Fluorescent methods and materials for directed biomarker signal amplification

• US 8,802,450 B2
• Filed: 08/01/2011
• Issued: 08/12/2014
• Est. Priority Date: 10/06/2006
• Status: Active Grant

• Current Assignee: Sirigen, Inc. (Becton, Dickinson & Co)
• Original Assignee: Sirigen, Inc. (Becton, Dickinson & Co)
• Inventors: Gaylord, Brent S., Hong, Janice W., Fu, Tsu-Ju, Sun, Cheng-Jun, Baldocchi, Russell
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/195,747
• Publication Number:
  US 20120028828A1
• Time in Patent Office: 1,107 Days
• US Class Current: 436/546
• CPC Class Codes: C08G 2261/3142 fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
• CPC Class Codes: C08G 2261/3221 containing one or more nitrogen atoms as the only heteroatom, e.g. pyrrole, pyridine or triazole
• CPC Class Codes: C08G 2261/3241 containing one or more nitrogen atoms as the only heteroatom, e.g. carbazole
• CPC Class Codes: C08G 2261/3246 containing nitrogen and sulfur as heteroatoms
• CPC Class Codes: C08G 2261/522 fluorescent
• CPC Class Codes: C08G 2261/78 Complexation
• CPC Class Codes: C08G 2261/94 Applications in sensors, e.g. biosensors
• CPC Class Codes: C08G 61/02 Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
• CPC Class Codes: C08G 61/12 Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: H10K 85/115 Polyfluorene; Derivatives thereof
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

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.

Claims

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;

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-Br₂, 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.