Fluorescent methods and materials for directed biomarker signal amplification
Methods and compositions are provided that include a multichromophore and/or multichromophore complex for identifying a target biomolecule. A sensor biomolecule, for example, an antibody can be covalently linked to the multichromophore. Additionally, a signaling chromophore can be covalently linked to the multichromophore. The arrangement is such that the signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore. Since the sensor biomolecule is capable of interacting with the target biomolecule, the multichromophore and/or multichromophore complex can provide enhanced detection signals for a target biomolecule.
1. A multichromophore complex for identifying a target cell protein comprising:
a multichromophore comprising a water-soluble, light harvesting π conjugated polymer comprising substituted or unsubstituted multiple units in a π conjugated state wherein each unit has the structure:
and a linker having a bioconjugation site,
an antibody covalently linked to the multichromophore by the bioconjugation site on the linker of the π conjugated polymer,
an optional signaling chromophore covalently bound to the multichromophore or covalently bound to the antibody,
wherein
the optional signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore and
wherein the antibody is capable of recognizing cells with the target cell protein expressed and specifically bind to the protein,
wherein A is independently an aromatic, heteroaromatic, optionally substituted conjugated polymer segment or oligomeric structure, and where at least one A is a phenyl linked to the linker,
wherein R 1 is a water solubilizing group,
wherein * is a site for π conjugation and
wherein said water solubilizing group is elected from the group consisting of ethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkyl salts, ω-ammonium alkoxy salts, ω-ammonium oligoether salts, ω-sulfonate alkyl salts, ω-sulfonate alkoxy salts, and ω-sulfonate oligoether salts and combinations thereof.
2. The multichromophore complex of claim 1 , wherein the signaling chromophore is present and covalently bound to the multichromophore.
3. The multichromophore complex of claim 1 , wherein the signaling chromophore is present and covalently bound to the antibody.
4. The multichromophore complex of claim 1 , wherein the bioconjugation site comprises a reactive group selected from the group consisting of maleimide/thiol, succimidylester (NHS ester)/amine, azide, carboxy/EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride)/amine, amine/Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol, and amine/BMPH(N-[β-Maleimidopropionic acid]hydrazide)/thiol.
5. The multichromophore complex of claim 1 , wherein the signaling chromophore is present and is an organic dye.
6. The multichromophore complex of claim 5 , wherein the organic dye is selected from the group consisting of fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and -6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br 2 , BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 576/589, BODIPY® 581/591, BODIPY® 630/650, BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugates thereof, and combinations thereof.
7. The multichromophore complex of claim 1 , wherein the conjugated polymer is a charge-neutral conjugated polymer.
8. The multichromophore complex of claim 1 , wherein the conjugated polymer further comprises π-conjugated repeat units selected from the group consisting of benzene, naphthalene, anthracene, fluorene, indenofluorene, thiophene, thienothiophene, dithienothiophene, 3,4-ethylenedioxythiophene, furan, pyridine, pyrrole, fused pyrrole, tetrahydropyrene, perylenediimide, paracyclophane and oxadiazole, each optionally substituted.
9. The multichromophore complex of claim 1 , wherein the conjugated polymer further comprises π-conjugated repeat units selected from the group consisting of benzene, naphthalene, anthracene, fluorene, indenofluorene, thiophene, thienothiophene, dithienothiophene, 3,4-ethylenedioxythiophene, furan, pyridine, pyrrole, fused pyrrole, tetrahydropyrene, perylenediimide, paracyclophane, oxadiazole, vinyl olefin, cyano-substituted olefin, tri- and tetra-substituted ethylene, ethynyl, 2,1,3-benzothiadiazole, benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole, 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dye, quinoxaline, pyrazine, triazinyl, biphenyl, indene, indole, naphthalene, quinoline, isoquinoline, quinazoline, pyridopyridine, pyrrolopyridine, purine, pyridazinyl, triazole, tetrazole, imidazole, bipyridine, tripyridine, tetrapyridine, phenazine, phenanthrolene, benzofuran, phenylene vinylene, triarylamine, biarylamine, binaphthalene, alkyne, phenyl alkyne, naphthalene bisimide, tetrahydropyrene, thiophene, thiophene alkyne, furan alkyne, pyrrole alkyne, carbazole alkyne, phenylene alkyne, naphthalene vinylene, paracyclophane, paracyclophane phenylene, paracyclophane vinylene, paracyclopane vinylene phenylene, thiophene naphthalene, furan naphthalene, pyrrole naphthalene, thiophene vinylene, furan vinylene, pyrrole vinylene, and 3,4-ethylenedioxythiophene each optionally substituted.
10. The multichromophore complex of claim 1 , wherein the antibody is a goat-anti-mouse antibody or a donkey-anti-mouse antibody.
11. The multichromophore complex of claim 1 , wherein the antibody is an anti-digoxigenin antibody.
12. The multichromophore complex of claim 1 , wherein the antibody is a mouse-anti-human CD22 antibody.
13. The multichromophore complex of claim 1 , wherein the antibody is a primary antibody.
14. The multichromophore complex of claim 1 , wherein the antibody is a secondary antibody.
15. The multichromophore complex of claim 1 , wherein the multichromophore complex consists essentially of the multichromophore and the antibody.
16. The multichromophore complex of claim 1 , wherein the multichromophore complex further comprises one or more multichromophore(s) covalently linked to one or more antibody(ies) to monitor for expression of two or more different proteins on different cell types simultaneously.