# US 10,481,161 B2 · Reagents for directed biomarker signal amplification

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- Number: US 10,481,161 B2
- Document ID: US10481161B2
- Status: Granted
- Kind: B2
- Application number: 16/359,473
- Filed: 2019-03-20
- Published: 2019-09-26
- Granted: 2019-11-19
- Earliest claimed: 2010-01-19
- Assignees: Sirigen II Limited
- Applicants: Sirigen II Limited
- Inventors: Brent S. Gaylord; Glenn P. Bartholomew; Russell A. Baldocchi; Janice W. Hong; William H. Huisman; Yongchao Liang; Trung Nguyen; Lan T. Tran; Jean M. Wheeler; Adrian Charles Vernon Palmer; Frank Peter Uckert
- Web page: https://patentgrep.com/patent/US10481161B2

## CPC

- C08G 61/10: only aromatic carbon atoms, e.g. polyphenylenes
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- C07K 16/2812: {against CD4}
- C07K 16/2815: {against CD8}
- C08G 61/02: Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
- C09B 57/00: Other synthetic dyes of known constitution
- C09B 68/41: {Polymers attached to the pigment surface (C09B68/444, C09B68/446 take precedence)}
- C09B 69/00: Dyes not provided for by a single group of this subclass
- 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
- C09K 11/06: containing organic luminescent materials
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/682: Signal amplification
- G01N 33/582: {with fluorescent label}
- H10K 85/151: {Copolymers}
- C08G 2261/1424: containing ether groups, including alkoxy
- C08G 2261/143: Side-chains containing nitrogen
- C08G 2261/1432: containing amide groups
- C08G 2261/146: Side-chains containing halogens
- C08G 2261/148: Side-chains having aromatic units
- C08G 2261/164: comprising organic end groups
- C08G 2261/1642: comprising reactive double bonds or triple bonds
- C08G 2261/3142: fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
- C08G 2261/334: containing heteroatoms
- C08G 2261/3422: conjugated, e.g. PPV-type
- C08G 2261/411: Suzuki reactions
- C08G 2261/94: Applications in sensors, e.g. biosensors
- H10K 85/115: {Polyfluorene; Derivatives thereof}
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- C09D: COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR (cosmetics A61K; processes for applying liquids or other fluent materials to surfaces, in general, B05D; staining wood B27K5/02; glazes or vitreous enamels C03C; natural resins, French polish, drying-oils, driers, turpentine, per se, C09F; polishing compositions other than French polish, ski waxes C09G; adhesives or use of materials as adhesives C09J; materials for sealing or packing joints or covers C09K3/10; materials for stopping leaks C09K3/12; processes for the electrolytic or electrophoretic production of coatings C25D)
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- H10K: ORGANIC ELECTRIC SOLID-STATE DEVICES

## 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

### Claim 1

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, C1-C12 alkyl, C2-C12 alkene, C2-C12 alkyne, C3-C12 cycloalkyl, C1-C12 haloalkyl, C1-C12 alkoxy, C2-C18 (hetero)aryloxy, C2-C18 (hetero)arylamino, a C2-C18 (hetero)aryl group and (CH2)x′(OCH2CH2)y′OCH3 where x′ is independently an integer from 0-20 and y′ is independently an integer from 0 to 50;
optional linkers L1 and L2 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;
G1 and G2 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:
n is the number of repeat units and is an integer selected so that the water-soluble conjugated polymer has a molecular weight ranging from 10,000 g/mol to 100,000 g/mol;
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 G1, G2, L1 or L2, or a conjugated organic dye or biomolecule; 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%.

### Claim 2 (depends on 1)

The water-soluble conjugated polymer 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.

### Claim 3 (depends on 1)

The water-soluble conjugated polymer 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.

### Claim 4 (depends on 3)

The water-soluble conjugated polymer according to claim 3, wherein MU is selected from one of the following:
wherein * is site for covalent attachment to unsaturated backbone.

### Claim 5 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein MU is a phenyl.

### Claim 6 (depends on 5)

The water-soluble conjugated polymer according to claim 5, wherein MU is
wherein *=site for covalent attachment to unsaturated backbone.

### Claim 7 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein the non-ionic side group comprises an ethylene glycol oligomer.

### Claim 8 (depends on 7)

The water-soluble conjugated polymer according to claim 7, wherein the non-ionic side group comprises mPEG5, mPEG8, mPEG11 or mPEG24.

### Claim 9 (depends on 7)

The water-soluble conjugated polymer according to claim 7, wherein Ar is substituted with one or more groups selected from (CH2)x′(OCH2CH2)y′OCH3 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, C1-C12 alkoxy, or (OCH2CH2)zOCH3 where z is independently an integer from 0 to 50.

### Claim 10 (depends on 7)

The water-soluble conjugated polymer according to claim 7, wherein Ar is substituted with one or more (CH2)x′(OCH2CH2)y′OCH3 where x′ is independently an integer from 0-20, y′ is independently an integer from 0 to 50.

### Claim 11 (depends on 1)

The water-soluble conjugated polymer according to claim 1, 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 ( - - - )CH2CH2O)jCH3 and ( - - - )(OCH2CH2)jCH3 where j is an integer from 0 to 25 and the dotted lines ( - - - ) are each independently selected from any one or a combination of, C1-C12 alkyl, C1-C12 alkoxy, C2-C12 alkene, amido, amino, aryl, (CH2)r(OCH2CH2)s(CH2)r where each r is independently an integer from 0-20, s is independently an integer from 0 to 50, carbamate, carboxylate, C3-C12 cycloalkyl, imido, phenoxy and C4-C18(hetero)aryl group.

### Claim 12 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein Ar is a substituted fluorene unit.

### Claim 13 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein L1 and/or L2 are present.

### Claim 14 (depends on 13)

The water-soluble conjugated polymer according to claim 13, wherein L1 and/or L2 is conjugated to a signaling chromophore.

### Claim 15 (depends on 13)

The water-soluble conjugated polymer according to claim 13, wherein L1 and/or L2 have the structure:
wherein:
*=site for covalent attachment to backbone;
each R25 is independently a bond, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkene, C2-C20 alkyne, C3-C20 cycloalkyl, C1-C20 haloalkyl; (CH2)x(OCH2CH2)p(CH2)x where each x is independently an integer from 0-20, p is independently an integer from 0 to 50; aryl, C2-C18(hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate, carbonates, sulfide, disulfide, or imido groups; and
at least one R25 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.

### Claim 16 (depends on 15)

The water-soluble conjugated polymer according to claim 15, wherein L1 and/or L2 have the structure:

### Claim 17 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein at least one of G1 and G2 comprises a functional conjugation site.

### Claim 18 (depends on 17)

The water-soluble conjugated polymer according to claim 17, wherein at least one of G1 and G2 has the structure:
wherein:
*=site for covalent attachment to backbone; and
R11 is a bond, C1-C2 alkyl, C1-C2 alkoxy, C2-C20 alkene, C2-C2 alkyne, C3-C20 cycloalkyl, C1-C20 haloalkyl, (CH2)x(OCH2CH2)p(CH2)x wherein each x is independently an integer from 0-20 and p is an integer from 0 to 50, aryl, C2-C18(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.

### Claim 19 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein at least one of G1 and G2 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
R15 is selected from the group consisting of 1-t having the structures:

### Claim 20 (depends on 19)

The water-soluble conjugated polymer according to claim 19, wherein at least one of G1 and G2 is selected from one of the following structures:

### Claim 21 (depends on 1)

The water-soluble conjugated polymer according to claim 1, wherein the water-soluble conjugated polymer has a molecular weight ranging from 30,000 g/mol to 90,000 g/mol.

### Claim 22 (depends on 21)

The water-soluble conjugated polymer according to claim 21, wherein the water-soluble conjugated polymer has a molecular weight ranging from 40,000 g/mol to 80,000 g/mol.

### Claim 23 (depends on 22)

The water-soluble conjugated polymer according to claim 22, wherein the water-soluble conjugated polymer has a molecular weight ranging from 50,000 g/mol to 70,000 g/mol.