantification of the corresponding analyte.

[0086] The faster acridinium labels generally complete emission within 5 seconds of chemiluminescent triggering. In some embodiments, the fast acridinium label of the present disclosure may have faster light emission kinetics as compared to other acridinium compounds such as emitting at least 90% of their light, measured over 5 seconds, within 2 seconds. Slower acridinium labels may complete emission within 120 seconds of chemiluminescent triggering (and have minimal light contribution during the triggering of faster labels). For example, slower acridinium labels may complete less than 30% or less than 20% or less than 10% of their emission within 5 seconds or within 2 seconds and emit at least 90% of their light measured over 120 second or over 60 seconds or over 30 seconds.

[0087] For example, the acridinium labels of U.S. Pat. No. 8,119,422, which is hereby incorporated by reference in its entirety, can be used as materials to form the wavelength separated and emission separated sets of acridinium labels. Table 2 provides the percentage of relative light units (RLU) of various acridinium labels measured at 0.5 s, 1.0 s, 2.0 s, 5.0 s, and 10 s similar to as described in the Examples.

Table 2

[0088] The structures of each compound in Table 2 are :

It will be understood that any inconsistency between the structure and of the compounds in U.S. Pat. No. 8,119,422, both compounds will be considered to be embraced by the disclosure. Using these acridinium labels coupled with the wavelength and kinetic distinctions offered herein may allow for the creation of various assays having the ability to detect multiple analytes. For example, each of these acridinium labels may be used in the assay formats described in FIGS. 2A-J and 3 A- J. Leveraging the differences in acridinium conjugation and conjugation at the phenyl ester, and the associated conjugations described herein (e.g., dialkyl substitution of the acridinium ring (e.g., 1,3 methyl substitution of the acridinium ring), positioning of electron donating groups at the 4’ position of the phenyl group), these compounds may be integrated into the multiple assay formats of the present disclosure. [0089] If the acridinium labels have relatively close emission kinetics properties that do not allow for a clear or “sufficient” separation of chemiluminescent signal, there may be an overlay in signals from different acridinium esters within a time frame. In this case, deconvolution of the overlapping signals can be made by an algorithm or artificial intelligence (Al) which can measure emission kinetics profiles of individual AEs at multiple different time points and obtain the signal of an acridinium ester of interest by subtracting the overlapping signal due to the stray light from the unwanted signal. The differentiation may be further improved by leveraging the different emission wavelengths and measuring the emission kinetics of the individual AEs at multiple points in time at different wavelengths. Through algorithms or Al the different emission wavelengths or spectral changes together with the kinetic measurements (measurements at different points in time) can further improve the differentiation of overlapping signals.

[0090] The compounds of the present disclosure may be characterized by their stability. The acridinium labels used in the assay methods disclosed herein, such as the fast acridinium labels and/or the slow acridinium labels may be characterized as being stable. For example, a compound may be considered stable if there is a minimal loss of chemiluminescent activity as measured by the loss of relative light units ("RLU") when the compounds or conjugates are stored in an aqueous solution typically, in the pH range of 6-9. Compounds having increased instability as compared to another compound may have a greater loss of chemiluminescent activity. For example, the compounds of the present disclosure (e.g, compounds having the structure of formula (I)-(VI) may be characterized as having increased stabilities at pH 6 and/or 7 and/or 8) at 4°C (common reagent storage temperature) and/or 37°C (accelerated temperature) over 33 days. The compounds may have increased stability as compared to an otherwise identical compound not having fused heterocycles conjugated to the acridinium system. In some embodiments, the compounds may be characterized as having a change in chemiluminescent activity of 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%) after 33 days of storage at 37°C and pH 7 and/or pH 8.

[0091] The compounds can be prepared from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods (in addition to those provided herein). Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It will be appreciated that where typical or preferred process conditions (e.g. , reaction temperatures, times, mole ratios of reactants, solvents, pressures) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented may be varied for the purpose of optimizing the formation of the compounds described herein.

[0092] Synthetic chemistry transformations (including protecting group methodologies) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R.C. Larock, Comprehensive Organic Transformations, 2d. Ed., Wiley-VCH Publishers (1999); P.G.M. Wuts and T.W. Greene, Protective Groups in Organic Synthesis, 4th Ed., 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, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof, each of which are hereby incorporated by reference in their entirety.

[0093] The processes described herein can be monitored according to 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.,

or ¹³C), infrared spectroscopy (FT- IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatography such as high pressure liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0001] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0094] The reactions of the processes described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

[0095] Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. For example, the absolute configuration of the stereoisomers may be determined by ID and 2D NMR techniques such as COSY, NOESY, HMBC and HSQC. Specific implementations of these NMR techniques may be found in Hauptmann, H et al., Bioconjugate Chem. 11 (2000): 239-252 or Bowler, J. Steroids 54/1 (1989): 71-99, each hereby incorporated by reference in their entirety. Another example method includes preparation of the Mosher’s ester or amide derivative of the corresponding alcohol or amine, respectively. The absolute configuration of the ester or amide is then determined by proton and/or ¹⁹F NMR spectroscopy. An example method includes fractional recrystallization using a “chiral resolving acid’' which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g.. dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0096] Typically, zwitterionic acridinium esters (“ZAE”) comprising a reactive functional group for forming covalent linkages as described in U.S. Pat Nos. 6,664,043 to Natrajan et al., 7,309,615 to Natrajan et a/., 9.575.062 to Natrajan et al., or 9.487.480 to Natrajan, each hereby incorporated by reference in their entirety and in particular with respect to the zwitterionic acridinium esters described therein and their syntheses, may be used for synthesizing the compounds disclosed herein. For example, the zwitterionic acridinium ester starting materials may comprise an N-sulfopropyl (“NSP”) group in a zwitterionic moiety and/or comprise a charged nitrogen atom connected to the charged acridinium nucleus (“DIZAE”) and/or comprise a sterically stabilized dimethyl acridinium ester (“DMAE”) and/or comprise an isopropoxy functionalized acridinium nucleus (“ISO”) and/or comprise a zwitterionic (“Z”) and/or hexa(ethylene) glycol derived (“HEG") and/or glutarate derived (e.g., -C(O)-(CH2)3- C(O)-) linking moieties between the acridinium ester and the reactive functional group. The reactive functional group may by NH2 or N-hydroxysuccinimidyl ester (“NHS”). For example, the compound (e.g, a compound for conjugating with an analyte or binding partner of an analyte such as a peptide, a protein, or a macromolecule including an antibody) may have the structure of formula (IV):

wherein RFG is a reactive functional group for conjugating to the analyte or binding partner for an analyte,

L is absent (i.e., it is a bond) or a linker, and is a chemiluminescent acridinium. The chemiluminescent conjugates or compounds for forming the conjugates may also be synthesized through the use of acridinium sulfonamide reactants. For example, the acridinium sulfonamides disclosed in US Pat No 5,543,524 to Mattingly et al., hereby incorporated by reference in its entirety, are useful starting materials for the preparation of the chemiluminescent compounds disclosed herein.

[0097] The assay may be, for example, a competitive immunoassay which typically involves the detection of a large molecule, also referred to as macromolecular analyte, using binding molecules such as antibodies. The antibody is immobilized or attached to a solid phase such as a particle, bead, membrane, microtiter plate, or any other solid surface.

[0098] In an example of a competitive heterogeneous assay, a support having an antibody for an analyte (e.g. , 3C3. 3H10, 4G8 bovine monoclonal antibodies) bound thereto is contacted with a medium containing a sample suspected of containing the analyte and the chemiluminescent conjugates (or “labeled analogs”) described herein. Analyte from the sample competes for binding to the analyte antibody with the labeled analog. After separating the support and the medium, the label activity of the support or the medium is determined by conventional techniques and is related to the amount of analyte in the sample. In a variation of the above competitive heterogeneous assay, the support comprises the analyte analog, which competes with analyte of the sample for binding to an antibody reagent in accordance with the principles described herein. The labeled analyte analog may be covalently attached with a chemiluminescent or fluorescent molecule often referred to as a label or tracer.

[0099] When the solid phase with the immobilized antibody is mixed with a sample containing the analyte and the labeled analyte, a binding complex is typically formed between the analyte or the labeled analyte. This type of assay is often called a heterogeneous assay because of the involvement of a solid phase. 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. Usually, the binding complex is separated from the rest of the binding reaction components such as excess, labeled analyte, prior to signal generation. For example, if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution.

[0100] In an example of a sandwich assay format employing two antibodies (or fragments thereof), a solid support with a first immobilized antibody or fragment thereof for an analyte is mixed with a sample containing the analyte and a labelled conjugate comprising a second antibody or fragment thereof. A binding complex is formed between the solid particle and the labelled conjugate via the analyte in the sample. The different chemiluminescent moi eties may be conjugated to the same binding partner for an analyte. In some embodiments, the different chemiluminescent moieties are conjugated to different binding partners for an analyte. In some embodiments, the different chemiluminescent moieties are conjugated to different binding partners for different analytes (all of which may be detected on the same solid support). The signal associated with the binding complex and can the measured and the presence or absence or amount of analyte can be inferred. Usually, the binding complex is separated from the rest of the binding reaction components such as excess, labeled analyte, prior to signal generation. For example, if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution. In some embodiments, the first immobilized antibody is a biotinylated mouse monoclonal antibody bound to coated (e.g., streptavidin coated) optionally paramagnetic particles. In some embodiments, the second antibody is a mouse monoclonal antibody fragment labelled with acridinium (e.g., acridinium ester).

[0101] By using a series of ‘"standards,’ that is, known concentrations of the analyte, a “dose- response” curve can be generated for the known labeled analyte. These dose response curves may be identified individually for any acridinium label or identified based on combinations of the acridinium labels used in the assay. Thus, the dose-response curve correlates a certain amount of measured signal with a specific concentration of analyte. In a competitive assay, as the concentration of the analyte increases, the amount of signal decreases if the chemiluminescence from the binding complex is measured. The concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated by an unknown sample containing the macromolecular analyte, with the dose-response curve.

[0102] The methodology' of the attachment of binding molecules such as antibodies to solid phases ty pically involves a mixing of the requisite components to induce attachment. For example, an antibody can be covalently attached to a particle containing amines on its surface by using a cross-linking molecule such as glutaraldehyde. The attachment may also be non- covalent and may involve simple adsorption of the binding molecule to the surface of the solid phase, such as polysty rene beads and microtiter plate. Labeling of binding molecules such as antibodies and other binding proteins are also well known in the prior art and are commonly called conjugation reactions and the labeled antibody is often called a conjugate. Typically, an amine-reactive moiety on the label reacts with an amine on the antibody' to form an amide linkage. Other linkages, such as thioether, ester, carbamate, and the like between the antibody and the label may also be used.

[0103] In another aspect of the invention, a reagent may be provided for the detection of an analyte comprising a chemiluminescent acridinium compound bound the analyte or binding partner. The reagent may comprise from 0.1 to 100 ng/mL of the chemiluminescent acridinium compound or from 1 to 50 ng/mL of the chemiluminescent acridinium compound or from 5 to 30 ng/mL of the chemiluminescent acridinium compound. In some embodiments, the compound is provided in a reagent which further comprises a buffer.

[0104] Typically, the assay for the detection or quantification of an analyte in a sample comprises:

(a) providing a first set of chemiluminescent labels and a second set of chemiluminescent labels, wherein the first set of chemiluminescent labels comprise at least two chemiluminescent labels having emission spectra separated in the wavelength domain, and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first set and the second set may overlap (e.g., a chemiluminescent label in the first set may also be in the second set), wherein each chemiluminescent label in the first and second set is capable of forming a binding complex with at least one of the multiple analytes;

(c) mixing the first set and said second set with said sample;

(f) measuring the chemiluminescence in the wavelength domain (e.g., measuring light intensity as a function of wavelength) and in the time domain (e.g.. measuring light intensity' as a function of time from the triggering step);

[0105] In some embodiments, the sample derived from a mammal (e.g, human). In some embodiments, the sample comprises saliva and/or blood and/or serum. In some embodiments, the sample is saliva and/or blood and/or serum.

[0106] In some assays, the sample to be analyzed is subjected to a pretreatment to release analyte from endogenous binding substances such as, for example, plasma or serum proteins that bind the analyte. The release of the analyte from endogenous binding substances may be carried out, for example, by addition of a digestion agent or a releasing agent or a combination of a digestion agent and a releasing agent used sequentially. The digestion agent is one that breaks down the endogenous binding substances so that they can no longer bind the analyte.

[0107] The conditions for conducting an assay on a portion of a sample in accordance with the principles described herein may include carrying out the assay in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity. The aqueous medium may be solely water or may include from 0.1 to 40 % by volume of a cosolvent. The pH for the medium may be in the range of 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8. Usually, the pH value of the solution will be a compromise between optimum binding of the binding members of any specific binding pairs, the pH optimum for other reagents of the assay such as members of the signal producing system, and so forth. Various buffers may be used to achieve the desired pH and maintain the pH during the assay. Illustrative buffers include borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE, for example.

[0108] Various ancillary materials may be employed in the assay methods. For example, in addition to buffers, the composition, reagents, or reaction medium may comprise stabilizers for the medium and for the reagents employed. In some embodiments, the medium may comprise proteins (e.g., albumins), organic solvents (e.g. formamide), quaternary ammonium salts, polyanions (e.g, dextran sulfate), binding enhancers (e.g, polyalkylene glycols), polysaccharides (e.g, dextran, trehalose), and combinations thereof.

[0109] Triggering the chemiluminescence of the analogs may be performed by the addition chemiluminescent triggering reagents. The chemiluminescent triggering reagents may be acidic or basic. Multiple chemiluminescent triggering reagents may be added sequentially. For example, an acidic solution may first be added followed by a basic solution. In some embodiments, the chemiluminescent triggering reagents comprise hydrogen peroxide, hydrogen peroxide salts, nitric acid, nitric acid salts, sodium hydroxide, ammonium salts, or combinations thereof.

[0110] A kit according to the present disclosure may involve an immunoassay reagent composition comprising one or more AE conjugated to an binding partner such as an antibody (or fragment thereof). The kit may comprise accessory ingredients such a buffers, blocking reagents, ions, e.g. bivalent cations or monovalent cations, calibration proteins, secondary antibodies, detection reagent such as detection dyes and any other suitable compound or liquid necessary for the performance of analyte detection. Additionally, the kit may comprise an instruction leaflet and/or may provide information (or access to) as to the relevance of the obtained results and/or additional tests that may be prescribed for additional risk assessment. In some embodiments, the kit further comprises the solid phase reagent. In some embodiments, the kit further comprises chemiluminescence triggering reagents. EXAMPLES

[0111] The following Examples illustrate the synthesis of a representative number of compounds, characterization of parameters implicated in assay development, and the use of these compounds in the measurement of samples in heterogeneous competitive assay. Accordingly, the Examples are intended to illustrate but not to limit the disclosure. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.

[0112] Example 1 : Synthesis of stable, slow light emission acridinium ester 1 ,3-Dimethyl-

DMAE-Bz (10)

[0113] 4-hydroxybenzoic acid (A, 7.8 g) was dissolved in 50 mL of methanol and the insoluble material was filtered off. To the solution was dropwise added a solution of 2.3 g of potassium hydroxide in 15 mL of water until pH 7.6. It was allowed to stir at room temperature for 1 hour and evaporated by rotovap under vacuum to dryness to product B (9.35 g). Synthesis of C

[0114] A suspension of compound B (9.35 g) and dibenzo- 18-crown-6 (1.975 g) in 210 mL of mixed acetonitrile/DMF (2: 1) was stirred at 80-90 °C for 30 minutes, followed by addition of benzyl bromide (6.1 mL). It was allowed to stir at 80-90 °C for 3 hours, and then cooled to room temperature and stored in refrigerator over weekend. The resulting mixture was filtered. The filtrate was concentrated under reduced pressure to a brown oil. The rude material was diluted with chloroform and applied to a flash silica column packed with silica gel (600 mL) in hexanes. The column was eluted with 5%, 10% and 15% ethyl acetate/hexanes. The fractions containing the desired product was combined and evaporated by rotovap under reduced pressure to dryness to yield 7. 12 g of compound C.

Synthesis of F

[0115] A solution of isatin (D, 5 g) in anhydrous N, /V-di methyl formamide (DMF,150 mL) was treated with sodium hydride (60% suspension, 1.632 g) for about 20 minutes until no gas was released. To the mixture was added 5-bromo-xylene (E, 9.24 mL), followed by copper (I) iodide (12.9 g). The mixture was heated at 150 °C under stirring for 18 hours. The mixture was cooled to room temperature and diluted with -750 mL of chloroform and filtered to remove the off-white precipitates (Cui). The filtrate was evaporated by rotovap under reduced pressure to give a crude material F as a brown material (15 g).

Synthesis of G

[0116] A mixture of compound F in the above in 100 mL of 10% sodium hydroxide in water was heated under reflexing at 120 °C 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 wash solution was acidified with concentrated hydrochloride in an ice-water bath to pH 2-3. The resulting precipitate was collected and washed with water (2 x 50 mL). The material was air-dried overnight and to give 4. 1 g of compound G.

Synthesis of H

[0117] A mixture of G (110 mg) in pyridine (5 mL) was treated with Tosyl chloride (146 mg) at room temperature for 5 minutes followed by addition of C (98 mg). The solution was heated at 60 °C for 24 hours and cooled to room temperature. It was continued to stir at room temperature for two days. The resulting mixture was evaporated by rotovap under reduced pressure to dryness. The solid was dissolved with chloroform (50 mL), and the chloroform layer was washed with IN sodium hydroxide solution (2 x 20 mL), brine (1 x 20 mL) and dried over sodium sulfate. Removal of the solvent gave a crude product, which was purified by two 2mm-thick (20x20 cm) preparative silica gel plates eluted with ethyl acetate/hexanes (1: 1). The major band was collected and extracted with ether (200 mL). Removal of the ether by rotovap under reduced pressure gave 29 mg of H.

Synthesis of 1,3-Dimethyl-DMAE-Bz (10, Lot# QJ4-30-3)

[0118] A solution of H (23 mg) in 2 mL of methylene chloride was stirred with methyl fluorosulfonate (38 uL) under nitrogen at room temperature for two days. The reaction was stopped by blowing nitrogen gas to remove the solvent. The resulting crude product was purified by preparative HPLC (Solvent A 0.5% TFA/water and Solvent B 0.5% acetonitrile, flow rate 16 mL/min, detection 260 nm) to give 20 mg of 1,3-Dimethyl-DMAE-Bz (10, Lot# QJ4-30-3).

[0119] Example 2: Synthesis of stable, slow light emission acridinium ester ?-CE-DMPAE (12, lot# WJW4-188A)

Synthesis of J

[0120] A solution of 2,6-dimethylpheol (I, 10 g) and acrylonitrile (8.08 mL) was cooled to 15 °C in a water bath and aluminum chloride (10.91 g) was added slowly over a period of 15 minutes. After the addition, the mixture was heated with stirring at 100 °C for 45 minutes. The resulting solid was added with small amount of methanol to 200 g of crashed ice. The precipitate was collected and recrystallized from methanol to give 4.944 g of J.

Synthesis ofK

[0121] To a round-bottom flash containing 3.6 of J was added a solution of potassium hydroxide (23 G) in 35 mL of water, followed by addition of 50 mL of ethanol. It was stirred at 110 °C overnight. The mixture was cooled to room temperature and evaporated by rotovap under reduced pressure to remove most of the ethanol and then diluted with 300 mL of water. It was acidified with 50% sulfuric acid to pH 4. The resulting precipitate was filtered and washed with water (2 x 100 mL), The white solids were dried under vacuum to produce 3.07 g of K.

Synthesis of L

[0122] To a solution of 2.69 g of K in 25 mL of methanol was added potassium hydroxide (0.848 g) in 7 mL of water so that it was slightly basic. It was allowed to stir at room temperature for 1 hour, then evaporated by rotovap under reduced pressure to give a solid material K. which was used in the next step of reaction.

Synthesis of M

[0123] A suspension of K (all material obtained in the above step) and dibenzo-8-crown-6 (511 mg) in the mixed solvent of acetonitrile (30 mL) and DMF (15 mL) was stirred at 80 °C for 15 minutes before benzyl bromide (2.6 g) was added. The suspension was stirred at 80 °C for 4 hours and then at room temperature overnight. The resulting mixture was filtered and the solids washed with ethyl acetate. The evaporation by rotovap under reduced pressure gave an oil. It was purified by flash silica column eluted with 15% ethyl acetate/ hexanes to afford 2. 11 g of product M as an oil.

Synthesis of O

[0124] 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 cooled and concentrated to a small volume by blowing with nitrogen stream. The suspension was quenched with 200 mL of anhydrous ether and the precipitate was filtered, washed with more ether and then dried under high vacuum to give O as a yellow solid in 1.96 g.

Synthesis of P

[0125] To a solution of M ( 2.01 g) in pyridine (25 mL) was added DMAP (188 mg). It was stirred at 0 °C for 5 minutes followed by addition of O (1.96 g). The suspension was stirred at 100 °C for 3 hours and then room temperature overnight. It was evaporated under reduced pressure to dryness with the help of co-evaporation with toluene. It was purified by flash silica column eluted with 20% ethyl acetate in hexanes to produce 1.84 g of the product P. Synthesis of Q

[0126] A solution of P (969 mg) and methyl fluorosulfonate (1.56 mL) in 25 mL of methylene chloride was stirred under nitrogen at room temperature overnight. The reaction was stopped by blowing the solvent with nitrogen steam. The resulting solid was dissolved in methylene chloride and the solution was added to 450 mL of anhydrous ether. The yellow precipitate was re-dissolved in methylene chloride (75 mL) and the solution was added to 350 mL of anhydrous ether to give 715 mg of the product Q.

Synthesis of i-CE-DMPAE (12, lot# WJW4-188A)

[0127] A solution of Q (572.9 mg) in 3 mL of 30% hydrogen bromide in acetic acid was stirred at 100 °C under nitrogen for 2 hours. It was blow n with nitrogen gas to a small volume, and then poured into anhydrous ether. The resulting precipitate was filtered, collected and washed with ether (6 x 10 mL). The yellow material was dried under high vacuum to give 373 mg of /i-CE-DMPAE (11, lot# WJW4-188A).

[0128] Example 3: Chemiluminescence wavelength and kinetics measurements

[0129] General procedure for light emission kinetics measurements: All light measurements were performed on a MLA1™. For measurement of RLUs from the acridinium esters, 1 mg/mL DMF solutions of the acridinium ester were sequentially diluted 10⁶-fold into 10 mM phosphate, 150 mM NaCl pH 8 also containing 0.05% BSA and 0.1% sodium azide. Light measurements used 25 pL of sample and were initiated in the instrument with the addition of 0.350 mL of reagent 1 which contained 0.5% hydrogen peroxide in 0.1 N nitric acid followed by the addition of 0.35 mL reagent 2 which contained a surfactant in 0.25 N NaOH. A very short delay time of 0. 1 second was used between addition of reagent 1 and reagent 2.

1,3 Acridinium Ring Conjugation Alters Chemiluminescence Kinetics

[0130] Kinetics measurements of 1,3-Dimethyl-DMAE-Bz 10 and DMAE-Bz 11 were compared. Measuring time was varied from 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 to 10.0 seconds. The structures for 1,3-Dimethyl-DMAE-Bz 10 and DMAE-Bz 11 are:

[0131] The amount of light emitted at each measuring time was reported as RLUs by the instruments, which were then converted to percentages by assigning a value of 100% for the RLUs recorded at 10 s for the acridinium esters. Table 3 and FIG. 4 show the percentage of the light measured for 1,3-Dimethyl-DMAE-Bz and DMAE-Bz over 10 seconds under the standard flash condition.

Table 3

[0132] In FIG. 4, the 1,3-dimethyl-DMAE-Bz is shown on the left (black) and DMAE-Bz is shown on the right (white) for each As can be seen, 1,3 conjugation of the acridinium label resulted in slower emission kinetics as compared to an acridinium label without conjugation at these positions.

Conjugation of Electron Donating Groups at the 4 position of the phenyl ester

[0133] Kinetics measurements of p-CE-DMPAE 12 and DMAE-Bz 11 were also performed. The measuring time was varied from 2.0, 5.0, 10.0, 25.0 to 60.0 seconds. The structures of p- CE-DMPAE 12 and DMAE-Bz 11 are:

[0134] The amount of light emitted at each measuring time was reported as RLUs by the instruments, which were then converted to percentages by assigning a value of 100% for the RLUs recorded at 60.0 seconds for the acridinium esters. Table 4 and FIG. 5, show the show the percentage of the light measured for 1,3-Dimethyl-DMAE-Bz and DMAE-Bz over 10 seconds under the standard flash condition.

Table 4

[0135] p-CE-DMPAE (12) takes approximately 60 seconds to emit all the light while DMAE-Bz (11) takes only 2 seconds to emit all the light (98% light emitted).

[0136] Using these compounds, a possible sequence of the detection of individual signals is shown below in Table 5. The first time frame is to collect light from 0 to 2 seconds where 8% of the signal from p-CE-DMPAE (12) and 98% of the signal from DMAE-Bz (11) can be obtained, respectively. Because of the vast difference in emission speeds of two compounds, the light collection in time frame 2 can start anywhere after 2 seconds without the need of a delay. The significant slow emission of fight of DMAE-Bz (11) provides an option to start measuring the light at much later time, e.g., after 10 seconds. This provides an option to allow a third AE to be used along the time domain. Table 5

NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0137] Non-limiting illustrative embodiments are provided below, each of which should be considered to be part of the disclosure of the present application. These embodiments may apply to any embodiment described herein.

[0138] Illustrative Embodiment 1. A method for the detection or quantification of multiple analytes in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) comprising:

(a) providing a first set of chemiluminescent labels and a second set of chemiluminescent labels, wherein the first set of chemiluminescent labels comprise at least two chemiluminescent labels having emission spectra separated in the wavelength domain, and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first set and the second set may overlap (e g., a chemiluminescent label in the first set may also be in the second set), wherein each chemiluminescent label in the first and second set is capable of forming a binding complex with at least one of the multiple analytes;

(c) mixing the first set and said second set with said sample;

(f) triggering chemiluminescence from the first set and second set of chemiluminescent labels following preparation of the mixture (e.g., by the addition of one or more triggering compositions which trigger chemiluminescence of the acridinium labels); (f) measuring the chemiluminescence in the wavelength domain (e.g., measuring light intensity as a function of wavelength) and in the time domain (e.g.. measuring light intensity as a function of time from the triggering step);

[0139] Illustrative Embodiment 2. The method according to Illustrative Embodiment 1, wherein the two chemiluminescent labels in the first set are capable of forming a binding complex with different analytes in the sample.

[0140] Illustrative Embodiment 3. The method according to Illustrative Embodiment 1 or 2, wherein the two chemiluminescent labels in the second set are capable of forming a binding complex with different analytes in the sample.

[0141] Illustrative Embodiment 4. The method according to any one of Illustrative Embodiments 1-3, wherein each of the two chemiluminescent labels in the first set has a corresponding chemiluminescent label of different emission speed (e.g., to form two different second sets of chemiluminescent labels separated in emission speed from four different chemiluminescent labels), and the four chemiluminescent labels are capable of forming a binding complex with different analytes in the sample.

[0142] Illustrative Embodiment 5. The method according to any one of Illustrative Embodiments 1-4. wherein the first set of chemiluminescent labels comprise at least three chemiluminescent labels having emission spectra separated in the wavelength domain.

[0143] Illustrative Embodiment 6. The method according to Illustrative Embodiment 5, wherein the three chemiluminescent labels in the first set are capable of forming a binding complex with three different analytes in the sample.

[0144] Illustrative Embodiment 7. The method according to Illustrative Embodiment 5, wherein each of the three chemiluminescent labels in the first set having a corresponding chemiluminescent label with a different emission speed (e.g., to form three different second sets of chemiluminescent labels separated in emission speed from six different chemiluminescent labels), and each of the six chemiluminescent labels are capable of forming a binding complex with six different analytes in the sample.

[0145] Illustrative Embodiment 8. The method according to any one of Illustrative Embodiments 1-7, wherein said preparing step comprises:

(e2) separating said solid support from said mixture.

[0146] Illustrative Embodiment 9. The method according to Illustrative Embodiment 8, wherein said measuring step comprises the addition of one or more chemiluminescence triggering reagents to said separated solid support and/or said separated mixture.

[0147] Illustrative Embodiment 10.The method according to Illustrative Embodiment 8 or 9, wherein said solid support comprises at least two molecules (e.g., two, three, four, five, six, seven, eight, nine ten), each capable of forming a binding complex with different analytes and capable of forming a binding complex with at least two different chemiluminescent labels in the first set and/or second set of chemiluminescent labels.

[0148] Illustrative Embodiment 11. The method according to any one of Illustrative Embodiments 1-10, wherein the chemiluminescent labels independently have the structure of formula (I):

wherein A is an analyte or binding partner for an analyte,

^VP is a chemiluminescent acridinium comprising the structure:

“f and “K* are independently 0 (e.g., all Ra groups are hydrogen, all R₃ groups are hydrogen), 1, 2, 3, or 4;

R2 and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e g., with 1 to 20 heteroatoms, with 1-20 substituents);

Z^L is a zwitterionic linker group having the structure:

“/n” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

X¹ and X^b are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g, alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g, 1-20, 1-10, 1-5) points of substitution (e.g, with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl (e.g, methyl, ethyl, propyl); and

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof (e.g, a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0149] Illustrative Embodiment 12.The method according to Illustrative Embodiment 11 or

12, wherein the chemiluminescent labels independently have the structure of formula (la):

wherein Ω is O or N;

Y’ is either absent (i.e. it is a bond), or is selected from -L_l- -R^L-, -R^L-L_l-, -L1-L1-, -LL-R^L-, -L_l-R^L-L_l, and -R^L-L_l-R^L-. [0150] Illustrative Embodiment 13. The method according to Illustrative Embodiment 11, wherein at least one (eg., one, two, three, four, five, six) of the chemiluminescent labels has the structure of formula (lb) or (Ic):

wherein R4-R7 are independently hydrogen, an electron donating group, or C1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and

Y” is either absent (i.e., it is abond) or-L^c-, -L_l-, -R^L- or -R^L-L_l-.

[0151] Illustrative Embodiment 14. The method according to Illustrative Embodiment 13, wherein at least one of

) are an electron donating group (eg., and forms a chemiluminescent label with different emission speed as compared to an otherwise identical label without the electron donating group).

[0152] Illustrative Embodiment 15. The method according to any one of Illustrative Embodiments 1-14, wherein the two chemiluminescent labels in the first set are each conjugated to a binding pair of one of the analytes, and the chemiluminescent labels is formed from a chemiluminescent compound or salt having a reactive functional group for conjugation to the binding pair.

[0153] 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-Imidate, 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-b- Alanine, Texas Red-3-AM-DMAE-b-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.

[0154] 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 in the first set is a zwitterionic acridinium (e.g., N-sulfopropyl zwitterionic acridinium, a compound having the structure of formula I, la, lb, or Ic wherein Ri

[0155] 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 zwitterionic acridinium e.g., N-sulfopropyl zwitterionic acridinium, a compound having the structure of formula I, la, lb, or Ic wherein Ri is selected from -X^b, -R^L-X^b, or -L^c-X^b such as -L_l-X^b, Ri is selected from -SOs’, -R^L- SO.v such as - (CH2)_1-5- SOS’. or -L^c- SCE" such as -L_l- SO3 ).

[0156] 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 an acridinium salt (e.g., acridinium carboxylate salts such as fluoro alkyl carboxylate salts, acridinium sulfonate salts such a fluoroalkyl sulfonate salts, acridinium halide salts such as acridinium chloride salts, a compound having the structure of formula I, la, lb, or Ic wherein Ri is selected from -R, -L^c-R, -Z, -R^L-Z, -L^c-Z,-L_l-Z, -R^L-L^C-R^L-Z, - R^L-L_l-R^L-Z with a negative counterion such as R-COO’, R-SO3", Cl"). [0157] 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 in the first set is an acridinium salt (e.g., acridinium carboxylate salts such as halocarboxylate salts, haloalkyl carboxylate salts, fluoroalkyl carboxylate salts, acridinium sulfonate salts such a halo sulfonate salts, haloalkyl sulfonate salts, fluoroalkyl sulfonate salts, acridinium halide salts such as acridinium chloride salts, a compound having the structure of formula I, la, lb, or Ic wherein Ri is selected from — R, -L^c-R, — Z, -R^L— Z, - L^c-Z,-L_l-Z, -R^L-L^C-R^L-Z, -R^L-L_l-R^L-Z with a negative counterion such as R-COO", R- SO3 , Cl , F).

[0158] Illustrative Embodiment 21. The method according to any one of Illustrative Embodiments 1-20, wherein at least one chemiluminescent label (e.g., a chemiluminescent label in the second set) has the structure of formula (II):

wherein A is an analyte or binding partner for an analyte,

L is absent (i.e., it is a bond) or a linker optionally comprising a group L^c or Z^L, and ψ is a chemiluminescent acridinium comprising the structure:

“j” and “k” are independently 0 (e.g., all Rz groups are hydrogen, all Rz groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, -R, -X?¹, R¹' X^b, -L^c-R, -L^c-X^b (e.g., -L_l-X^b), -Z, -R^L-Z, -L^c-Z (e.g. L_l-Z), or — R^L— L^c— R^L-Z (e.g., -R^L-L_l-R^L-Z); R2a and R2b are independently selected from alkyl optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);

R2c is hydrogen, -R, an electron donating group, or -Z; R₃ is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 groups may together form a fused heterocyclic group (e.g., 5-7 membered fused heterocyclic group) and wherein R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

Z^L is a zwitterionic linker group having the structure:

“m ” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

“r" is independently an integer from 0 to 10 (e.g., from 1 to 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10);

X¹ and X^b are independently at each occurrence an anionic group;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g, 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g, 1-20, 1-10, 1-5) points of substitution (eg, with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

[0159] Illustrative Embodiment 22.The method according to Illustrative Embodiment 21, wherein R2a and Rab are independently alkyl (e.g., lower alkyl such including CM alkyl as methyl, ethyl, propyl, butyl).

[0160] Illustrative Embodiment 23.The method according to any one of Illustrative Embodiments 1-22, wherein one chemiluminescent label in the first set has the structure of

wherein A1 and Aa are independently an analyte or binding partner for an analyte and A1 is different than A₂;

Ω is O orN;

Y is selected from - R or -R^L— Z, or in the case where Ω is O then Y is absent; and

Y’ is either absent (i.e. it is a bond), or is selected from -L_l- -R^L-, -R^L-L_l-, -L1-L1-, -LL-R^L- — L_l— R^L-L_l, and -R^L-L_l-R^L-;

Rz and Rs are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Rz or Rs groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent (LA) such as a fluorophore (e.g., rhodamine); and at least one Rz group is not hydrogen;

Z^L is a zwitterionic linker group having the structure:

Z is a zwitterionic group independently at each occurrence has the structure:

is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

X^a and X^b are independently at each occurrence an anionic group; L_l is independently at each occurrence -O-, -S-, -NH-, -N(R^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)-

-N(R^N)-(CH₂)_1-4- -(CH₂)_1-4-N(R^N)-, -(OCH₂)_1-10- -(CH₂0)_1-10-, -(OCH₂CH₂)_1-10- or -(CH₂CH₂0)_1-10-;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alky l, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g.. 1-10. 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);

R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alky l (e.g., methyl, ethyl, propyl); and

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (eg., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0161] Illustrative Embodiment 24. The method according to Illustrative Embodiment 23, wherein the chemiluminescent label of formula (IIIb) has the structure of formula (IIIbl):

[0162] Illustrative Embodiment 25. The method according to Illustrative Embodiment 23 or

24, wherein at least one R3 is not hydrogen (eg., an electron donating group such as alkoxy).

[0163] Illustrative Embodiment 26.The method according to Illustrative Embodiment 23 or 24, wherein at least one R₃ and/or at least one Rs group is an electron donating group (e.g., alkoxy).

[0164] Illustrative Embodiment 27.The method according to any one of Illustrative Embodiments 23-26, wherein the first set of chemi luminescent labels further comprise a compound having the structure of formula (IIIc):

wherein IA is an imaging agent (e.g., a fluorophore such as rhodamine or phenol modified rhodamine); and

A3 is a different analyte or binding partner for an analyte than A1 and As. [0165] Illustrative Embodiment 28.The method according to Illustrative Embodiment 27, wherein the chemiluminescent label of formula (IIIc) has the structure of formula (IIIcl) or (IIIc2):

[0166] Illustrative Embodiment 29.The method according to Illustrative Embodiment 23, wherein the chemiluminescent label of formula (IIIb) has the structure of formula (IIIb3):

wherein IA is an imaging agent (e.g., a fhiorophore such as rhodamine or phenol modified rhodamine).

[0167] Illustrative Embodiment 3O.The method according to Illustrative Embodiment 29, wherein the chemiluminescent label of formula (IIIb3) has the structure of formula (IIbI4) or (IIIb5):

[0168] Illustrative Embodiment 31. The method according to any one of Illustrative Embodiments 23-30, wherein the difference between compounds of formula (IIIa), formula (IIIb), and formula (IIIe) is the different A1, Aa, and A1 groups and in the conjugation of the acridinium ring system (eg., Ri, Ω, L, Y, Y”, IA in formulas (IIIa), (IIIb), and (IHc) are identical).

[0169] Illustrative Embodiment 32.The method according to any one of Illustrative Embodiments 23-31, wherein the chemiluminescent label of formula (IIIb) (e.g., formula (IIIbl), formula (IIIb 2), formula (IIIb3), formula (IIbI4) is a salt (e.g., a carboxylate salt such as a halocarboxylate salt, halo alkyl carboxylate salt, fluorocarboxylate salt, fluro alkyl carboxylate salt F, ₃CCOO- salt, Ri in formula (IIbI2) is alkyl and the counterion is carboxylate, halocarboxylate, halo alkyl carboxylate, fluorocarboxylate, fluoroalkyl carboxylate, F₃CCOO- ).

[0170] Illustrative Embodiment 33. The method according to any one of Illustrative Embodiments 1-31, wherein one chemiluminescent label in the first set has the structure of formula (IVa):

and another chemiluminescent label in the first set has the structure of formula (IVb):

wherein A1 and A2 are independently an analyte or binding partner for an analyte and A1 is different than Aa;

Ω is O orN;

R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or Rs groups may together form a fused cyclic group (e.g., 5- 7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g, rhodamine).

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); Z^L is a zwitterionic linker group having the structure:

“m ” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

‘r’ is independently an integer from 0 to 10 (e.g., from 1 to 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10);

X¹ and X^b are independently at each occurrence an anionic group;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e g, 1-10, 1-5) points of substitution (e.g, with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g, alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (eg, with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

[0171] Illustrative Embodiment 34. The method according to Illustrative Embodiment 33, wherein the difference between the chemiluminescent labels of formula (IVa) and formula (IVv) is located in the conjugation of the acridinium ring system (e.g., Ri, Ω, L, Y, Y”, IA in formulas (IVa) and (IVb) are identical) and the different A1 and M groups.

[0172] Illustrative Embodiment 35. The method according to any one of Illustrative Embodiments 1-34, wherein one chemiluminescent label in the second set has the structure of formula (Va):

wherein A1 and A₂ are independently an analyte or binding partner for an analyte and A1 is different than Aa;

Ω is O orN;

“k” is 0 (e.g., all Ra groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4;

Ri is hydrogen, -R, -X?¹, R¹' X^b, -L^c-R, -L^c-X^b (e.g., -L_l-X^b), -Z, -R^L-Z, -L^c-Z (e.g., L_l-Z), or — R^L— L^c— R^L-Z (e.g., -R^L-L_l-R^L-Z);

Rz and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (eg., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein Ra or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

Z^L is a zwitterionic linker group having the structure: “ m” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

is independently an integer from 0 to 10 (e.g., from 1 to 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10);

X¹ and X^b are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g, alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e g, with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or Ci-s alkyl (e.g., methyl, ethyl, propyl); and

[0173] Illustrative Embodiment 36.The method according to any one of Illustrative Embodiments 1-35, wherein one chemiluminescent label in the second set has the structure of formula (Vc):

wherein A1 and A2 are independently an analyte or binding partner for an analyte and A1 is different than A₂;

Ω is O orN;

Rz and Rs are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Ra or Ri groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or Rs may comprise a linkage to an imaging agent such as a fluorophore (eg., rhodamine);

Z^L is a zwitterionic linker group having the structure:

Z is a zwitterionic group independently at each occurrence has the structure:

-C(O)-N(R^N)-(CH₂)_1-3- -(CH₂)_1-3-C(O)-N(R^N)- -(CH₂)_1-3-N(R^N)-C(O)-

R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). [0174] Illustrative Embodiment 37.The method according to any one of Illustrative Embodiments 1-36, wherein the at least two chemiluminescent acridiniums with wavelength separated chemiluminescence have similar chemiluminescence emission rates (e.g., the percentage of light measured at a time point such as 1 second or 2 seconds or 4 seconds or 6 seconds following triggering as compared to total emission is within 10% or within 5% or within 1%).

[0175] Illustrative Embodiment 38.The method according to any one of Illustrative Embodiments 1-37, wherein the at least two chemiluminescent acridiniums with time domain separated chemiluminescence have similar chemiluminescence wavelengths (e.g., A™ within 10% or within 5% or within 1%).

[0176] Illustrative Embodiment 39.The method according to any one of Illustrative Embodiments 1-38, wherein the first set of chemi luminescent labels are provided in a composition to be mixed with the sample.

[0177] Illustrative Embodiment 4O.The method according to any one of Illustrative Embodiments 1-39, wherein the second set of chemi luminescent labels are provided in a composition to be mixed with the sample

[0178] Illustrative Embodiment 41. The method according to any one of Illustrative Embodiments 1-40, wherein the first and second set of chemiluminescent labels are provided in a composition to be mixed with the sample.

[0179] Illustrative Embodiment 42.A compound having the structure of formula (V)

wherein A is an analyte or binding partner for an analyte,

R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and — Z; wherein two vicinal Rs groups may together form a fused heterocychc group (e.g., 5-7 membered fused heterocychc group) and wherein R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

Z^L is a zwitterionic linker group having the structure:

R'

“m” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

X¹ and X^b are independently at each occurrence an anionic group;

R is independently at each occuncnce hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g, with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0180] Illustrative Embodiment 43.A compound having the structure of formula (VI)

wherein RFG is a reactive functional group for conjugating to an analyte or a binding partner for an analyte, L is absent (i.e., it is a bond) or a linker optionally comprising a group L or Z ,

Ri is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Rz groups may together form a fused heterocyclic group (e.g., 5-7 membered fused heterocyclic group) and wherein R₃ may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);

L^c is a divalent Ci-as alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms);

Z^L is a zwitterionic linker group having the structure:

“m” is 0 (i.e. it is a bond) or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

X³ and X^b are independently at each occurrence an anionic group; L_l is independently at each occurrence -O-, -S-, -NH-, -N(R^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)I-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- -(CH₂0)_1-10-, -(OCH₂CH₂)_1-10- or (CH₂CH₂0)_1-10 ;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alky l substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g., 1-10. 1-5) points of substitution (e.g.. with 1-10 heteroatoms, with 1-10 substituents);

R is independently’ at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents);

R’ and R” are independently' at each occurrence hydrogen or a C1-10 alkyl;

R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g, a halide salt such as a chlonde salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0181] Illustrative Embodiment 44. A composition comprising at least three chemiluminescent acridiniums, wherein each chemiluminescent acridinium is conjugated to a different analyte or a different binding pair for an analyte, and the chemiluminescence of at least two chemiluminescent acridiniums are separated in the wavelength time domain and the chemiluminescence of a different set of at least two chemiluminescent acridiniums are separated in the time domain. [0182] Illustrative Embodiment 45. The composition according to Illustrative Embodiment 44, wherein the composition comprises at least four chemiluminescent acridiniums conjugated to a different analyte or binding pair for an analyte, wherein the chemiluminescence of three chemiluminescent acridiniums is separated in the wavelength domain.

[0183] Illustrative Embodiment 46. The composition according to Illustrative Embodiment 44 or 45, wherein the at least two chemiluminescent acridiniums with wavelength separated chemiluminescence have similar chemiluminescence emission rates.

[0184] Illustrative Embodiment 47. The composition according to any one of Illustrative Embodiments 44-46, wherein the at least two chemiluminescent acridiniums with time domain separated chemiluminescence have similar chemiluminescence wavelengths.

[0185] Illustrative Embodiment 47. The composition according to any one of Illustrative Embodiments 44-47, wherein chemiluminescent acridiniums (e.g., the at least three chemiluminescent acridiniums, the at least four chemiluminescent acridiniums) are independently selected from compounds having the structure of Formula (I) e.g., (la), (lb), (Ic)), (II), (Illa), (Illb) (e.g., formula (Illb I), formula (IIIb2), formula (IIIb3), formula (IIIb4)), (IV) (e.g., (IVa), (IVb)).

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

[0186] All references including patent applications and publications cited herein are incorporated herein by reference and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A method for the detection or quantification of multiple analytes in a sample comprising:

(a) providing a first set of chemiluminescent labels and a second set of chemiluminescent labels, wherein the first set of chemiluminescent labels comprise at least two chemiluminescent labels having emission spectra separated in the wavelength domain, and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first set and the second set may overlap, wherein each chemiluminescent label in the first and second set is capable of forming a binding complex with at least one of the multiple analytes:

(c) mixing the first set and said second set with said sample:

(e) preparing the mixture of said first set. second set, and sample to measure chemiluminescence from the first set and the second set of chemiluminescent labels;

(f) triggering chemiluminescence from the first set and second set of chemiluminescent labels following preparation of the mixture;

(f) measuring the chemiluminescence in the wavelength domain and in the time domain;

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

3. The method according to any one of claims 1-2, wherein each of the two chemiluminescent labels in the first set has a corresponding chemiluminescent label of different emission speed, and the four chemiluminescent labels are capable of forming a binding complex with different analytes in the sample.

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

5. The method according to any one of claims 1 -4, wherein the chemiluminescent labels independently have the structure of formula (I):

wherein A is an analyte or binding partner for an analyte,

L is absent (Le., it is a bond) or a linker optionally comprising a group L^c or Z^L, and

T is a chemiluminescent acridinium comprising the structure:

R2 and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Ra or R3 groups may together form a fused cyclic group and wherein Ra or Ri may comprise a linkage to an imaging agent such as a fhiorophore;

L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted;

Z^L is a zwitterionic linker group having the structure:

Tn” is 0 or 1 ; are independently at each occurrence an integer from 0 to 10;

Z is a zwitterionic group independently at each occurrence has the structure:

is independently an integer from 0 to 10;

X^a and X^b are independently at each occurrence an anionic group; L_l is independently at each occurrence -O-, -S-, -NH-, -N(R^N)-, -(CH₂)_1-10-, — S(=O)_1-2— , -C=C- -C=C-(CH₂)_1-3- -CIO)-. -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-

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical, optionally having one or more points of substitution;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon radical, optionally having one or more (e.g, 1-20, 1-10, 1-5) points of substitution;

R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and

R’ is hydrogen or a C1-10 alkyl; or a salt thereof.

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

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-Imidate, 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-b-Alanine, Texas Red-3-AM-DMAE- b-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):

wherein A is an analyte or binding partner for an analyte,

T is a chemiluminescent acridinium comprising the structure:

“j" and are independently 0, 1 , 2, 3, or 4;

Ri is hydrogen, -R, -X^b, -R^L-X^b, -L^c-R, -L^c-X^b (e.g., -L_l-X*), -Z, -R^L-Z, -L^c-Z, or-R^L- L^C-R^L-Z; R2a and Rzb are independently selected from alkyl optionally substituted;

Ric is hydrogen, -R, an electron donating group, or -Z; R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 groups may together form a fused heterocyclic group and wherein R3 may comprise a linkage to an imaging agent such as a fluorophore;

Z^L is a zwitterionic linker group having the structure:

“n” and “p” are independently at each occurrence an integer from 0 to 10;

Z is a zwitterionic group independently at each occurrence has the structure:

“

“r" is independently an integer from 0 to 10;

X¹ and X^b are independently at each occurrence an anionic group;

R^L is independently at each occurrence a C1-20 bivalent hydrocarbon radical, optionally having one or more points of substitution; R is independently at each occurrence hydrogen or C1 35 hydrocarbon radical, optionally having one or more points of substitution;

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof.

9. The method according to any one of claims 1-8, wherein one chemiluminescent label in the first set has the structure of formula (IIIa):

Ω is O orN;

Y’ is either absent, or is selected from -L_l-, -R^L- -R^L-L_l— , -L1-L1-, -L_l-R^L— , -L_l-R^L-L_l, and -R^L-L_l-R^L-; is 1, 2, 3, or 4; is 0, 1, 2, 3, or 4;

Ri is hydrogen, -R, -X^b, -R^L-X^b, -L^c-R, -L^c-X^b (e.g., -L_l-X¹¹), -Z, -R^L-Z, -L^c-Z, or-R^L- L^C-R^L-Z;

R2 and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group and wherein R2 or R3 may comprise a linkage to an imaging agent (IA) such as a fluorophore; and at least one lb group is not hydrogen;

Z^L is a zwitterionic linker group having the structure:

“m” is 0 or 1;

“ri” and “p” are independently at each occurrence an integer from 0 to 10;

Z is a zwitterionic group independently at each occurrence has the structure:

is independently an integer from 0 to 10;

X³ and X^b are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon radical, optionally having one or more points of substitution;

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof.

10. The method according to any one of claims 1-9, wherein one chemiluminescent label in the first set has the structure of formula (IVa):

and another chemiluminescent label in the first set has the structure of formula (IVb): Ω

wherein A1 and A₂ are independently an analyte or binding partner for an analyte and A1 is different than A₂; Ω is O orN;

Y’ i ith b t (i it i b d) i l t d fr L R^L R^L L L L

R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group and wherein Ra or R3 may comprise a linkage to an imaging agent such as a fluorophore.

Z^L is a zwitterionic linker group having the structure:

“m” is 0 or 1;

Z is a zwitterionic group independently at each occurrence has the structure:

“r” is independently an integer from 0 to 10;

X¹ and X^b are independently at each occurrence an anionic group;

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof.

11. The method according to any one of claims 1-10, wherein one chemiluminescent label in the second set has the structure of formula (Vc):

wherein A1 and Aa are independently an analyte or binding partner for an analyte and A1 is different than A2;

Ω is O orN;

Y’ is either absent, or is selected from -L_l- -R^L- -R^L-L_l— , -L1-L1-, -L_l-R^L— , -L_l-R^L-L_l, and -R^L-L_l-R^L-;

“A” is 0, 1, 2, 3, or 4;

Ri 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;

L^c has an electron withdrawing linker with respect to the phenyl;

I ,^cb is an electron donating linker with respect to the phenyl;

Z^L is a zwitterionic linker group having the structure:

Z is a zwitterionic group independently at each occurrence has the structure:

is independently an integer from 0 to 10;

X¹ and X^b are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution;

R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and R’ is hydrogen or a C_1-10 alkyl; or a salt thereof.

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

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

14. A compound having the structure of formula (V)

wherein A is an analyte or binding partner for an analyte,

L is absent (Le., it is a bond) or a linker optionally comprising a group L^c or Z^L,

“k” is independently 0 1 2 3 or 4;

Ric is hydrogen, -R, an electron donating group, or -Z; R₃ is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal R2 groups may together form a fused heterocyclic group and wherein Ri may comprise a linkage to an imaging agent such as a fluorophore; L^c is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted;

Z^L is a zwitterionic linker group having the structure:

Z is a zwitterionic group independently at each occurrence has the structure:

“r” is independently an integer from 0 to 10;

X¹ and X*’ are independently at each occurrence an anionic group;

R is independently at each occurrence hydrogen or C1-35 hydrocarbon radical, optionally having one or more points of substitution; R’ and R” are independently at each occurrence hydrogen or a C_1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and

R’ is hydrogen or a C_1-10 alkyl; or a salt thereof.

15. A compound having the structure of formula (VI)

L is absent or a linker optionally comprising a group L^c or Z^L,

R3 is independently selected at each occurrence from hydrogen, -R, an electron donating group, and -Z; wherein two vicinal Ra groups may together form a fused heterocyclic group and wherein Ra may comprise a linkage to an imaging agent such as a fluorophore;

Z^L is a zwitterionic linker group having the structure:

independently at each occurrence an integer from 0 to 10;

Z is a zwitterionic group independently at each occurrence has the structure:

is independently an integer from 0 to 10;

-C(O)-N(R^N)-(CH₂)_1-3- -(CH₂)_1-3-C(O)-N(R^N)- -(CH₂)_1-3-N(R^N)-C(O)-

R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;

R^N is independently at each occurrence from hydrogen or C1-5 alkyl; and

R’ is hydrogen or a C1-10 alkyl; or a salt thereof.

16. A composition comprising at least three chemiluminescent acridiniums, wherein each chemiluminescent acridinium is conjugated to a different analyte or a different binding pair for an analyte, and the chemiluminescence of at least two chemiluminescent acridiniums are separated in the wavelength time domain and the chemiluminescence of a different set of at least two chemiluminescent acridiniums are separated in the time domain.

17. The composition according to claim 16, wherein the composition comprises at least four chemiluminescent acridiniums conjugated to a different analyte or binding pair for an analyte, wherein the chemiluminescence of three chemiluminescent acridiniums is separated in the wavelength domain.

18. The composition according to claim 16 or 17, wherein the at least two chemiluminescent acridiniums with 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 acridiniums with time domain separated chemiluminescence 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 a solid phase reagent.