Novel reagents for directed biomarker signal amplification

• US 20110256549A1
• Filed: 03/17/2011
• Published: 10/20/2011
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: Sirigen Ii Limited (Becton, Dickinson & Co)
• Original Assignee: Sirigen, Inc. (Becton, Dickinson & Co)
• Inventors: Huisman, William H., Bartholomew, Glenn P., Wheeler, Jean M., Hong, Janice W., Gaylord, Brent S., Uckert, Frank Peter
• Granted Patent: US 8,455,613 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2815 against CD8
• CPC Class Codes: C08G 2261/1424 containing ether groups, including alkoxy
• CPC Class Codes: C08G 2261/143 Side-chains containing nitrogen
• CPC Class Codes: C08G 2261/1432 containing amide groups
• CPC Class Codes: C08G 2261/146 Side-chains containing halogens
• CPC Class Codes: C08G 2261/148 Side-chains having aromatic units
• CPC Class Codes: C08G 2261/164 comprising organic end groups
• CPC Class Codes: C08G 2261/1642 comprising reactive double bonds or triple bonds
• CPC Class Codes: C08G 2261/3142 fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
• CPC Class Codes: C08G 2261/334 containing heteroatoms
• CPC Class Codes: C08G 2261/3422 conjugated, e.g. PPV-type
• CPC Class Codes: C08G 2261/411 Suzuki reactions
• 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 61/10 only aromatic carbon atoms, e.g. polyphenylenes
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09B 68/41 Polymers attached to the pigment surface C09B68/444, C09B68/446 take precedence
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C09D 165/00 Coating compositions based on macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain C09D107/00 - C09D157/00, C09D161/00 take precedence; Coating compositions based on derivatives of such polymers
• CPC Class Codes: C09K 11/06 : containing organic luminescent materials
• CPC Class Codes: C12Q 1/6818 : involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/682 : Signal amplification
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: H10K 85/115 : Polyfluorene; Derivatives thereof
• CPC Class Codes: H10K 85/151 : Copolymers

Abstract

Described herein are methods, compositions and articles of manufacture involving neutral conjugated polymers including methods for synthesis of neutral conjugated water-soluble polymers with linkers along the polymer main chain structure and terminal end capping units. Such polymers may serve in the fabrication of novel optoelectronic devices and in the development of highly efficient biosensors. The invention further relates to the application of these polymers in assay methods.

Claims

1. A water soluble conjugated polymer having the structure of Formula:

2. The water soluble conjugated polymer of claim 1 wherein the linker L₁ is

3. The water soluble conjugated polymer of claim 1 having the structure:

4. The water soluble conjugated polymer of claim 1, further comprising a signaling chromophore selected from the group consisting of fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®

, Cy3®

, Cy3.5®

, Cy5®

, Cy5.5®

, Cy7®

, Cy-Chrome, DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4′

,5′

-dichloro-2′

,7′

-dimelhoxyfluorescein), 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, BODPY®

R6G, BODIPY®

TMR, BODIPY®

TR, conjugates thereof, and combinations thereof.

5. The water soluble conjugated polymer of claim 1, further comprising a signaling chromophore that is a rhodamine or cyanine dye.

6. The water soluble conjugated polymer of claim 1, further comprising a signaling chromophore that is selected from the group consisting of California Red, Cy3®

, Cy5® and

Cy7®

.

7. The water soluble conjugated polymer of claim 1, wherein the polymer has a purity of at least 80%.

8. The water soluble conjugated polymer of claim 1, wherein the polymer has a purity of at least 95%.

9. The water soluble conjugated polymer of either claim 7 or claim 8, wherein G₁ and G₂ are each independently selected from the group consisting of 5-9.

10. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to an antibody.

11. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to an antibody and wherein the polymer-antibody conjugate has a purity of at least 95%.

12. An assay method for detecting a target biomolecule in a sample comprising cells comprising:

providing a sample that is suspected of containing a target biomolecule;

providing a sensor protein conjugated to a water soluble conjugated polymer of claim 1 that is further conjugated to at least one signaling chromophore and wherein the sensor protein is capable of interacting with the target biomolecule or a target-associated biomolecule;

contacting the sample with the sensor protein and the conjugated polymer in a solution under conditions in which the sensor protein can bind to the target biomolecule or a target-associated biomolecule if present;

applying a light source to the sample that can excite the conjugated polymer; and

detecting whether light is emitted from the signaling chromophore.

13. A flow cytometry system comprising:

a flow cytometer;

a sample that is suspected of containing a target biomolecule;

a sensor protein conjugated to a water soluble conjugated polymer of claim 1 that is further conjugated at least one signaling chromophore and wherein the sensor protein is capable of interacting with the target biomolecule or a target-associated biomolecule.