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 the formula:
wherein:
Ar is an aryl or heteroaryl unit substituted with a non-ionic 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, a C 2 -C 18 (hetero)aryl group and (CH 2 ) x′ (OCH 2 CH 2 ) y′ OCH 3 where x′ is independently an integer from 0-20 and y′ is independently an integer from 0 to 50;
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 ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, 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, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, 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 ester, N-hydroxysuccinimidyl, hydrazines, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol 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 MU is selected from optionally substituted benzothiadiazole, benzoxidazole, benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole, 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxaline, perylene, perylene diimide, diketopyrrolopyrrole, thienopyrazine low bandgap commercial dyes, olefin, and cyano-substituted olefins and isomers thereof.
3. The method according to claim 1 , wherein MU is selected from the group consisting of a′-k′ having the structure:
wherein * is a site for covalent attachment to unsaturated backbone and each R is a non-ionic side group capable of imparting solubility in water.
4. The method according to claim 3 , wherein MU is selected from one of the following:
wherein * is site for covalent attachment to unsaturated backbone.
5. The method according to claim 1 , wherein MU is a phenyl.
6. The method according to claim 5 , wherein MU is
wherein *=site for covalent attachment to unsaturated backbone.
7. The method according to claim 1 , wherein the non-ionic side group comprises an ethylene glycol oligomer.
8. The method according to claim 7 , wherein Ar is substituted with one or more non-ionic side groups comprising mPEG5, mPEG8, mPEG11 or mPEG24.
9. The method according to claim 7 , wherein Ar is substituted with one or more groups selected from (CH 2 ) x′ (OCH 2 CH 2 ) y′ OCH 3 where x′ is independently an integer from 0-20 and y′ is independently an integer from 0 to 50, and a benzyl substituted with one or more halogen, hydroxyl, C 1 -C 12 alkoxy, or (OCH 2 CH 2 ) z OCH 3 where z is independently an integer from 0 to 50.
10. The method according to claim 7 , wherein Ar is substituted with one or more (CH 2 )x(OCH 2 CH 2 ) y′ OCH 3 where x′ is independently an integer from 0-20, y′ is independently an integer from 0 to 50.
11. The method according to claim 7 , wherein Ar is substituted with a dendrimer of PAMAM, PEA, PEHAM, PPI, tri-branched benzoate, or glycerol with a generation of 1 to 4 and optional terminal substitutions selected from (- - - - -)CH 2 CH 2 O) j CH 3 and (- - - - -)(OCH 2 CH 2 ) j CH 3 where j is an integer from 0 to 25 and the dotted lines (- - - - -) are each independently selected from any one or a combination of, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkene, amido, amino, aryl, (CH 2 ) r (OCH 2 CH 2 ) s (CH 2 ) r where each r is independently an integer from 0-20, s is independently an integer from 0 to 50, carbamate, carboxylate, C 3 -C 12 cycloalkyl, imido, phenoxy and C 4 -C 18 (hetero)aryl group.
12. The method according to claim 1 , wherein Ar is a substituted fluorene unit.
13. The method according to claim 1 , wherein L 1 and/or L 2 is conjugated to a signaling chromophore.
14. The method according to claim 1 , wherein L 1 and/or L 2 are present and 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 ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde and thiols, or a conjugated organic dye or biomolecule.
15. The method according to claim 1 , wherein at least one of G 1 and G 2 comprises a functional conjugation site.
16. The method according to claim 15 , 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.
17. The method according to claim 1 , 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-t having the structures:
18. The method according to claim 1 , wherein the sensor is an antibody.
19. The method according to claim 1 , wherein the method is configured for intracellular staining.
20. The method according to claim 1 , wherein the method is configured for flow cytometry.