Antibody conjugated ion indicators and method of detecting proteins

Abstract

Described herein, is a platform for immunofluorescence imaging, specifically designed to enable the visualization of multiple proteins in a single sample using a single fluorescence channel. The platform uses ion-sensitive fluorescent indicators conjugated to antibodies to assess protein expression by selective activation or deactivation of the fluorophore component of the indicator using different activating solutions (e.g., ion-containing buffers). Using fluorescent ion indicator antibody conjugates, the imaging of multiple target proteins can be significantly streamlined using a single fluorescence channel.

Description

ANTIBODY CONJUGATED ION INDICATORS AND METHOD OF DETECTING PROTEINSTECHNICAL FIELD

[0001] The present invention is related fluorescent indicators and methods for detecting multiple proteins in a single sample using a single fluorescence channel.BACKGROUND

[0002] Immunofluorescence (IF) is an essential tool in drug discovery that combines antibody labeling with fluorescence microscopy to study the effects of compounds on a population of cells or tissues. More specifically, multiplexed IF or spatial proteomics combines IF with fluorescent compounds and advanced imaging to acquire a detailed snapshot of cell morphology, cell phenotypes, and protein expression at the single cell level to provide critical insights into a drug’s mechanism of action or potency. A major limitation of this platform is the number of fluorescent compounds and channels, where each fluorescent compound occupies a single fluorescence channel. In this scenario, the best set up can combine six fluorescent compounds across five channels to visualize up to five features in a single frame.

[0003] The use of multiple fluorescent compounds and multiple fluorescence channels in this manner is inefficient. Analysis using different fluorescence channels requires complex and expensive multiplexing equipment. It is therefore desirable to simplify and improve the throughput and scale of immunofluorescence imaging.SUMMARY OF THE INVENTION

[0004] The problems associated with immunofluorescent imaging of multiple proteins in a single sample can be resolved by conjugating antibodies with fluorescent ion indicators that can be selectively activated or deactivated through changes in the surrounding solution. Such changes can be induced by exposing the fluorescent ion indicators to an activating solution. Activating solutions contain ions that interact with the fluorescent ion indicators to cause a fluorophore component of the indicator to exhibit or change fluorescence. In one aspect, the activating solution comprises an ion that can be bound by an ion chelating component of the fluorescent ion indicator. Activating solutions can also induce fluorescence, or a change in fluorescence, by altering the ion concentrations, ionic strength, pH, or adding additional chemical components to the solution surrounding the fluorescent ion indicator.

[0005] In one aspect of the present disclosure, a fluorescent ion indicator comprises: a fluorophore component; an ion chelating component coupled to the fluorophore component; and a linker coupled to the fluorophore component. The linker comprises a coupling agent configured to connect to an amino acid, or complementary coupling agent, of an antibody. The fluorophore component exhibits or changes fluorescence in the presence of an activating solution. In one aspect, the activating solution comprises an ion that is bound by the ion chelating component.

[0006] In an aspect of the present disclosure, a fluorescent ion indicator antibody conjugate (also referred to herein as a “conjugate”) for imaging a protein in a sample, comprises: a fluorophore component; an ion chelating component coupled to the fluorophore component; a linker coupled to the fluorophore component, and an antibody for a protein of interest coupled to the fluorophore component by the linker. The fluorophore component exhibits or changes fluorescence in the presence of an activating solution. In one aspect, the activating solution comprises an ion that is bound by the ion chelating component of the fluorescent ion indicator.

[0007] In an aspect of the present disclosure, a method of detecting multiple proteins in a biological sample (e.g., cells or a tissue sample) comprises an initial step of contacting the biological sample with a plurality of distinct fluorescent ion indicator antibody conjugates. Each fluorescent ion indicator comprises a fluorophore component and an ion chelating component. Each distinct fluorescent ion indicator antibody conjugate comprises an antibody and / or an ion chelating component that is different than antibodies and / or ion chelating components coupled to other distinct fluorescent ion indicator antibody conjugates.

[0008] The method further comprises contacting the biological sample with a first activating solution. In one aspect, the first activating solution comprises a first ion that can be captured by an ion chelating component of at least one of the distinct fluorescent ion indicator antibody conjugates.

[0009] The method further comprises analyzing the proteins in the biological sample, in the presence of the first activating solution, with a fluorescence imaging device using a single fluorescence channel. The biological sample is then washed to remove the first activating solution.

[0010] The method further comprises contacting the biological sample with a second activating solution. The second activating solution comprises a second ion, different from thefirst ion. The second ion can be captured by an ion chelating component of at least one of the fluorescent ion indicator antibody conjugates. The biological sample is then analyzed, in the presence of the second activating solution, with a fluorescence imaging device using the same single fluorescence channel that was used to analyze the biological sample in the presence of the first activating solution.

[0011] In another aspect of the present disclosure, a method of detecting multiple distinct proteins in a sample solution comprises an initial step of coupling a plurality of the distinct proteins in the sample solution to a solid substrate, directly or indirectly. The solid substrate is then washed to remove to remove proteins that are not bound to the solid substrate.

[0012] The solid substrate is then contacted with a plurality of distinct fluorescent ion indicator antibody conjugates. Each distinct fluorescent ion indicator antibody conjugate comprises a fluorophore component, an ion chelating component, and a unique antibody for at least one of the distinct proteins in the sample solution.

[0013] The solid substrate is then contacted with a first activating solution. In one aspect, the first activating solution comprises a first ion that can be captured by an ion chelating component in at least one of the fluorescent ion indicator antibody conjugates. The proteins bound to the solid substrate are analyzed with a fluorescence imaging device using a single fluorescence channel. The solid substrate is then washed to remove the first activating solution.

[0014] The solid substrate is contacted with a second activating solution. The second activating solution comprises a second ion, different from the first ion. The second ion can be captured by an ion chelating component of at least one of the fluorescent ion indicator antibody conjugates. The proteins bound to the solid substrate are analyzed, in the presence of the second activating solution, with a fluorescence imaging device using the same single fluorescence channel that was used to analyze the proteins bound to the solid substrate in the presence of the first activating solution.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Advantages of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description of embodiments and upon reference to the accompanying drawings in which:

[0016] FIG. 1 depicts a schematic diagram of a method of analyzing multiple proteins coupled to a solid support using fluorescent ion indicator antibody conjugates having different metal ion chelating component.

[0017] FIG. 2 shows a schematic diagram of streptavidin-biotin binding of a fluorescent ion indicator to a solid support (FIG. 2B).

[0018] FIG. 3A shows the relative fluorescence unit (RFU) value for each fluorescent ion indicator, coupled to a well of a solid support, in the presence of the appropriate activating solution, compared to uncoated wells.

[0019] FIG. 3B shows the normalized fluorescence for each fluorescent ion indicator, coupled to a well of a solid support, in the presence of the appropriate activating solution, compared to uncoated wells.

[0020] FIG. 4A shows the relative fluorescence unit (RFU) value for each fluorescent ion indicator, coupled to a well of a solid support, when each indicator is cycled between “on” and “off’ states.

[0021] FIG. 4B shows the normalized fluorescence for each fluorescent ion indicator, coupled to a well of a solid support, when each indicator is cycled between “on” and “off’ states.

[0022] FIGS 5A shows the relative fluorescence unit (RFU) value for each fluorescent ion indicator, coupled to a well of a solid support, when each fluorescent ion indicator is sequentially exposed to (1) ’’Off’ solution, (2) K+solution, (3) Na+solution, and (4) Ca2+solution.

[0023] FIGS 5B shows the normalized fluorescence for each fluorescent ion indicator, coupled to a well of a solid support, when each fluorescent ion indicator is sequentially exposed to (1) ’’Off’ solution, (2) K+solution, (3) Na+solution, and (4) Ca2+solution.

[0024] FIG. 6A shows the relative fluorescence unit (RFU) value for the Na+Dye indicator as the concentration of Na+ Dye indicator is increased.

[0025] FIG. 6B shows the normalized fluorescence for the Na+Dye indicator as the concentration of Na+ Dye indicator is increased.

[0026] FIG. 7A shows the relative fluorescence unit (RFU) value for the Na+Dye indicator as the concentration of sodium in an activating solution is increased.

[0027] FIG. 7B shows the normalized fluorescence for the Na+Dye indicator as the concentration of sodium in an activating solution is increased.

[0028] FIG. 8A shows the relative fluorescence unit (RFU) value for the Ca2+Dye indicator as the concentration of sodium in an activating solution is increased.

[0029] FIG. 8B shows the normalized fluorescence for the Ca2+Dye indicator as the concentration of sodium in an activating solution is increased.

[0030] FIG. 9A shows an absorption spectrum of TAMRA - Donkey anti -rabbit IgG antibody (“Dk a-Rabbit IgG TAMRA) and Compound VIII-7 (“Dk a-Rabbit IgG Sodium Ion Indicator - Compound VIII-7).

[0031] FIG. 9B shows an absorption spectrum of Compound VIII-8 (“Dk a-Rabbit IgG Sodium Ion Indicator - Compound VIII-8).

[0032] FIG. 10A shows the fluorescence imaging of the control cells when treated with 4’, 6-diamidino-2-phenylindole (DAPI).

[0033] FIG. 10B shows the fluorescence imaging of the cells treated with anti -histone H3 and Compound VIII-7.

[0034] FIG. 10C depicts an overlay of the cell fluorescence in FIG. 10B with the cell fluorescence in FIG. 10A.

[0035] FIG. 11 depicts quantification of the relative fluorescence of anti-Histon H3 / Compound VIII-7 treated cells in numerous activating solutions compared to an “Off” solution.DETAILED DESCRIPTION OF THE INVENTION

[0036] An aspect of the present disclosure is to develop a method for imaging multiple proteins in a single sample using a single fluorescence channel. This can be achieved by use of distinct fluorescent ion indicator antibody conjugates that can be selectively activated or deactivated through changes in the ion composition of the surrounding solution.

[0037] Described herein are fluorescent ion indicator antibody conjugates and a method of imaging proteins in a sample using these conjugates. The use of a fluorescent ion indicator antibody conjugates, as described herein streamlines multi-antibody labeling without requiring complex multiplexing instruments. Additionally, the number of proteins that can be assessed in a single sample can be increased. Another advantage of using the distinct fluorescent ion indicator antibody conjugates described herein is that the versatility andaccessibility of the immunofluorescence imaging can be enhanced. Additionally, the described distinct fluorescent ion indicator antibody conjugates can be used to improve the throughput and scale of immunofluorescence imaging.

[0038] The fluorescent ion indicator includes a fluorophore component coupled to an ion chelating component, and a linker for coupling the fluorophore component to an antibody. The linker can include a coupling agent that can be used to couple the fluorophore component to an antibody, or an antibody may be preloaded on the fluorophore component though the linker. The ion chelating component in combination with the florescence component allows the detection of changes of ion concentration in the vicinity of the fluorescent ion indicator.

[0039] While the present description generally describes one fluorescent ion indicator coupled to an antibody, it should be understood that more than one fluorescent ion indicator can be coupled to the antibody. For example, one to twelve fluorescent ion indicators can be coupled to an antibody. The fluorescent ion indicators, bound to an antibody, can all be the same fluorescent ion indicators or can be different fluorescent ion indicators. In one aspect of the present disclosure, the intensity of the signal generated by the fluorescent ion indicator antibody conjugate can be increased by adding multiple fluorescent ion indicators of the same structure to the antibody.

[0040] The fluorophore component will exhibit or change fluorescence in the presence of an activating solution. In one aspect, the activating solution comprises an ion that is bound by an ion chelating component of the fluorescent ion indicator. For example, the activating solution can include metal ions that can be captured by the ion chelating component of the fluorescent ion indicator. Exemplary metal ions include, but are not limited to, lithium, sodium, potassium, and calcium. Activating solutions can also induce fluorescence, or a change in fluorescence, by altering the ion concentrations, ionic strength, pH, or adding additional chemical components to the solution surrounding the fluorescent ion indicator.

[0041] In some embodiments, the fluorophore component is tethered to a coupling agent through a linker. The coupling agent allows for the coupling of the fluorescent ion indicator to an antibody or a solid substrate (e.g., formed from glass or a polymer).

[0042] Fluorescent ion indicators and fluorescent ion indicator antibody conjugates, as described in some embodiments herein, have the general structures (Fl) or (F2).IonChelatingIonChelating > Linker Coupling Agent / AntibodyComponentThe fluorescent ion indicator and conjugates are composed of: a fluorophore component, an ion chelating component, and linker having a coupling agent for connecting to an antibody.Fluorophore Component

[0043] The fluorophore component can be any compound that exhibits or changes fluorescence. When coupled to an ion chelating component, the fluorophore component exhibits or changes fluorescence in the presence of an activating solution containing an ion that is captured by the ion chelating component. For example, if an activating solution comprises a metal ion, the metal ion is bound to the ion chelating component which induces, or changes, fluorescence of the fluorescent component.

[0044] In a preferred embodiment, the fluorophore component is a xanthene fluorescence compound. Xanthene fluorescence compounds useful in the present fluorescent indicators include fluorescence compounds having the general structure (FC-1):where:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, or OC(O)R; R’ is H or a countercation; each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibodyeach W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl. In the structures set forth in this application the use of a dashed line | == | in the structures denotes that the bond can be either a single bond or a double bond. The term “countercation” refers to a positively charged ion that is associated with a negatively charged oxygen group. Examples of countercations include, but are not limited to, positively charged metal ions (e.g., Li+, Na+, K+, Ca+2, etc. and ammonium salts. Ammonium salts include: ammonium (NH₄⁺), monalkyl ammonium (NRH3+), dialkyl ammonium (NRR'H2+), trialkyl ammonium (NRR'R" H+), and tetraalkyl ammonium (NRR'R" R"'+), wherein the alkyl groups may be identical (NR2H2+for dialkyl, NR3H+for trialkyl, and NR₄⁺ for tetraalkyl ammonium (e.g., NMe₄+), two identical and the remainder different (NR2R'H+for trialkyl and NR2R'R"+for tetraalkyl ammonium), three identical and the remainder different (NR3R'+for tetraalkyl), two identical and two others identical but different from the other two (NR2R'2+for tetraalkyl), or each independently different (NRR'H2+for dialkyl, NRR'R" H+for trialkyl, and NRR'R" R"'+ for tetralkyl).

[0045] Specific examples of xanthene fluorescence compounds include the compounds set forth in Table 1.TABLE 1

[0046] Many xanthene fluorescent compounds can exist in tautomeric forms. The specific tautomeric form adopted by the xanthene dye can depend on the solution conditions that the compound is exposed to (e.g., pH). For example, for fluorescein and similar xanthene fluorescent compounds having a carboxylate group in the R1position (e.g., Oregon Green), the fluorescent compound can exist in either an open form (FC-2) or a spirolactone form (FC-3).

[0047] Xanthene fluorescent compounds having an amine group in the W1and / or the W2position, such as rhodamine B, and a carboxylate group in the R1position, can exist in either an open form (FC-4), (FC-6) or a spirolactone form (FC-5), (FC-7).Throughout the specification, for readability and efficiency of describing the fluorescent compounds, only one of the various tautomeric chemical structures will be displayed.However, it should be understood that the non-displayed tautomeric chemical structure is also encompassed by the displayed chemical structure.

[0048] In one aspect of the present disclosure, xanthene fluorescent ion indicators can be used which have the general structures (I), (II), (III) or a salt thereof.Ionwhere:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, or OC(O)R; R’ is H or a countercation; each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6alkyl, CH2OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody; andW1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.Ionwhere:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a countercation; each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;W1represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; andW2represents NH, NMe, NEt, or O.Ionwhere:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, or OC(O)R; R’ is H or a countercation; each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6alkyl, CH2OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody; andW1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

[0049] Examples of fluorescent ion indicators having coupling agents for coupling to an antibody are described in PCT Publication No. WO 2024 / 243471, which is incorporated herein by reference.Ion Chelating Component

[0050] The fluorescent ion indicators can function as an ion sensor when an ion bound to the ion chelating component alters the electronic structure or the molecular structure of the fluorophore component. Changes in the electronic structure can lead to a change in the intensity or wavelength of light absorption or emission. In the fluorescent ion indicators disclosed herein, the ion chelating component is attached to the fluorophore component such that the fluorescence of the fluorophore component is changed in the presence of the appropriate ion. In preferred embodiments, the ion chelating component can bind to metal ions, including, but not limited to thallium, calcium, sodium or potassium.Thallium Ion Chelating Component and Conjugates

[0051] Examples of groups capable of chelating thallium cations include derivatives of 2,2'-((2-alkoxyphenyl)azanediyl)diacetic acid and their cell-permeable, esterase-hydrolyzable ester forms, preferably methyl, ethyl, and acyloxyalkyl ester derivatives (e.g., acetoxymethyl). An example of a thallium ion chelating compound is depicted as (T- 1 ).Each R of (T-l) can independently represent H, Me, Et, CH₂OAc, or a countercation. R’ is typically methyl. However, it has been surprisingly discovered that the thallium ion chelating component retains the ability to bind to thallium ions when R’ is a methylene (CH2) group which can be used to connect a coupling agent to the thallium ion chelating component (as shown schematically in structure (F-2)).

[0052] In an aspect of the disclosure, thallium selective fluorescent ion indicator antibody conjugates have the structure (IV):where:each R independently represent H, Me, Et, -CH₂OAc, or a countercation;R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a countercation each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH₂OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;MU represents or5-5andW1and W2each independently represents NH2, N(Me)2, N(Et)2, OH, OAc, or OAlkyl.

[0053] Exemplary thallium selective fluorescent ion indicator antibody conjugates have the structure (IV- 1) and (IV-2):(IV-2).Calcium Ion Chelating Component and Conjugates

[0054] Calcium ion chelating components include derivatives of l,2-bis(o-aminophenoxy)ethane-N, N, N’-N’-tetraacetic acid and 5-methyl l,2-bis(o-aminophenoxy)ethane-N, N, N’-N’-tetraacetic acid and their cell-permeable, esterase-hydrolyzable ester forms, with methyl, ethyl, and acyloxyalkyl ester (e.g., acetoxymethyl) derivatives preferred. Examples of calcium ion chelating components that are suitable for use in the described fluorescent indicators are described in U. S. Patent Application Publication No. 2012 / 0183986 and U. S. Patent No. 9,810,700, both of which are incorporated herein by reference.

[0055] Embodiments of a calcium ion chelating components have the structures (CA-1) or (CA-2).(CA-1) (CA-2)whereeach R independently represent H, Me, Et, -CH₂OAc, or a countercation; andR12represents H, halogen, or Me.

[0056] In an aspect of the disclosure, calcium selective fluorescent ion indicator antibody conjugates have the structure (V):where:each R independently represent H, Me, Et, -CH₂OAc, or a countercation (e.g., Li+, Na+, K+, Me4N+)R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, or OC(O)R; R’ is H or a countercation; each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, Ci-6 alkyl, CH₂OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;W1represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;W2represents N or O.

[0057] Exemplary calcium selective fluorescent ion indicator antibody conjugates have the structures (V- 1), (V-2), and (V-3):Compound (V-l): R2is Cl.Compound (V-2): R2is F.Compound (V-3): R2is H.

[0058] Other exemplary calcium selective fluorescent ion indicator antibody conjugates have the structures (V-4), (V-5), and (V-6):Compound (V-4): R2is Cl.Compound (V-5): R2is F.Compound (V-6): R2is H.

[0059] A specific example of a calcium selective fluorescent ion indicator antibody conjugate is a compound having the structure (V-7):(V-7)Lithium. Sodium and Potassium Ion Chelating Components

[0060] Lithium, sodium and potassium ion chelating groups include cryptands and crown ethers that are specific for lithium, sodium or potassium ions. Additional sodium and potassium ion chelating groups include ethylene glycol tetracetic acid and 2,2'-((2-(2-(2-(bis(carboxymethyl)amino)ethoxy) ethoxy)phenyl)-azanediyl)diacetic acid, and ester derivates thereof.

[0061] Cryptand lithium, sodium and potassium ion chelating groups include cryptands having the structures (C-l), (C-2), (C-3), or (C-4).

[0062] Additional lithium, sodium and potassium ion chelating groups include crown ethers and aza-crown ethers. Exemplary crown ethers and aza-crown ethers that can be coupled to the fluorescent indicator include, but are not limited to, monoaza-15-crown-5, monoaza-18-crown-6, diaza- 15 -crown-5, diaza- 18-crown-6. Crown ethers and aza-crown ethers can be tailored to a specific metal cation (e.g., lithium, sodium, or potassium) by altering the size of the crown ether. Exemplary crown ether sodium or potassium ion chelating groups include crown ethers having the structures (C-5), (C-6), or (C-7)R6is H, alkyl, or –(CH₂CH₂O)p–CH₃; p = 1-4; andR7is H or Me.

[0063] In an aspect of the disclosure, potassium selective fluorescent ion indicator antibody conjugates have the structure (VI):each R3independently represent H, F, Br, Cl, or –C₁₋₆ alkyl–CO₂R4;R4is H, Ci-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;R8is -OH, -O', or -OC1-6 alkyl;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

[0064] An exemplary potassium selective fluorescent ion indicator antibody conjugate has the structures (VI- 1) and (VI-2):

[0065] Potassium selective fluorescent ion indicator conjugate precursors include a fluorescent ion indicator, a Linker, and a Coupling Agent. The conjugate precursors can be reacted with an antibody to form a fluorescent ion indicator antibody conjugate. Exemplary potassium selective fluorescent ion indicator conjugate precursors have the structures (VI-2),(VI-4)

[0066] In an aspect of the disclosure, potassium selective fluorescent ion indicator antibody conjugates have the structure (VII):where:each R3independently represent H, F, Br, Cl, or –C₁₋₆ alkyl–CO₂R4;R4is H, Ci-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;R8is -OH, -O'-countercation, or -OC1-6 alkyl;R9is C1-6 alkyl or CHO;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

[0067] Exemplary potassium selective fluorescent ion indicator antibody conjugates have the structures (VII- 1) or VII-2:(VII-2) - IPG -1

[0068] In an aspect of the disclosure, sodium selective fluorescent ion indicator antibody conjugates have the structure (VIII):where:each R3independently represent H, F, Br, Cl, or –C₁₋₆ alkyl–CO₂R4;R4is H, Ci-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;R8is -OH, -O', or -OC1-6 alkyl;R9is C1-6 alkyl or CHO;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

[0069] Exemplary sodium selective fluorescent ion indicator antibody conjugates have the structures (VIII- 1) or (VIII-2):(VIII-2)R4is H, -Ci-6 alkyl, CH₂OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody.

[0070] Sodium selective fluorescent ion indicator conjugate precursors include a fluorescent ion indicator, a Linker, and a Coupling Agent. The conjugate precursors can be reacted with an antibody to form a fluorescent ion indicator antibody conjugate. Exemplary sodiumselective fluorescent ion indicator conjugate precursors have the structures (VIII-3), (VIII-4), (VIII-5) or (VIII-6):(VIII-5): R4is a countercation(VIII-6): R4is -CH2OAC.Coupling Agent

[0071] The coupling agent represents a means for connecting the fluorescent component / ion chelating component to a biomolecule or a solid substrate. The term “biomolecule” encompasses polypeptides, polynucleotides, and polysaccharides. Exemplary biomolecules include proteins, enzymes, antibodies, DNA and RNA. Solid substrates can include glass or polymeric substrates in the form of beads or plates.

[0072] A “coupling agent” is a functional group that can be used to couple the fluorescent component / ion chelating component to a biomolecule or a solid substrate. For example, a coupling agent can include a functional group capable of reacting with an amino acid side chain of an antibody, protein or polypeptide, or a complementary functional group attached to an amino acid side chain of an antibody, protein or polypeptide, to couple the antibody, protein or polypeptide to the fluorescent component / ion chelating component. Alternatively, the coupling agent can include a functional group capable of reacting with a complementary functional group on a solid substrate (e.g., a polymeric substrate) to couple the fluorescent component / ion chelating component to the solid substrate.

[0073] In one embodiment, the coupling agent represents a ligand for a self-labeling enzyme. The introduction of a ligand for a self-labeling enzyme enables the precise localization of thefluorescent component / ion chelating component on antibodies, proteins, or polypeptides. Examples of self-labeling enzymes include HALOTAG® ligands, SNAP -tag ligands, and CLIP -tag ligands.

[0074] HALOTAG® ligands have the general structure:where t is 2-8 and u is 2 to 10. Exemplary HALOTAG® ligands include [2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamino group (Amine O2chloroalkane) and 21-chloro- 3,6,9, 12, 15-pentaoxahenicosan-l-amino group (Amine O4chloroalkane)].e O4chloroalkane).

[0075] SNAP -tag ligands include benzylguanine and chloropyrimidine derivatives.Exemplary SNAP-tag ligands include:(benzylguanine) (chloropyrimidine).

[0076] CLIP-tag ligands include benzylcitosine derivatives. The benzylcitosine ligand is typically attached to the coupling group, -C(O)-, as an amide.(benzylcytosine).

[0077] An alternate method of binding the fluorescent component / ion chelating component onto a biomolecule is through the use of a covalent bond. A number of covalent bond forming ligands can be used to couple the fluorescent compound / metal ion chelatingcomponent to a biomolecule. In an embodiment, the coupling agent is a maleimide group. Maleimide ligands have the general structure:Maleimide groups react with thiol groups on the side chain of cysteine amino acids of an antibody to form a covalent bond linking the antibody to the fluorescent component / ion chelating component (“Indicator”), as shown in the reaction scheme below:

[0078] In an embodiment, the coupling agent is an N-hydroxy succinimide group (NHS). NHS ligands have the general structure:NHS groups activate the adjacent carbonyl coupling group to allow amine side chain groups of amino acids of an antibody to form a covalent bond (an amide) linking the antibody to the fluorescent component / ion chelating component (“Indicator”), as shown in the reaction scheme below:

[0079] In an embodiment, the coupling agent is an alkyne group or an azido group. The copper(I) catalyzed azide alkyne cycloaddition reaction (CuAAC) between an alkyne group and an azide forms a 1,4-disubstituted 1,2,3-triazole, which can be used to couple thefluorescent component / ion chelating component to the biomolecule (e.g., an antibody). The antibody and the fluorescent component / ion chelating component have complementary groups, azide and alkyne, attached which react, in the presence of a copper catalyst, to form a disubstituted 1,2, 3 -triazole. Exemplary coupling schemes based on azide-alkyne cycloaddition reaction are shown below. In one embodiment, the alkyne group is coupled to the fluorescent component / ion chelating component and an azido group is coupled to the antibody. In an alternate embodiment, the azido group is coupled to the fluorescent component / ion chelating component and the alkyne group is coupled to the antibody.Ruthenium catalyzed azide-alkyne cycloaddition can also be used as a coupling scheme with the only difference being the generation of the isomeric 1,5-disubstituted 1,2, 3 -triazole.IndicatorAntibodyThe 1,4-disubstituted 1,2,3-triazole forms a stable covalent attachment between the fluorescent component / ion chelating component and the antibody, as shown in the reaction scheme (1).

[0080] In an embodiment, the coupling agent comprises a biotin group:Biotin is a small hapten molecule with high binding affinity for streptavidin. The streptavidin-biotin complex is one of the strongest non-covalent interactions between a protein and its ligand. This strong binding can be used to couple the fluorescentcomponent / ion chelating component to an antibody or a solid-support through streptavidin which is coupled to the antibody or solid support.

[0081] Click chemistry can also be used to couple the fluorescent component / ion chelating component to a biomolecule (e.g., an antibody) or a solid substrate. Click chemistry uses the formation of a heterocycle from highly reactive precursors coupled to the fluorescent component / ion chelating component and the biomolecule or solid substrate.

[0082] Heterocycle groups can be synthesized by reaction of complementary chemical handle groups (T, I”) coupled to the fluorescent component / ion chelating component and the biomolecule (e.g., an antibody). Alternatively, as depicted in the reaction scheme below, a chemical handle (T) can be coupled to a linker (X) of a coupling agent (e.g., a ligand for a self-labeling enzyme)OIndicator — X — T+T' — X' — Indicator X Het XL Lwhere T and T’ combine to form the Het group. Examples of chemical handles (T, I”) include, but are not limited to:

[0083] The click chemistry reaction forms a heterocycle ring that couples the fluorescent component / ion chelating component to the biomolecule. In one embodiment, a 1,2, 3 -triazole heterocycle is formed by the reaction of an azido group with an ethynyl group, a cyclooctyne,a cyclopropylcyclooctyne, or a dibenzoazacyclooctyne. Exemplary 1,2, 3 -triazole heterocyles formed using click chemistry include, but are not limited to:In other embodiments, a pyridazine or dihydropyridazine heterocycle is formed by the reaction of a tetrazine (e.g., 1,2,4,5-tetrazine; and 6-methyl-l,2,4,5-tetrazine) with a strained alkene or alkyne (e.g., norbomene, (methyl)cyclopropene, cyclopropyloctyne, and transcyclooctene). Exemplary pyridazine or dihydropyridazine heterocycles formed using click chemistry include, but are not limited to:where R10is H or Me.

[0084] In some embodiments, the coupling agent is a functional group capable of reacting with a complementary functional group on the biomolecule (e.g., an antibody) or solid substrate to form a covalent bond linking the biomolecule or solid substrate to the fluorescent component / ion chelating component. Exemplary reactive functional groups include, but are not limited to: -OH, -NHR11, -O-C(O)-OR9, -O-C(O)-NHRn, -C(O)-OR9, -C(O)-NHRn, -NRn-C(O)-OR9, or-NRn-C(O)-NHRn, where R9is H, alkyl, countercation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.Linking Groups

[0085] Linking groups, designated as “Linker” in the structures, can, optionally, be used to connect the fluorescent component / ion chelator component together. A “linking group” or “linker” describes one or more atoms that directly links components of the fluorescent component / ion chelator component to a coupling agent. Linking groups can be used to:connect the fluorescent component / ion chelator component to the coupling agent. Linking groups can be used to create space between the components and / or provide chemical handles to facilitate the formation of chemical bonds linking the fluorescent component / ion chelator component to the biomolecule or solid substrate.

[0086] Generally, linking groups have chemical structures that include, but are not limited to: — (CH2)m—, — (CH2)m— Y1— (CH2)n—, -(CH2)m-Y1-(CH2)n-Y2-(CH2)0-, -(CH2)m-(CH2CH2O)P-, -(CH2)m-(CH2CH2O)p-(CH2)n-Y1-(CH2)o-(CH2CH2O)q-(CH2)r-,-(CH2)m-(CH2CH2O)p-(CH2)n-Y1-(CH2)o-(CH2CH2O)q-(CH2)r-Y2-(CH2)s-,-(CH2)m-(OCH2CH2)p-, -(CH2)m-(OCH2CH2)p-(CH2)n-Y1-(CH2)o-(OCH2CH2)q-(CH2)r-, or-(CH2)m-(OCH2CH2)p-(CH2)n-Y1-(CH2)o-(OCH2CH2)q-(CH2)r-Y2-(CH2)s-where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4;each Y1and Y2independently represent O, NR11, -C(O)-, -O-C(O)-, -C(O)-O-, -NR11-C(O)-, -C(O)-NR11-, or Het;R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.Linker-Coupling Agent / Antibody

[0087] A combination of a linker and coupling agent can be used to form a covalent coupling between the fluorescent component / ion chelator component and a biological molecule (e.g., an antibody) as shown above (see compounds (Fl) and (F2).

[0088] Exemplary Linker-Coupling Agent / Antibody include, but are not limited to:Fluorescent / lon ChelatorComponents Streptavidin - AntibodyFluorescent / lon ChelatorComponentsO Antibody Fluorescent / lon ChelatorComponentsBiological Samples

[0089] The fluorescent ion indicators described herein can be used to analyze proteins in biological samples. As used herein a “biological sample” is a sample from a human or from a non-human animal. A biological sample may comprise cells, a tissue sample, body fluid, or a combination thereof. The proteins studied in the biological sample may be intracellular, extracellular, part of a cell membrane; attached to a surface of a cell, or a combination thereof. A biological sample can be obtained from a body fluid. The body fluid may be extracellular fluid, which may comprise one or both of interstitial fluid, intravascular fluid, and transcellular fluid. The body fluid may be intracellular fluid. Non-limiting examples of body fluid include amniotic fluid, aqueous humor, bile, blood plasma, breast milk, cerebrospinal fluid, cerumen, chyle, exudates, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, or urine.Fluorescent ion indicator antibody conjugates

[0090] A fluorescent ion indicator antibody conjugate for imaging a cell or tissue sample includes: a fluorophore component; an ion chelating component coupled to the fluorophore component; a linker / coupling agent coupled to the fluorophore component, and an antibody for a protein present in the cell or tissue sample, coupled to the fluorescent component by the linker / coupling agent. The fluorophore component exhibits or changes fluorescence in thepresence of an activating solution. In one aspect, the activating solution comprises a metal ion that is bound by the ion chelating component of the fluorescent ion indicator.

[0091] The antibody can be any antibody designed to interact with a desired target. In some embodiments, the antibody target is a protein or a small molecule compound found in biological sample.

[0092] The antibody can be a general antibody that binds to proteins prevalent in most cells. For example the antibody can be an anti-histone antibody or an anti-IgG antibody. In other aspects of the present disclosure, the antibody can be an antibody that targets proteins associated with diseases and adverse physiological conditions. Antibodies targeting proteins associated with cancer can be coupled to a fluorescent ion indicator for diagnosis and study of cells that include proteins associated with cancer. The fluorescent ion indicators, coupled to the appropriate antibody, can be used to identify the presence of the protein in cells as a diagnostic tool. Use of a multiple fluorescent ion indicator conjugates that fluoresce in the presence of different ions, but can be studied in a single fluorescence channel, can allow multiple protein targets to be studied by changing the activating solution (i.e., the ion present in the solution) without the need for modification of the fluorescent channel.Activating Solutions

[0093] Activating solutions contain ions that interact with the fluorescent ion indicators to cause a fluorophore component of the indicator to exhibit or change fluorescence. In one aspect, the activating solution comprises an ion that can be bound by an ion chelating component of the fluorescent ion indicator. Activating solutions can also induce fluorescence, or a change in fluorescence, by altering the ion concentrations, ionic strength, pH, or adding additional chemical components to the solution surrounding the fluorescent ion indicator.

[0094] Activating solution can include metal ions that can be captured by the ion chelating component of the fluorescent ion indicator. Exemplary metal ions include, but are not limited to, lithium, sodium, potassium, thallium, and calcium.

[0095] Activating solutions can include the appropriate metal ion species and content and optional solution additives. Optional solution additives can include buffering agents.Buffering agents include, but are not limited to, amines, ammonium salts, phosphoric acids, phosphate salts, citric acid, citrate salts, carbonate salts, sulfonic acids, sulfate salts, sulfonate salts, glycinate salts, boronic acids, and borate salts. Specific buffering agents include, butare not limited to tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid) sodium salt (MES), 2-(bis(2-hydroxyethyl)amino)acetic acid (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, sodium salt (TES). Buffer agents can be used to maintain the pH of the activating solution to a pH compatible with the biological sample. The pH ranges for the activating solution can be between 5 and 9, preferably between 6 and 8, and more preferably between 6.5 and 7.5.

[0096] Optional solution additives that can also be present in the activating solution include organic chelating agents. Exemplary organic chelating agents include, but are not limited to, Ethylenediaminetetraacetic acid (EDTA), l,2-bis(o-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA), ethylene glycol-bis([3-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), citric acid, gluconic acid, sodium gluconate, ethylenediamine-N, N'-disuccinic acid (EDDS), dimercaptosuccinic acid (DMSA), tetrasodium glutamate diacetate (GLDA), and hydroxyethylethylenediaminetriacetic acid (HEDTA). Organic chelating agents can be added to activating solutions to sequester ions that may activate other fluorescent ion indicators. Additionally, organic chelating agents can sequester ions that may interfere with the fluorescence of the fluorescent ion indicator that is matched to the ion in the activating solution.

[0097] Activating solutions can include one optional additive, two optional additives, three optional additives, or may include more than three optional additives. Activating solutions can include one or more buffer agents and one or more chelating agents.

[0098] Activating solutions that contain a metal ion can include the metal ion at concentrations of 1 mM to 1 M, preferably from 10 mM to IM. The concentration of optional solution additives (e.g., buffer agents and / or organic chelating agents) in an activating solution can range from 1 mM to 1 M, preferably from 10 mM to IM.“Off’ solutions

[0099] ‘ ‘Off’ solution are solutions that can be used to prepare a sample for analysis with an activating solution. In the initial step of the analysis, the biological sample can be washed with an “off’ solution to remove ions that may interfere with the subsequent analysis of the biological sample. In subsequent steps, the “off’ solution can be used to remove ions (e.g.,metal ion) from the biological sample that were used to activate one or more of the fluorescent ion indicators.

[0100] An “off’ solution may include one or more buffer agents and / or one or more chelator agents. Buffer agents and chelator agents that can be included are the same buffer agents and chelators that can be present in the activating solution. The “off’ solution is free of ions that will interact with any of the fluorescent ion indicators present in the biological sample. The concentration of buffer agents and / or organic chelating agents in an activating solution can range from 1 nM to 1 M, preferably from 100 nM to IM.Methods of Detecting Proteins in a Sample

[0101] In an aspect of the present disclosure, a method of detecting multiple proteins in a biological sample comprises an initial step of contacting the biological sample with a plurality of distinct fluorescent ion indicator antibody conjugates. A fluorescent ion indicator antibody conjugate comprises a fluorescent ion indicator coupled to an antibody. The fluorescent ion indicator comprises a fluorophore component and an ion chelating component. As used herein the term “distinct fluorescent ion indicator antibody conjugate” is meant to describe different conjugates that have different antibodies and / or different ion chelator components. For example, a first distinct fluorescent ion indicator antibody conjugate can have a first fluorescent ion indicator coupled to a first antibody. A second distinct fluorescent ion indicator antibody conjugate can have a second fluorescent ion indicator coupled to a second antibody. The conjugates are distinct because the first and second fluorescent ion indicators bind to different ions and the first and second antibodies bind to different targets. In this manner, each distinct fluorescent ion indicator antibody conjugate will interact with a different target and interact with a different ion.

[0102] Contact of the biological sample with the plurality of distinct fluorescent ion indicator antibody conjugates allows the antibody of each distinct conjugate to bind with one or more proteins present in the biological sample. Each of the distinct fluorescent ion indicator antibody conjugates will bind to a different target (e.g., different protein targets).

[0103] After the biological sample is contacted with a plurality of distinct fluorescent ion indicator antibody conjugates, the biological sample is then contacted with a first activating solution. The first activating solution includes a first ion which can be captured by an ion chelating component of at least one of the distinct fluorescent ion indicator antibody conjugates.

[0104] The biological sample is analyzed with a fluorescence imaging device using a single fluorescence channel in the presence of the first activating solution. In the presence of the first activating solution, the distinct fluorescent ion indicator antibody conjugates that capture the first ion will fluoresce. Fluorescent imaging devices generally include an excitation source and an emission detector. The excitation source produces light at wavelengths that will produce fluorescence in the fluorescent component. In the present disclosure, a change in fluorescence is produced when a metal ion is captured by the metal ion chelating component and light of the appropriate wavelength is received by the fluorophore component. The emission detector will capture the fluorescent emission from the fluorescent component. To improve excitation and detection of fluorescence in the fluorophore component, the excitation light can be filtered such that a narrow band of wavelengths of light pass through the sample, around the peak excitation wavelength for the fluorescent component. Similarly, the emission light from the fluorescent component can also be filtered to allow only the desirable fluorescence from the sample to reach the detector while blocking unwanted traces of excitation light. Like the excitation filter, this filter only allows a narrow band of wavelengths to pass through it, around the peak fluorophore emission wavelength. Excitation filters and emission filters are matched and together are often referred to as a “fluorescence channel”. Channels are named according to the color of light being used for the fluorescent imaging, as is known in the art. Typical fluorescence channels include the “blue fluorescence channel”, the “green fluorescence channel”, and the “red fluorescence channel”. The “blue fluorescence channel” as used herein, refers to fluorescent imaging that uses an excitation light of about 420 nm to about 500 nm and an emission filter that collects light of about 400 nm to about 460 nm. The “green fluorescence channel” as used herein, refers to fluorescent imaging that uses an excitation light of about 480 nm to about 540 nm and an emission filter that collects light of about 500 nm to about 550 nm. The “red fluorescence channel” as used herein, refers to fluorescent imaging that uses an excitation light of about 540 nm to about 680 nm and an emission filter that collects light of about 610 nm to about 750 nm. Other channels can be created using an appropriate combinations of excitation filters and emission filters. The fluorophore component of the fluorescent ion indicators can be selected for operation in a specific channel. As used herein the term “single fluorescence channel” refers to a combination of matched excitation filters and emission filters that produce a narrow band of wavelengths of light. For example the blue channel, the red channel, and the green channel of a fluorescent imaging device are considered examples of single fluorescence channels.

[0105] After analysis in the presence of the first activating solution. The biological sample is washed to remove the first activating solution. The biological sample can be washed, for example, with an “off’ solution to remove all ions present in the biological sample. In an embodiment, the “off” solution can be a buffered solution that does not contain any ions that can be captured by the ion chelating component of the fluorescent ion indicator antibody conjugates.

[0106] The biological sample is contacted with a second activating solution, which is different from the first activating solution. The second activating solution comprises ions that are different from the ions present in the first activating solution. The ions in the second activating solution are bound by an ion chelating component of at least one of the fluorescent ion indicator antibody conjugates. The conjugates that bind to the ions in the second activating composition are different from the conjugates that bind to the ions in the first activating composition. Since the biological sample was washed to remove the ions present in the first activating composition, most of the conjugates that are bound to an ion are different from the conjugates that were bound to an ion in the presence of the first activating solution.

[0107] The biological sample, in the presence of the second activating solution, is analyzed with a fluorescence imaging device using the same single fluorescence channel that was used to analyze the biological sample in the presence of the first activating solution. In the presence of the second activating solution, the distinct conjugates that capture the second ion will fluoresce. The conjugates that captured ions in the presence of the first activating solution will have minimal or no fluorescence. Thus, without changing the fluorescence channel, at least two different targets in the biological sample can be separately analyzed by simply changing the activating solution.

[0108] In another aspect of the present disclosure, a method of detecting multiple proteins in a sample solution comprises an initial step of contacting a biological sample with a primary antibody. The primary antibody binds to one or more proteins or small molecules present in the biological sample. The biological sample is subsequently contacted with a plurality of distinct fluorescent ion indicator antibody conjugates. Each of the plurality of distinct fluorescent ion indicator antibody conjugates comprises a fluorescent ion indicator coupled to a secondary antibody. A secondary antibody binds to one or more primary antibodies present in the biological sample. Contact of the biological sample with the plurality of distinctfluorescent ion indicator antibody conjugates allows the secondary antibody of each distinct conjugate to bind with one or more primary antibodies present in the biological sample.

[0109] After the biological sample is contacted with a plurality of distinct fluorescent ion indicator antibody conjugates, the biological sample is then contacted with a first activating solution. The first activating solution includes a first ion which can be captured by an ion chelating component of at least one of the distinct fluorescent ion indicator antibody conjugates. The biological sample is analyzed with a fluorescence imaging device using a single fluorescence channel in the presence of the first activating solution. In the presence of the first activating solution, the distinct fluorescent ion indicator antibody conjugates that capture the first ion will fluoresce.

[0110] After analysis in the presence of the first activating solution. The biological sample is washed to remove the first activating solution. The biological sample can be washed, for example, with an “off’ solution to remove all ions present in the biological sample. In an embodiment, the “off” solution can be a buffered solution that does not contain any ions that can be captured by the ion chelating component of the fluorescent ion indicator antibody conjugates.

[0111] The biological sample is contacted with a second activating solution, which is different from the first activating solution, as discussed above. The biological sample, in the presence of the second activating solution, is analyzed with a fluorescence imaging device using the same single fluorescence channel that was used to analyze the biological sample in the presence of the first activating solution. In the presence of the second activating solution, the distinct conjugates that capture the second ion will fluoresce.

[0112] In another aspect of the present disclosure, a method of detecting multiple proteins in a sample solution comprises an initial step of coupling proteins or small molecules from a biological sample to a solid substrate. The solid substrate is then washed to remove any proteins, small molecules, or other matter that is not captured by the solid substrate. The solid substrate is then contacted with a plurality of distinct fluorescent ion indicator antibody conjugates, as described herein, wherein each distinct fluorescent ion indicator antibody conjugate comprises a unique antibody for at least one of the proteins or small molecules bound to the solid substrate. Each distinct fluorescent ion indicator antibody conjugate comprises a different ion chelating component. The solid substrate is then contacted with a first activating solution. In one aspect, the first activating solution comprises an ion that isbound by an ion chelating component in at least one of the fluorescent ion indicator antibody conjugates. The solid substrate is analyzed with a fluorescence imaging device using a single fluorescence channel (e.g., a blue fluorescence channel, a green fluorescence channel, or a red fluorescence channel). The solid substrate is then washed to remove the first activating solution. The solid substrate is then contacted with a second activating solution, which is different from the first activating solution. In one aspect, the second activating solution comprises ions that are bound by an ion chelating component of at least one of the fluorescent ion indicator antibody conjugates. The ions in the second activating solution are different from the ions in the first activating solution. The solid substrate, in the presence of the second activating solution, is analyzed with a fluorescence imaging device using the same single fluorescence channel to analyze the proteins in the presence of the first metal ion solution.

[0113] FIG. 1 depicts a schematic diagram of a method of analyzing a biological sample using fluorescent ion indicator antibody conjugates (“conjugates”) having different ion chelating components and different antibodies. As shown in FIG. 1, three different targets (e.g., proteins) from a sample are contacted with three distinct fluorescent ion indicator antibody conjugates. Each of the distinct conjugates includes a different antibody and a different ion chelating component. In the depicted example, a first distinct conjugate has an antibody which binds to protein (A) and an ion chelator component that binds to potassium ions (K). A second distinct conjugate has an antibody which binds to protein (B) and an ion chelator component that binds to sodium ions (N). A third distinct conjugate has an antibody which binds to protein (C) and an ion chelator component that binds to calcium ions. The fluorophore components of each of the conjugates are matched fluorophores. The term “matched fluorophores” as used herein, refers to fluorophores that fluorescence in the same single fluorescence channel. For example, the fluorophores can be selected such that all of the conjugates have fluorophore components that fluorescence in the green fluorescence channel.

[0114] Initially, the biological sample is contacted with the three distinct fluorescent ion indicator antibody conjugates under conditions that allow the conjugates to bind to the appropriate targets (i.e., proteins (A), (B), and (C). The biological sample is washed with an “off” solution. The “off’ solution is a buffer solution that aids in the removal of bound ions from the fluorescent ion indicator antibody conjugates. In the presence of the off solution, no or little fluorescence is detected.

[0115] When the biological sample is contacted with a potassium activating solution (K+Activating Solution) the sample is analyzed under a single fluorescence channel (e.g., the red fluorescence channel). Only the fluorescent ion indicators having a potassium ion chelating component (K) will fluoresce. In this example, the potassium activated fluorophores are coupled to antibodies that bind to protein (A). The portions of the biological sample (e.g., cells) that contain protein (A) will be indicated by fluorescence from the potassium selective fluorescent ion indicator.

[0116] When the biological sample is contacted with a sodium activating solution (Na+Activating Solution) the sample is analyzed under the same single fluorescence channel (e.g., the red fluorescence channel) used during the analysis of the sample with the potassium activating solution. Only the fluorescent ion indicators having a sodium ion chelating component (N) will fluoresce. In this example, the sodium activated fluorophores are coupled to antibodies that bind to protein (B). The portions of the biological sample (e.g., cells) that contain protein (B) will be indicated by fluorescence from the sodium selective fluorescent ion indicator.

[0117] When the biological sample is contacted with a calcium activating solution (Ca2+Activating Solution) the sample is analyzed under the same single fluorescence channel (e.g., the red fluorescence channel) used during the analysis of the sample with the potassium activating solution and the sodium activating solution. Only the fluorescent ion indicators having a sodium ion chelating component (C) will fluoresce. In this example, the calcium activated fluorophores are coupled to antibodies that bind to protein (C). The portions of the biological sample (e.g., cells) that contain protein (C) will be indicated by fluorescence from the calcium selective fluorescent ion indicator.

[0118] . Since each of the fluorescent ion indictor antibody conjugates have a different ion chelating component, a different antibody, and a fluorophore that fluoresces in the same fluorescence channel, multiple proteins can be sequentially analyzed using a single fluorescence channel. The different fluorescent indicators can be activated for analysis by changing the properties of the activating solution, allowing each of the immobilized proteins to be separately analyzed.EXAMPLESFluorescence Study of Conjugated Fluorescent Indicators Coupled to a Solid Support

[0119] In a proof of concept experiment, fluorescent ion indicators were coupled to a solid support using a streptavidin protein - biotin conjugation. FIG. 2 shows a schematic diagram of the biotin-streptavidin coupling technique. In this example, the fluorescence ion indicator is coupled to biotin through a linker. The solid support includes regions that have the protein streptavidin bound to the support. When the fluorescent component having biotin is incubated with the solid support having streptavidin, the fluorescent component is bound to the solid support through the streptavidin-biotin interaction.

[0120] Three different fluorescent ion indicators having different ion chelating components were coupled to different wells of a 384-well plate using streptavidin protein - biotin conjugation. The three fluorescent indicators used are: Compound VI-2 (K+Indicator), Compound VIII-3 (Na+Indicator), and Compound V-7 (Ca2+Indicator). Each fluorescent ion indicator was coupled, through a linker, to biotin.

[0121] Some of the wells of the plate were coated with streptavidin protein such that the coated wells had about 5 pmol biotin binding sites. Some wells were uncoated and used for control experiments. The coated wells were incubated overnight at 4 °C with 25 pL of PBS containing 3 pM of biotinylated K+Indicator, biotinylated Na+Indicator, or biotinylated Ca2+Indicator. After the incubation period was complete, the wells were washed five times with an “off’ buffer using a plate washer.

[0122] A number of experiments were used after the plate was loaded with the K+Indicator, Na+Indicator, and Ca2+Indicator. In these experiments, potassium, sodium, and calcium stock solutions were prepared, as well as an ’’Off’ solution. All of the solutions included one or more buffer agents and one or more organic chelating agents, as discussed previously. ’’Off’ Solution: No metal ions that bind to ion indicators present.K+Solution: K+ions (140 mM).Na+Solution: Na+ions (1000 mM).Ca2+Solution: Ca2+ions (10 mM).

[0123] In the first experiment, conjugation of the fluorescent ion indicators to the plates was studied. Each of the ion containing solutions was added to a well having a fluorescent ion indicator corresponding to the metal ion in the solution. The ’’Off’ solution was added to uncoated wells to provide background readings. FIGS. 3A and 3B show results of the test with each fluorescent ion indicator compared to background reading obtained from uncoatedwells. As can be seen from the data, the fluorescent ion indicators were successfully attached to streptavidin-coated 384-well plate through streptavidin-biotin interaction. Furthermore, these tests show that fluorescent ion indicators, attached to the plate through a protein, are activated in the presence of the appropriate ions in the added solutions. This shows that the fluorescence activity of the fluorescent ion indicators is not affected by coupling to a protein.

[0124] In the second experiment, fluorescent ion indicator cycling from “on” to “off’ was studied. Each of the ion containing solutions was added to a well having a fluorescent ion indicator corresponding to the metal ion in the solution. The fluorescence of the well was measured and the plate was washed with an “off’ solution to clear out the ion containing solutions. Fluorescence was measured again. The plates were washed again with an “off’ solution and ion containing solutions were added to the wells for a second time. The fluorescence of the well was measured after the second addition of the ion containing solutions and the plate was washed with an “off’ solution to clear out the ion containing solutions. Fluorescence was measured again. FIGS. 4A and 4B show results of the test with each fluorescent ion indicator as the indicator is cycled between “on” and “off’ states. As can be seen from the data, the fluorescent ion indicators, coupled to a protein, can be repeatedly turned “on” and “off’ by changing the solution.

[0125] In a third experiment, fluorescent ion indicator selectivity was studied by sequentially adding (1) ’’Off’ solution, (2) K+activating solution, (3) Na+activating solution, and (4) Ca2+activating solution to all of the wells containing an indicator, regardless of the indicator present in the well. Between the addition of each solution, the wells were washed with an “off” solution. The results of these tests are depicted in FIGS 5 A and 5B. The tests show that the fluorescent ion indicators will only show significant fluorescence in the presence of a metal ion that binds to the ion chelating component. In the presence of the other metal ions, the fluorescence of the well is substantially equivalent to the baseline fluorescence.

[0126] A fourth experiment was performed to determine the optimal Na+Indicator concentration in the wells. A streptavidin-coated 96-well plate was used. Each well was incubated with 100 pL of PBS containing varying concentrations of the Na+Indicator for 30 minutes at 4 °C. After incubation, the wells were manually washed, and the Na+Indicator was activated using a modified Na+activating solution excluding chelators. The results of these tests are depicted in FIGS. 6A and 6B. The tests show that increasing fluorescence was detected by conjugating more of the Na+ Indicator to the well. This data indicates that the fluorescence of the Na+Indicator increases when more of the Na+is present and activated.This data indicates that protein concentration level can be quantified by using a sodium selective fluorescent ion indicator.

[0127] A fifth experiment was performed to optimize the sodium ion concentration in the Na+Activating Solution. A streptavidin-coated 96-well plate was used. Each well was incubated with 100 pL of PBS containing 3 pM of the Na+Indicator for 30 minutes at 4 °C. After incubation, the wells were manually washed, and the Na+Indicator was activated using a mixture of a modified Na+Activating Solution (solution excluding chelators) and a modified ’’Off’ Solution to generate an ion binding curve. RFUs were calculated from top reads instead of imaging. The results of these tests are depicted in FIGS. 7A and 7B. The tests show that increasing fluorescence was detected as the concentration of sodium ions is increased.

[0128] A sixth experiment was performed to optimize the calcium ion concentration in the Ca2+Activating Solution. A streptavidin-coated 96-well plate was used. Each well was incubated with 100 pL of PBS containing 3 pM of the Ca2+Indicator for 30 minutes at 4 °C. After incubation, the wells were manually washed, and the ion sensitive fluorescent dyes were activated using a mixture of a modified Ca2+Activating Solution and “Off’ solution to generate an ion binding curve. RFUs were calculated from top reads instead of imaging. Images were taken with an integration time of 1000 instead of 400. The results of these tests are depicted in FIGS. 8A and 8B. The tests show that increasing fluorescence was detected as the concentration of calcium ions is increased.Fluorescence Study of Conjugated Fluorescent Indicators in Cells

[0129] In a seventh experiment, a sodium selective fluorescent ion indicator conjugate was made by coupling sodium selective fluorescent ion indicator conjugate precursor Compound VIII-5 with Donkey antirabbit IgG antibody to give Compound VIII-7.

[0130] As a control indicator, 5-Carboxytetramethylrhodamine (TAMRA) was also coupled to Donkey anti-rabbit IgG antibody. TAMRA is a dye, not an ion indicator, therefore it remains fluorescent in all buffer compositions tested.

[0131] FIG. 9A shows an absorption spectrum of TAMRA - Donkey anti -rabbit IgG antibody (“Dk a-Rabbit IgG TAMRA) and Compound VIII-7 (“Dk a-Rabbit IgG Sodium Ion Indicator - Compound VIII-7). The absorption spectra are similar, showing that conjugation of the sodium ion selective fluorescent ion indicator to an antibody using NHS esters was successful.

[0132] Conjugation with azide / dibenzocyclooctyne (DBCO) click chemistry was also studied. A sodium selective fluorescent ion indicator conjugate was made by coupling sodium selective fluorescent ion indicator conjugate precursor Compound VIII -4 (having an azide coupling group) with Donkey antirabbit IgG antibody (having an attached DBCO group) to give Compound VIII-8.

[0133] FIG. 9B shows an absorption spectrum of Compound VIII-8 (“Dk a-Rabbit IgG Sodium Ion Indicator - Compound VIII-8). The absorption spectra is similar to the absorption spectra for Compound VIII-7, showing that conjugation of the sodium ion selective fluorescent ion indicator to an antibody using azide-DBCO chemistry was also successful.

[0134] In an eighth experiment, a sodium selective fluorescent ion indicator conjugate, Compound VIII-7 was tested for the ability to label cells by through indirect protein binding. A sample containing cells was treated with anti-histone H3 as a primary antibody. The cells were then treated with Compound VIII-7 as a secondary antibody. In a control experiment, a group of cells were labeled with anti-histone H3 antibody, but not Compound VIII-7 secondary antibody.

[0135] FIG. 10A shows the fluorescence imaging of the control cells when treated with 4’, 6-diamidino-2-phenylindole (DAPI) which can be studied using the blue fluorescence channel. DAPI binds to DNA in the cell’s nucleus allowing most, if not all, of the cells present in the sample to be visualized. The image shown in FIG. 10A depicts most of the cells present in the sample.

[0136] FIG. 10B shows the fluorescence imaging of the cells treated with anti-histone H3 and Compound VIII-7. The cells were treated with an activating solution containing sodium ions. In the presence of sodium ions the sample treated with Compound VIII-7 exhibitsfluorescence in the red fluorescence channel. An overlay of the cell fluorescence in FIG. 10B with the cell fluorescence in FIG. 10A is depicted in FIG. 10C. This analysis confirms that Compound VIII-7 binds to the anti-histone H3 antibodies and fluoresces in the presence of sodium ions.

[0137] The anti-Histon H3 / Compound VIII-7 treated cells were studied in the presence of six different solutions: (1) “Off” solution - Buffered solution, no pertinent metal ions; (2) Na+Activating Solution: Na+(1000 mM) (3) Li+ Activating Solution: (140 mM); (4) K+Activating Solution: K+(140 mM); (5) Ca2+Activating Solution: Ca2+(10 mM); (6) Hank’s Balanced Salt Solution: Contains primarily sodium salts, with lesser amounts of potassium, calcium, and magnesium salts.

[0138] FIG. 11 depicts quantification of the relative fluorescence of anti-Histon H3 / Compound VIII-7 treated cells in numerous activating solutions compared to an “Off” solution. As shown in FIG. 11, the fluorescence is greatest in sodium activating solution and HBSS control (predominantly sodium ions). In K+, Ca2+and Li+, the fluorescence is significantly lower than the fluorescence in the presence of sodium. This shows that, at least, K+and Ca2+ion selective fluorescent ion indicators are “off” in the presence of sodium ions. Cells which include sodium selective fluorescent ion indicators can be selectively analyzed in the presence of other fluorescent ion indicators.

[0139] A potassium selective fluorescent ion indicator antibody conjugate was made by coupling potassium selective fluorescent ion conjugate precursor Compound VI-4 to Donkey anti-rabbit IgG antibody, to make potassium selective fluorescent ion antibody conjugate (VI-5).

[0140] Anti-Histon H3 / Compound VI-5 treated cells were studied in the presence of six different solutions: (1) “Off’ solution - Buffered solution, no metal ions; (2) Na+Activating Solution: Na+(1000 mM) (3) Li+ Activating Solution: Li+(140 mM); (4) K+Activating Solution: K+(140 mM); (5) Ca2+Activating Solution: Ca2+(10 mM); (6) Hank’s Balanced Salt Solution: Contains primarily sodium salts, with lesser amounts of potassium, calcium, and magnesium salts. Similar to the results seem with the sodium selective fluorescent ion indicators, the fluorescence is greatest in potassium activating solution. In Na+, Ca2+and Li+, the fluorescence is significantly lower than the fluorescence in the presence of potassium. This test shows that, at least, Na+, Li+and Ca2+ion selective fluorescent ion indicators are “off’ in the presence of potassium ions. The cells which include potassium selective fluorescent ion indicators to be selectively analyzed in the presence of other fluorescent ion indicators by use of the proper activating solution.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting multiple distinct proteins in a biological sample, the method comprising:contacting the biological sample with a plurality of distinct fluorescent ion indicator antibody conjugates, wherein each distinct fluorescent ion indicator antibody conjugate comprises a fluorescent ion indicator coupled to an antibody for at least one of the proteins or antibodies in the biological sample, wherein the fluorescent ion indicator comprises a fluorophore component and an ion chelating component; wherein each distinct fluorescent ion indicator antibody conjugate comprises an antibody and / or an ion chelating component that is different than antibodies and / or ion chelating components coupled to other distinct fluorescent ion indicator antibody conjugates;contacting the biological sample with a first activating solution, the first activating solution comprising a first ion which can be captured by an ion chelating component of at least one of the distinct fluorescent ion indicator antibody conjugates; analyzing the proteins in the biological sample, in the presence of the first activating solution, with a fluorescence imaging device using a single fluorescence channel; washing the biological sample to remove the first activating solution; contacting the biological sample with a second activating solution, wherein the second activating solution comprises a second ion, different from the first ion, wherein the second ion can be captured by an ion chelating component of at least one of the fluorescent ion indicator antibody conjugates;analyzing the biological sample, in the presence of the second activating solution, with a fluorescence imaging device using the same single fluorescence channel that was used to analyze the biological sample in the presence of the first activating solution.

2. The method of claim 1, wherein the antibody of one or more of the distinct fluorescent ion indicator antibody conjugates is an antibody that binds to a protein target of interest.

3. The method of claim 1, further comprising contacting the biological sample with one or more primary antibodies for at least one of the proteins in the biological sample, wherein the antibody of one or more of the distinct fluorescent ion indicator antibody conjugates is a secondary antibody that binds to one or more of the primary antibodies introduced into the biological sample.

4. The method of any one of claims 1-3, wherein the biological sample is washed with an “off’ solution to remove the ions from the first activating solution.

5. The method of any one of claims 1-4, wherein the single fluorescence channel is selected from the group consisting of: the red fluorescence channel, the blue fluorescence channel, or the green fluorescence channel.

6. The method of any one of claims 1-5, wherein one or more of the distinct fluorescent ion indicator antibody conjugates have the structure (Fl):IonChelatingComponentFl.

7. The method of claim 6, wherein one or more of the distinct fluorescent ion indicator antibody conjugates have the structure (I):ChelatingI Component(I)where:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, or OC(O)R; R’ is H or a countercation each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH2OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

8. The method of claim 7, wherein at least one of the distinct fluorescent ion indicator antibody conjugates is a potassium chelator antibody conjugate having the chemical structure (VI):where:each R3independently represent H, F, Br, Cl, or -C1-6 alkyl-CO2R4;R4is H, C1-6 alkyl, CH₂OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;R8is -OH, -O', or -OC1-6 alkyl;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

9. The method of claim 8, wherein at least one of the distinct fluorescent ion indicator antibody conjugates is a potassium chelator antibody conjugate having the chemical structure (VII):where:each R3independently represent H, F, Br, Cl, or –C₁₋₆ alkyl–CO₂R4;R4is H, Ci-6 alkyl, CH2OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;R8is -OH, -O', or -OC1-6 alkyl;R9is C1-6 alkyl or CHO;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

10. The method of claim 8, wherein at least one of the distinct fluorescent ion indicator antibody conjugates is a sodium chelator antibody conjugate having the chemical structure (VIII):each R3independently represent H, F, Br, Cl, or –C₁₋₆ alkyl–CO₂R4;R4is H, Ci-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / Antibody;R8is -OH, -O', or -OC1-6 alkyl;R9is C1-6 alkyl or CHO;W1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

11. The method of claim 7, wherein one or more of the distinct fluorescent ion indicator antibody conjugates have the structure (II):IonChelatingwhere:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a countercation each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH₂OAc. NH2, a countercation, or an additional -Linker-Coupling Agent / AntibodyW1represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl, andW2represents NH, NMe, NEt, or O.

12. The method of claim 11, wherein at least one of the distinct fluorescent ion indicator antibody conjugates is a calcium chelator antibody conjugate having the chemical structure:where:each R independently represent H, Me, Et, -CH₂OAc, or a countercation;R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, or OC(O)R; R’ is H or a countercation; each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH₂OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / AntibodyW1represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;W2represents N or O.

13. The method of any one of claims 1-5, wherein one or more of the distinct fluorescent ion indicator antibody conjugates have the structure (F-2):IonChelating >Linker Coupling Agent / AntibodyComponentF2.

14. The method of claim 13, wherein one or more of the distinct fluorescent ion indicator antibody conjugates have the structure (III):where:R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a countercation each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH2OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / AntibodyW1and W2each independently represents NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl.

15. The method of claim 14, wherein at least one of the distinct fluorescent ion indicator antibody conjugates is a thallium chelator antibody conjugate having the chemical structure (IV):where:each R independently represent H, Me, Et, -CH₂OAc, or a countercation;R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a countercation each R2and R3independently represent H, F, Br, Cl, or –C1-6alkyl–CO2R4;R4is H, C1-6 alkyl, CH₂OAc, NH2, a countercation, or an additional -Linker-Coupling Agent / AntiboU representsW1and W2each independently represents NH2, N(Me)2, N(Et)2, OH, OAc, or OAlkyl.

16. The method of any one of claims 1-15, wherein the group:-Linker-Coupling Agent / Antibodyis selected from the group consisting of:Fluorophore Streptavidin - AntibodyFluorophore17. The method of any one of claims 1-16, wherein each distinct fluorescent ion indicator antibody conjugate binds to a different metal ion selected from the group consisting of sodium ions, potassium ions, lithium ions, calcium ions, and thallium ions.

18. The method of any one of claims 1-17, wherein at least one of the distinct fluorescent ion indicator antibody conjugates binds to sodium ions, and wherein at least one of the distinct fluorescent ion indicator antibody conjugates binds to potassium ions, and wherein at least one of the distinct fluorescent ion indicator antibody conjugates binds to calcium ions.

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