Reagents for directed biomarker signal amplification
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.
1. A method of determining whether a target is present in a sample, the method comprising:
contacting the sample with a conjugated polymer complex comprising a sensor for the target conjugated to a water soluble conjugated polymer having the structure of Formula (Ia):
wherein:
each R is independently a side group capable of imparting solubility in water;
MU is a polymer modifying unit or band gap modifying unit that is evenly or randomly distributed along the polymer main chain and is optionally substituted with one or more optionally substituted substituents selected from halogen, hydroxyl, C 1 -C 12 alkyl, C 2 -C 12 alkene, C 2 -C 12 alkyne, C 3 -C 12 cycloalkyl, C 1 -C 12 haloalkyl, C 1 -C 12 alkoxy, C 2 -C 18 (hetero)aryloxy, C 2 -C 18 (hetero)arylamino, (CH 2 ) x′ (OCH 2 CH 2 ) y′ OCH 3 where each x′ is independently an integer from 0-20, y′ is independently an integer from 0 to 50, or a C 2 -C 18 (hetero)aryl group;
optional linkers L 1 and L 2 are each independently an aryl or a heteroaryl group evenly or randomly distributed along the polymer main chain and are substituted with one or more pendant chains terminated with: i) a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazids, hydrazones, azide, alkyne, aldehydes, thiols, and protected groups thereof for conjugation to a molecule or biomolecule; or ii) a conjugated organic dye or biomolecule;
G 1 and G 2 are each independently selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, boronic acid substituted aryl, boronic ester substituted aryl, boronic ester, boronic acid, optionally substituted fluorene and aryl or heteroaryl substituted with one or more pendant chains terminated with: i) a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazids, hydrazones, azide, alkyne, aldehydes, thiols, and protected groups thereof for conjugation to a molecule or biomolecule; or ii) a conjugated organic dye or biomolecule;
wherein the polymer comprises at least 1 functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazids, hydrazones, azide, alkyne, aldehydes, and thiols within G 1 , G 2 , L 1 or L 2 , or a conjugated organic dye or biomolecule;
n is an integer from 1 to about 10,000; and
a, b, c and d define the mol % of each unit within the structure which each can be evenly or randomly repeated and where a is a mol % from 10 to 100%, b is a mol % from 0 to 90%, and each c and d are mol % from 0 to 25%.
2. The method according to claim 1 , wherein the at least 1 functional group is conjugated to a signaling chromophore.
3. The method according to claim 2 , wherein the signaling chromophore is selected from the group consisting of fluorescent dyes, semiconductor nanocrystals, lanthanide chelates and fluorescent proteins.
4. The method according to claim 1 , wherein each R is a non-ionic side group capable of imparting solubility in water in excess of 10 mg/mL.
5. The method according to claim 1 , wherein each R is independently (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 wherein each x is independently an integer from 0-20 and each y is independently an integer from 0 to 50, or a benzyl substituted with at least one (OCH 2 CH 2 ) z OCH 3 group wherein each z is independently an integer from 0 to 50.
6. The method according to claim 5 , wherein each R is a benzyl substituted with at least two (OCH 2 CH 2 ) 10 OCH 3 groups.
7. The method according to claim 6 , wherein each R is (CH 2 ) 3 (OCH 2 CH 2 ) 11 OCH 3 .
8. The method according to claim 1 , wherein MU is selected from optionally substituted 2,1,3-benzothiadiazole, benzoxidazole, benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole, 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines, perylene, perylene diimides, diketopyrrolopyrrole, thienopyrazine low bandgap commercial dyes, olefins, and cyano-substituted olefins and isomers thereof.
9. The method according to claim 8 , wherein MU is selected from the group consisting of a′-k′ having the structure:
wherein *=a site for covalent attachment to unsaturated backbone and each R is a non-ionic side group capable of imparting solubility in water.
10. The method according to claim 9 , wherein MU is selected from one of the following:
wherein *=site for covalent attachment to unsaturated backbone.
11. The method according to claim 1 , wherein MU is selected from one of the following:
wherein *=site for covalent attachment to unsaturated backbone.
12. The method according to claim 1 , wherein L 1 and/or L 2 are present.
13. The method according to claim 12 , wherein linkers L 1 or L 2 have the structure:
wherein:
*=site for covalent attachment to unsaturated backbone;
each R′ is different from R and is independently selected from H, halogen, C 1 -C 12 alkyl, (C 1 -C 12 alkyl)NH 2 , C 2 -C 12 alkene, C 2 -C 12 alkyne, C 3 -C 12 cycloalkyl, C 1 -C 12 haloalkyl, C 2 -C 18 (hetero)aryl, C 2 -C 18 (hetero)arylamino, —[CH 2 —CH 2 ] r′ —Z 1 wherein Z 1 is —OH or —COOH and r′ is an integer from 1 to 20, (C 1 -C 12 )alkoxy-X 1 wherein X 1 is —NH 2 , —NHCOOH, —NHCOOC(CH 3 ) 3 , —NHCO(C 3 -C 12 )cycloalkyl(C 1 -C 4 )alkyl-N-maleimide or —NHCO[CH 2 —CH 2 —O] s′ (CH 2 ) s′ NH 2 and each s′ is independently an integer from 1 to 20, (CH 2 ) 3 (OCH 2 CH 2 ) x″ OCH 3 wherein x″ is an integer from 0 to 50, and a benzyl optionally substituted with one or more halogen, hydroxyl, C 1 -C 12 alkoxy or (OCH 2 CH 2 ) y″ OCH 3 wherein y″ is an integer from 0 to 50.
14. The method according to claim 12 , wherein linkers L 1 or L 2 have the structure:
wherein:
*=site for covalent attachment to backbone;
each R 25 is independently a bond, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkene, C 2 -C 20 alkyne, C 3 -C 20 cycloalkyl, C 1 -C 20 haloalkyl; (CH 2 ) x (OCH 2 CH 2 ) p (CH 2 ) x where each x is independently an integer from 0-20, p is independently an integer from 0 to 50; aryl, C 2 -C 18 (hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate, carbonates, sulfide, disulfide, or imido groups; and
at least one R 25 is terminated with a functional group selected from amine, carbamate, carboxylate, carboxylic acid, maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazids, hydrazones, azide, alkyne, aldehydes and thiols, or a conjugated organic dye or biomolecule.
15. The method according to claim 14 , wherein at least one R 25 comprises an amine conjugation site.
16. The method according to claim 14 , wherein linkers L 1 or L 2 have the structure:
17. The method according to claim 14 , wherein at least one R 25 comprises a carboxylic acid sidechain.
18. The method according to claim 14 , wherein at least one R 25 is conjugated to a signaling chromophore.
19. The method according to claim 1 , wherein at least one of G 1 and G 2 comprises a functional conjugation site.
20. The method according to claim 19 , wherein at least one of G 1 and G 2 has the structure:
wherein:
*=site for covalent attachment to backbone; and
R 11 is a bond, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkene, C 2 -C 20 alkyne, C 3 -C 20 cycloalkyl, C 1 -C 20 haloalkyl, (CH 2 ) x (OCH 2 CH 2 ) p (CH 2 ) x wherein each x is independently an integer from 0-20 and p is an integer from 0 to 50, aryl, C 2 -C 18 (hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate, carbonate, sulfide, disulfide, or imido groups; and is terminated with: i) a functional group selected from amine, carbamate, carboxylate, carboxylic acid, maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazids, hydrazones, azide, alkyne, aldehydes, thiols, and protected groups thereof, or ii) a conjugated organic dye or biomolecule.
21. The method according to claim 20 , wherein at least one of G 1 and G 2 is selected from the group consisting of capping units 1-31 having the structures:
wherein:
*=site for covalent attachment to backbone;
k is 2, 4, 8, 12 or 24; and
R 15 is selected from the group consisting of 1-u having the structures:
22. The method according to claim 19 , wherein at least one of G 1 and G 2 is a terminal carboxylic acid capping unit.
23. The method according to claim 22 , wherein at least one of G 1 and G 2 is selected from one of the following structures:
24. The method according to claim 19 , wherein at least one of G 1 and G 2 comprises a terminal amine capping unit.
25. The method according to claim 1 , wherein the polymer has the structure of formula:
wherein L 1 comprises a functional conjugation site.
26. The method according to claim 1 , wherein the polymer has the structure of formula:
wherein at least one of G 1 or G 2 comprises a functional conjugation site.
27. The method according to claim 1 , wherein the sensor is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid or an aptamer.
28. The method according to claim 1 , wherein the sensor is an antibody.
29. The method according to claim 1 , wherein the sensor is an avidin, streptavidin, neutravidin, avidinDN or an avidinD.
30. The method according to claim 1 , wherein the target is a target protein of a cell.
31. The method according to claim 1 , wherein the method is configured for immunohistochemistry or FISH.
32. The method according to claim 1 , wherein the method is configured for intracellular staining.
33. The method according to claim 1 , wherein the method is configured for flow cytometry.