# US 9,752,186 B2 · Polymer coatings

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- Number: US 9,752,186 B2
- Document ID: US9752186B2
- Status: Granted
- Kind: B2
- Application number: 14/679,760
- Filed: 2015-04-06
- Published: 2015-09-17
- Granted: 2017-09-05
- Earliest claimed: 2012-06-08
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Wayne N. George; Andrew A. Brown; Daniel Bratton; Hongji Ren; Ryan C. Smith
- Web page: https://patentgrep.com/patent/US9752186B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- B05D 3/06: by exposure to radiation (B05D3/02 takes precedence {; plasma treatment B05D3/141})
- B05D 3/065: {After-treatment}
- C09D 133/26: Homopolymers or copolymers of acrylamide or methacrylamide
- C09D 135/00: Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least another carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Coating compositions based on derivatives of such polymers
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- Y10T 428/24802: Discontinuous or differential coating, impregnation or bond [e.g., artwork, printing, retouched photograph, etc.]
- Y10T 428/24917: including metal layer
- Y10T 428/24926: including ceramic, glass, porcelain or quartz layer
- Y10T 428/2998: including synthetic resin or polymer
- Y10T 428/31536: Including interfacial reaction product of adjacent layers
- Y10T 428/31935: Ester, halide or nitrile of addition polymer
- 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
- B05D: PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL (conveying articles or workpieces through baths of liquid B65G, e.g. B65G49/02)
- 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)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present disclosure relates to polymer coatings covalently attached to the surface of a substrate and the preparation of the polymer coatings, such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), in the formation and manipulation of substrates, such as molecular arrays and flow cells. The present disclosure also relates to methods of preparing a substrate surface by using beads coated with a covalently attached polymer, such as PAZAM, and the method of determining a nucleotide sequence of a polynucleotide attached to a substrate surface described herein.

## Claims

### Claim 1

A method of preparing an array of polynucleotides, said method comprising:
providing a substrate comprising a polymer coating present on a surface of the substrate, the polymer coating comprising a recurring unit of Formula (Ia) and a recurring unit of Formula (II):
wherein:
R1 is selected from hydrogen, halo, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
RA is selected from the group consisting of amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester, epoxy, aziridine, triazoline, and thiol;
each R4, R4′, R5 and R5′ is independently selected from H, R6, OR6,—C(O)OR6, —C(O)R6, —OC(O)R6, —C(O) NR7R8, or —NR7R8;
R6 is independently selected from H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
each R7 and R8 is independently selected from H or alkyl, or R7 and R8 are joined together with the atom or atoms to which they are attached to form a heterocycle;
each of the —(CH2)—o can be optionally substituted; and
o is an integer between 1-50; and
reacting a plurality of first oligonucleotides and a plurality of second oligonucleotides with reactive sites of the polymer coating.

### Claim 2 (depends on 1)

The method of claim 1, further comprising contacting the plurality of first oligonucleotides attached to the polymer coating with templates to be amplified, each template comprising at the 3′ end a sequence capable of hybridizing to the first oligonucleotides and at the 5′ end a sequence the complement of which is capable of hybridizing to the second oligonucleotides.

### Claim 3 (depends on 2)

The method of claim 2, further comprising amplifying the templates using the first oligonucleotides and the second oligonucleotides, thereby generating a clustered array of polynucleotides.

### Claim 4 (depends on 1)

The method of claim 1, wherein RA is azido.

### Claim 5 (depends on 4)

The method of claim 4, wherein R1 is H or alkyl.

### Claim 6 (depends on 5)

The method of claim 5, wherein R1 is hydrogen and wherein o is 5.

### Claim 7 (depends on 1)

The method of claim 1, wherein the polymer coating comprises a polymer of Formula (III) or (III′):
wherein Rl is selected from H or alkyl;
R5 is selected from H or alkyl;
n is an integer in the range of 1 to 50,000; and
m is an integer in the range of 1 to 100,000.

### Claim 8 (depends on 7)

The method of claim 7, wherein the polymer of Formula (III) or (III′) is also represented by Formula (IIIa) or (IIIb):
wherein n is an integer in the range of 1 to 20,000, and m is an integer in the range of 1 to 100,000.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first oligonucleotides or the second oligonucleotides comprise one or more functional groups selected from the group consisting of: alkyne, amine, and aldehyde.

### Claim 10

A method of preparing an array of polynucleotides, said method comprising:
providing a substrate having a polymer coating present on a surface of the substrate, the polymer coating comprising a recurring unit of Formula (Ia) and a recurring unit of Formula (II):
wherein:
R1 is selected from hydrogen, halo, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
RA is selected from the group consisting of amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester, epoxy, aziridine, triazoline, and thiol;
each R4, R4′, R5 and R5′ is independently selected from H, R6, OR6, —C(O)OR6, —C(O)R6, —OC(O)R6, —C(O) NR7R8, or —NR7R8;
R6 is independently selected from H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
each R7 and R8 is independently selected from H or alkyl, or R7 and R8 are joined together with the atom or atoms to which they are attached to form a heterocycle;
each of the —(CH2)—0 can be optionally substituted;
o is an integer between 1-50; and
reacting a plurality of first oligonucleotides with reactive sites of the polymer coating to produce a plurality of first oligonucleotides attached to the polymer coating.

### Claim 11 (depends on 10)

The method of claim 10, further comprising contacting the plurality of first oligonucleotides attached to the polymer coating with templates to be amplified, each template comprising at the 3′ end a sequence capable of hybridizing to the first oligonucleotides.

### Claim 12 (depends on 11)

The method of claim 11, further comprising amplifying the templates using the first oligonucleotides, thereby generating an array of polynucleotides.

### Claim 13 (depends on 10)

The method of claim 10, wherein RA is azido.

### Claim 14 (depends on 13)

The method of claim 13, wherein R1 is H or alkyl.

### Claim 15 (depends on 14)

The method of claim 14, wherein R1 is hydrogen and wherein o is 5.

### Claim 16 (depends on 10)

The method of claim 10, wherein the polymer coating comprises a polymer of Formula (III) or (III′) :
wherein R1 is selected from H or alkyl;
R5 is selected from H or alkyl;
n is an integer in the range of 1 to 50,000; and
m is an integer in the range of 1 to 100,000.

### Claim 17 (depends on 16)

The method of claim 16, wherein the polymer of Formula (III) or (III′) is also represented by Formula (IIIa) or (IIIb):
wherein n is an integer in the range of 1 to 20,000, and m is an integer in the range of 1 to 100,000.

### Claim 18 (depends on 12)

The method of claim 12, further comprising reacting a plurality of the second oligonucleotides with reactive sites of the polymer coating, thereby producing a plurality of second oligonucleotides attached to the polymer coating.

### Claim 19 (depends on 11)

The method of claim 11, wherein each template further comprises, at the 5′ end, a sequence the complement of which is capable of hybridizing to a plurality of second oligonucleotides and wherein the amplifying comprises amplifying the templates using the first oligonucleotides and the second oligonucleotides, thereby generating an array of polynucleotides.

### Claim 20 (depends on 18)

The method of claim 18, wherein the first oligonucleotides or the second oligonucleotides comprise aldehyde or alkyne groups.

### Claim 21

A flow cell comprising a substrate, said substrate having a surface comprising a polymer coating covalently attached thereto, wherein the polymer coating comprises a recurring unit of Formula (Ia) and a recurring unit of Formula (II):
wherein:
R1 is selected from hydrogen, halo, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
RA is selected from the group consisting of amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester, epoxy, aziridine, triazoline, and thiol;
each R4, R4′, R5 and R5′ is independently selected from H, R6, OR6, —C(O)OR6, —C(O)R6, —OC(O)R6, —C(O) NR7R8, or —NR7R8;
R6 is independently selected from H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
each R7 and R8 is independently selected from H or alkyl, or R7 and R8 are joined together with the atom or atoms to which they are attached to form a heterocycle;
each of the —(CH2)—o can be optionally substituted; and
o is an integer between 1-50.

### Claim 22 (depends on 21)

The flow cell of claim 21, wherein RA is azido.

### Claim 23 (depends on 22)

The flow cell of claim 22, wherein R1 is H or alkyl.

### Claim 24 (depends on 23)

The flow cell of claim 23, wherein R1 is hydrogen and wherein o is 5.

### Claim 25 (depends on 21)

The flow cell of claim 21, wherein the polymer coating comprises a polymer of Formula (III) or (III′):
wherein R1 is selected from H or alkyl;
R5 is selected from H or alkyl;
n is an integer in the range of 1 to 50,000; and
m is an integer in the range of 1 to 100,000.

### Claim 26 (depends on 25)

The flow cell of claim 25, wherein the polymer of Formula (III) or (III′) is also represented by Formula (IIIa) or (IIIb):
wherein n is an integer in the range of 1 to 20,000, and m is an integer in the range of 1 to 100,000.

### Claim 27 (depends on 21)

The flow cell of claim 21, further comprising polynucleotides attached to the surface of the substrate via the polymer coating.

### Claim 28 (depends on 27)

The flow cell of claim 27, wherein the polynucleotides are present in polynucleotide clusters.

### Claim 29 (depends on 28)

The flow cell of claim 28, wherein polynucleotides within a single polynucleotide cluster have the same nucleotide sequence.

### Claim 30 (depends on 28)

The flow cell of claim 28, wherein polynucleotides of different polynucleotide clusters have different nucleotide sequences.

### Claim 31

A method of determining a nucleotide sequence of a polynucleotide, said method comprising the steps of:
(a) contacting a polynucleotide polymerase with polynucleotide clusters attached to a surface of a substrate via a polymer coating present on the surface of the substrate;
wherein the polymer coating comprises a recurring unit of Formula (Ia) and a recurring unit of Formula (II):
wherein:
R1 is selected from hydrogen, halo, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
RA is selected from the group consisting of amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester, epoxy, aziridine, triazoline, and thiol;
each R4, R4′, R5 and R5 ′ is independently selected from H, R6, OR6, —C(O)OR6, —C(O)R6, —OC(O)R6, —C(O) NR7R8, or —NR7R8;
R6 is independently selected from H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, or optionally substituted variants thereof;
each R7 and R8 is independently selected from H or alkyl, or R7 and R8 are joined together with the atom or atoms to which they are attached to form a heterocycle;
each of the —(CH2)—o can be optionally substituted; and
o is an integer between 1-50;
(b) providing nucleotides to the surface of the substrate such that a detectable signal is generated when one or more nucleotides are utilized by the polynucleotide polymerase;
(c) detecting signals at one or more polynucleotide clusters; and
(d) repeating steps (b) and (c), thereby determining a nucleotide sequence of a polynucleotide present at the one or more polynucleotide clusters.

### Claim 32 (depends on 31)

The method of claim 31, wherein RA is azido.

### Claim 33 (depends on 32)

The method of claim 32, wherein Rl is H or alkyl.

### Claim 34 (depends on 33)

The method of claim 33, wherein Rl is hydrogen and wherein o is 5.

### Claim 35 (depends on 31)

The method of claim 31, wherein the polymer coating comprises a polymer of Formula (III) or (III′):
wherein R1 is selected from H or alkyl;
R5 is selected from H or alkyl;
n is an integer in the range of 1 to 50,000; and
m is an integer in the range of 1 to 100,000.

### Claim 36 (depends on 35)

The method of claim 35, wherein the polymer of Formula (III) or (III′) is also represented by Formula (IIIa) or (IIIb):
wherein n is an integer in the range of 1 to 20,000, and m is an integer in the range of 1 to 100,000.

### Claim 37 (depends on 31)

The method of claim 31, wherein the surface of the substrate is present within a flow cell.

### Claim 38 (depends on 31)

The method of claim 31, wherein the detectable signal comprises an optical signal.

### Claim 39 (depends on 31)

The method of claim 31, wherein the detectable signal comprises a non-optical signal.

### Claim 40 (depends on 39)

The method of claim 39, wherein the non-optical signal is a change in pH.