BR112014030465B1 - SUBSTRATE AND PREPARATION METHODS OF POLYMER COATING IMMOBILIZED ON SUBSTRATE SURFACE AND GRANULE MATRIX PREPARATION METHODS - Google Patents

Abstract

SUBSTRATE, METHOD OF PREPARATION OF POLYMER COATING IMMOBILIZED ON SUBSTRATE SURFACE AND METHOD OF PREPARATION OF GRANULE MATRIX. The present disclosure relates to polymer coatings covalently bonded to the surface of a substrate and the preparation of polymer coatings, such as poly(N-(5-azidaacetamidylpentyl) acrylamide-coacrylamide) (PAZAM), in forming and manipulating of substrates such as molecular matrices and flow cells. The present disclosure also relates to methods for preparing a substrate surface using covalently bonded polymer coated beads, such as PAZAM, and to a method of determining a nucleotide sequence of a polynucleotide bound to a substrate surface described herein. .

Description

REFERENCE TO RELATED REQUESTS

[0001] This Application claims the benefit of priority to US Application no. 61/657,508, filed on June 8, 2012; US order no. 61/753,833, filed on January 17, 2013; and US Order no. 13/784,368, filed March 4, 2013, all of which are incorporated herein by reference in their entirety.

FIELD

[0002] In general, the present invention relates to the fields of chemistry, biology and materials science. More specifically, the present invention relates to polymer coatings covalently bonded to a surface of a substrate that is used for the detection and/or analysis of molecules, such as nucleic acids and proteins.

BACKGROUND

[0003] Polymer-coated substrates are used in many technological applications. For example, implantable medical devices can be coated with biologically inert polymers. In another example, polymer-coated substrates are used for the preparation and/or analysis of biological molecules. Molecular analyses, such as certain nucleic acid sequencing methods, rely on attaching nucleic acid chains to a polymer-coated surface of a substrate. The sequences of the fixed nucleic acid chains can then be determined by a number of different methods that are well known in the art.

[0004] In certain sequencing-by-synthesis processes, one or more surfaces of a flow cell are coated with a polymer to which nucleic acids are fixed. The current process used for coating flow cells involves transferring a polymerization mixture into channels in the flow cell and incubating for a fixed period of time. This procedure is simple and results in reliable coatings that are consistently able to withstand all downstream chemical processing steps, including bridge amplification and sequencing.

[0005] There are, however, several limitations to many of the currently used polymer surface coatings. For example, (i) some of the current approaches limit the methods that can be used to coat surfaces because an air-sensitive polymer blend is required; (ii) some of the coatings formed have to be stored in a “wet” state, for example in an aqueous solution; and (iii) grafting conditions often need to be optimized to avoid intensity gradients.

[0006] Furthermore, there is also a need to create standardized flow cells with defined characteristics spaced periodically. One method of creating patterned flow cells is to chemically modify a bead and then apply that bead to a pre-coated surface with the surface containing open wells that can accommodate the beads. The beads can be coated with a polymer that can support cluster growth-based sequencing before placing them in the wells. Polymer coatings that are useful on planarly arranged surfaces may not be suitable for coating granules due to aggregation caused by the coating and requirements for storing the granules in aqueous buffers after coating. This results in some limitations in commercial applications. Consequently, there is a need for new polymer coatings that do not suffer from one or more of the disadvantages of current polymer coatings when used to coat the granules.

SUMMARY

[0007] Some embodiments of the compositions described herein refer to substrates with one or more surfaces comprising an improved polymer coating. In some embodiments, the substrate is a flow cell and the polymer coating is applied to one or more surfaces of one or more lanes of the flow cell.

[0008] Some embodiments of the compositions described herein refer to a substrate comprising a surface, in which a polymer coating is covalently bonded to the surface. Other embodiments of the compositions described herein relate to a substrate comprising a functionalized surface, wherein a polymer coating is covalently linked to a series of functional groups on the surface and wherein the functional groups are selected from the group consisting in alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether, amine and maleimide and benzoyl ester with a phosphine substituent in the ortho position for Staudinger Bond. Still other embodiments provide a substrate comprising a surface having a polymer coating comprising recurring units of Formulas (I) and (II). In certain embodiments, the recurring unit of Formula (I) is also represented by Formula (Ia). In other embodiments, the recurring unit of Formula (I) is also represented by Formula (Ib). In some embodiments, the polymer coating comprises a polymer of Formula (III) or (III'). In one embodiment, the polymer coating comprises a polymer of Formula (IIIa). In one embodiment, the polymer coating comprises a polymer of Formula (Illb). In another embodiment, the polymer coating comprises a polymer of Formula (IV).

[0009] Some embodiments of the methods described in this document refer to the preparation of a polymer coating immobilized on a surface of a substrate. In some embodiments the method comprises contacting a polymer with a surface of a substrate, wherein the surface comprises a plurality of functional groups, thereby forming a polymer coating layer on the surface. In some embodiments, the polymer coating is covalently linked to functional groups on the surface of the substrate. In some embodiments, the polymer is formed in situ on the surface of the substrate by polymerization of a polymerizable material on the surface of the substrate. In another embodiment, the polymer is preformed before coming into contact with the surface of the substrate. In certain embodiments, the functional groups are selected from the group consisting of alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether, amine, maleimide and benzoyl ester and with a phosphine substituent in the ortho position to the Staudinger call. In certain embodiments, the functional groups on the surface of the substrate optionally comprise substituted phenyl azide groups. In other embodiments, the functional groups on the surface of the substrate comprise alkyne groups. In still other embodiments, the functional groups on the surface of the substrate comprise alkene groups. In certain embodiments, the polymer coating comprises recurring units of Formulas (I) and (II). In certain embodiments, the recurring unit of Formula (I) is also represented by Formula (Ia). In certain embodiments, the recurring unit of Formula (I) is also represented by Formula (Ib). In some embodiments, the polymer coating comprises a polymer of Formula (III) or (III'). In one embodiment, the polymer coating comprises a polymer of Formula (IIIa). In one embodiment, the polymer of Formula (III') is also represented by Formula (Illb). In another embodiment, the polymer coating comprises a polymer of Formula (IV). In some embodiments, the polymer coating is dissolved in an aqueous solution before covalently bonding to surface functional groups.

[0010] Other embodiments of the methods described in this document refer to the preparation of a polynucleotide matrix. In such embodiments, the methods may comprise the steps of reacting a plurality of oligonucleotides with reactive sites present in the polymer coating of any of the compositions described herein or polymer coatings prepared by any of the methods described herein; contacting the plurality of oligonucleotides bound to the polymer coating with templates to be amplified, each template comprising a sequence capable of hybridizing to the oligonucleotides; and amplify the templates using the oligonucleotides, thus generating a pooled array of polynucleotides. In some embodiments, two initiators may be used, one or both of which may be attached to the polymer coating. For example, the methods may comprise the steps of reacting a plurality of first oligonucleotides with reactive sites present in the polymer coating of any of the compositions described herein or polymer coatings prepared by any of the methods described herein; contacting the plurality of first oligonucleotides attached to the polymer coating with templates to be amplified, each template comprising the 3' end of a sequence capable of hybridizing to the first oligonucleotides and the 5' end of a complement sequence which is capable of to hybridize to a second oligonucleotide; and amplifying the templates using the first oligonucleotides and the second oligonucleotides, wherein the second oligonucleotide is optionally attached to the polymer coating, thereby generating a pooled array of polynucleotides. In some embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise alkyne groups to be reacted with azido groups of the polymer coating. In other embodiments of such methods, the first or second oligonucleotides comprise amine groups to be reacted with cyanuric chloride present in the polymer coating. In still other embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise aldehyde groups to be reacted with activated amine groups, such as hydrazinyl or hydrazonyl groups, of the polymer coating. In still other embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise amine groups to react with carboxylic acid or thiocyanate groups of the polymer coating. In other embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with glycidyl groups of the polymer coating. In still other embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with amine groups of the polymer coating via a dialdehyde linker. In still other embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with activated ester or epoxy groups present in the polymer layer. In still other embodiments of such methods, the first oligonucleotides or second oligonucleotides comprise aldehyde groups to be reacted with the oxo-amine groups present in the coating polymer. In some embodiments of such methods, the polymer coating is washed with water or aqueous buffer before reacting with the plurality of first oligonucleotides and the plurality of second oligonucleotides. In one embodiment, the polymer coating comprises a polymer of Formula (III). In another embodiment, the polymer coating comprises a polymer of Formula (III').

[0011] Some embodiments of the compositions described herein refer to a flow cell comprising any of the substrate compositions described herein. Some of such embodiments further comprise polynucleotides fixed to the surface of the substrate through the polymer coating. In some embodiments, the polynucleotides are present in polynucleotide groups. In some such embodiments, the polynucleotides within a single polynucleotide cluster have the same nucleotide sequence. Individual polynucleotides in a cluster can be attached to the polymer coating at one end or both ends. The attachment(s) may be through the 5' and/or 3' end of a polynucleotide chain. Polynucleotides from different polynucleotide clusters generally have different nucleotide sequences, but this is not necessary in all embodiments.

[0012] Some embodiments of the methods described in this document refer to determining a nucleotide sequence of a polynucleotide. Some of such embodiments comprise the steps of: (a) contacting a polynucleotide polymerase with the polynucleotide clusters attached to a surface of any of the compositions described herein, through the polymer coating; (b) delivering nucleotides to the substrate surface such that a detectable signal is generated when one or more nucleotides are used by the polynucleotide polymerase; (c) detect signals in one or more polynucleotide clusters; and (d) repeating steps (b) and (c), thereby determining a nucleotide sequence of a polynucleotide present in one or more polynucleotide clusters. In some such embodiments, the substrate surface is present within a flow cell. In some such embodiments, only a single type of nucleotide is present in the flow cell during a single flow step. In such embodiments, nucleotide sequences can be selected from dATP, dCTP, dGTP, dTTP and analogs thereof. In other embodiments of methods of determining a nucleotide sequence of a polynucleotide, a plurality of different types of nucleotides are present in the flow cell during a single flow step. In such embodiments, nucleotides can be selected from dATP, dCTP, dGTP, dTTP and analogs thereof. According to other embodiments, methods of determining a nucleotide sequence of a polynucleotide comprise detectable signals and optical signal. In other embodiments, the detectable signal comprises a non-optical signal. In such embodiments, the non-optical signal may be a change in pH or a change in pyrophosphate concentration.

[0013] Some embodiments described in this document refer to a method of preparing a matrix of granules. In some embodiments, the method comprises forming a plurality of functional groups on the surface of one or more granules; contacting a polymer coating described herein with the granules so as to form a polymer coating layer on the surface of the granules, wherein the polymer coating is covalently linked to functional groups on the surface of the granules. The polymer coated granules can be loaded onto the surface of a substrate, before or after being coated. In some embodiments, the surface may include open wells and each well may have dimensions, which can accommodate one or more beads (i.e., in some embodiments the wells may not accommodate more than a single bead). In some embodiments, the functional groups on the surface of the beads comprise acrylamide groups. In some embodiments, the functional groups on the surface of the beads optionally comprise substituted phenyl azide groups. In some embodies, the functional groups on the surface of the beads comprise alkyne groups. In certain embodiments, the polymer coating comprises recurring units of Formulas (I) and (II). In certain embodiments, the recurring unit of Formula (I) is also represented by Formula (Ia). In certain embodiments, the recurring unit of Formula (I) is also represented by Formula (Ib). In some embodiments, the polymer coating comprises a polymer of Formula (III) or (III'). In one embodiment, the polymer of Formula (III) is also represented by Formula (IIIa). In one embodiment, the polymer of Formula (III') is also represented by Formula (Illb). In another embodiment, the polymer coating comprises a polymer of Formula (IV). In certain embodiments, the granules are completely coated with the polymer coating described herein. In other embodiments, the granules are partially coated with the polymer coating described herein. In some embodiments, the polymer coating is covalently bonded to the surface of the beads at an elevated temperature. In some other embodiments, the polymer coating is covalently attached to the surface of the beads through photoactivation. In some other embodiments, the beads are prearranged into wells and the polymer coating is then covalently bonded to the arrayed beads.

[0014] The granule can be fixed to the surface of a substrate (for example, a flow cell or well) through chemical, physioadsorption forces or both. In some embodiments, the polymer-coated beads are attached to the surface of the substrate by placing them in open wells on the surface of the substrate. Alternatively or additionally, the polymer-coated beads are attached to the substrate surface by covalently bonding to functional groups on the substrate surface. In certain embodiments, the methods described herein further comprise washing the polymer-coated beads to remove excess unbound polymer coating before loading the beads onto the surface of the substrate. In certain embodiments, the polymer coating is dissolved in a solution before contacting the pretreated granules. In some embodiments, the polymer coating is dissolved in an aqueous solution. In certain embodiments, the methods described herein further include additional steps to form arrayed polynucleotides using similar methods as described herein. In some embodiments, the surface of the substrate is patterned with pits, pads, or other features.

[0015] Some preferred embodiments described herein relate to a substrate that has a surface comprising a covalently bonded polymer coating, wherein the polymer coating comprises a polymer of Formula (III) or (III'). In one embodiment, the polymer of Formula (III) is also represented by Formula (IIIa). In one embodiment, the polymer of Formula (III') is also represented by Formula (IIIb). In one embodiment, the substrate is a granule.

[0016] Some preferred embodiments described herein refer to a method for preparing a surface of a substrate comprising: forming a plurality of functional groups on the surface of the substrate, contacting a polymer of Formula (III) or (III ') with the substrate surface to form a polymer coating layer on the substrate surface, wherein the polymer coating is covalently bonded to functional groups on the substrate surface. In one embodiment, the polymer of Formula (III) is also represented by Formula (IIIa). In one embodiment, the polymer of Formula (III') is also represented by Formula (IIIb). In some embodiments, the functional groups on the surface of the substrate are selected from optionally substituted alkene, alkyne, or phenyl azide. In some embodiments, the polymer coating is covalently bonded to the alkene groups at an elevated temperature. In one embodiment, the alkene groups are acrylamide groups. In one embodiment, the substrate is a granule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A shows the 1H NMR spectrum of poly (N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM).

[0018] FIG. 1B shows computed molecular weight distributions for PAZAM.

[0019] FIG. 1C shows the 1H NMR spectrum of a lightly cross-linked PAZAM.

[0020] FIGS 2A and 2B show a Typhoon scan of a typical glass flow cell coated using PAZAM (FIG. 2B) and the average Typhoon intensity (FIG 2A) over the eight lanes of the flow cell.

[0021] FIGS 3A and 3B show a Typhoon scan of a PAZAM-coated HiSeq glass flow cell. FIG. 3A shows the Typhoon fluorescence image of the grafted surface hybridized with an oligonucleotide sequence containing the complementary dye. FIG. 3B shows a plot of the average Typhoon intensity of PAZAM in situ.

[0022] FIGS 4A and 4B show a Typhoon scan of a glass substrate that has been spin-coated using PAZAM. FIG. 4A shows the Typhoon fluorescence image of the grafted surface hybridized with an oligonucleotide sequence containing the complementary dye. FIG. 4B shows a spin-coated PAZAM Typhoon average intensity plot.

[0023] FIGS 5A-C show clusters on the coated flow PAZAM surfaces. FIG. 5A is an enlarged version of the UV-illuminated region of the channels. FIG. 5B and FIG. 5C show the cluster number and filtered cluster intensity of the channels.

[0024] FIGS. 6A-C show clusters on a spin-coated PAZAM surface. FIG. 6A shows a plot of spin-coated PAZAM Typhoon average intensity in HiSeq flow cell. FIG. 6B shows the Typhoon fluorescence image of the grafted surface hybridized with an oligonucleotide sequence containing complementary dye. FIG. 6C shows enlarged images of clusters in lanes 3 and 5.

[0025] FIGS. 7A and 7B show a typical flow cell image (FIG. 7B) and average intensity (FIG. 7A) across the eight flow cell lanes.

[0026] FIGS. 8A-I show clustering and sequencing of the polynucleotides in a flux-coated PAZAM mixture and the data obtained.

[0027] FIG. 9 shows a spin-coated substrate functionalized with alkyne oligos hybridized with complementary fluorescent oligonucleotides.

[0028] FIGS 10A and 10B show the results of screening alternative photoactive crosslinkers. FIG. 10A shows a graph of the average intensity of Typhoon vs. Photo XL. FIG. 10B shows a graph of UV time vs. XL photo of different crosslinking agents.

[0029] FIGS 11A - 11B show a photoactive, patterned surface and polymer blocking.

[0030] FIG. 12 illustrates the percentage of surface remaining on a thermally crosslinked PAZAM layer six days after storage at room temperature with less than 10% humidity.

[0031] FIG. 13A illustrates the cluster grown with model 5 Phix on a thermally crosslinked PAZAM surface.

[0032] FIG. 13B illustrates sequencing metrics after a 2 x 26 cycle run in a thermally cross-linked flow cell.

[0033] FIG. 14 illustrates the cluster grown in a patterned matrix made by thermally cross-linking PAZAM to patterned silane patches.

[0034] FIG. 15 illustrates standardized PAZAM matrices prepared by PAZAM-coated beads.

[0035] FIG. 16 illustrates a split and combine approach using a photoactive polymer.

DETAILED DESCRIPTION

[0036] The present invention relates to substrates comprising a surface coated with a polymer that is covalently bonded with the surface. A preferred embodiment of such substrates includes poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (also known as PAZAM or DASFA) coatings. These polymer coatings are covalently attached to a functionalized surface of a substrate, such as a surface of a flow cell or a surface of a molecular matrix. The present disclosure also relates to methods of preparing such surfaces and methods of using substrates comprising such polymer-coated surfaces. In a preferred embodiment, substrates having PAZAM-coated surfaces are used to determine a nucleotide sequence of a polynucleotide.

[0037] This new polymer coating and approach generates an air-stable material that overcomes many of the currently known limitations of polymer coatings.

Definitions

[0038] Unless otherwise defined, all technical and scientific terms used in the present documents have the same meaning as is normally understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include”, “include” and “include”, is not limiting. The use of the term “having” as well as other forms, such as “has”, “ter” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claims, the terms “comprise(s)” and “comprising” should be interpreted as having an open meaning. That is, the terms should be interpreted synonymously with the phrases “having at least,” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the steps cited, but may include additional steps. When used in the context of a compound, composition or device, the term “comprising” means that the compound, composition or device includes at least the recited features or components, but may also include additional features or components.

[0039] As used herein, common organic abbreviations are defined as follows: Ac Acetyl Ac20 Acetic Anhydride APTS aminopropyl silane APTES (3-aminopropyl)triethoxy silane APTMS (3-aminopropyl)trimethoxy silane aq. Aqueous AzapaN-(5-azidoacetamidylpentyl) acrylamide APTMS 3-aminopropyl trimethoxy silane BHT Butylated hydroxyl toluene Bn Benzyl Brapa or BRAPA N-(5-bromoacetamidylpentyl) acrylamide Bz Benzoyl BOC orBoc tert-Butoxycarbonyl Bu n-Butyl cat. Catalytic Cbz Carbobenzyloxy CyCl Cyanuric chloride °C Temperature in degrees Centigrade dATP Deoxyadenosine triphosphate dTTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate DBU 1,8-Diazabicyclo[5,4,0]undec-7-ene DCA Dichloroacetic acid DCE 1,2- Dichloroethane DCM Methylene chloride DIEA Diisopropylethylamine DMA Dimethylacetamide DME Dimethoxyethane DMF N,N'-Dimethylformamide DMSO Dimethylsulfoxide DPPA Diphenylphosphoryl azide Et Ethyl EtOAc Ethyl acetate g Gram(s) GPC Gel permeation chromatography h or hr Hour(s) iPr Isopropyl KPi 10 mM potassium phosphate buffer at pH 7.0 KPS Phosphate persulfate IPA Isopropyl alcohol IPHA.HCl N-Isopropyl hydroxylamine hydrochloride LCMS Liquid chromatography-mass spectrometry LDA Lithium diisopropylamide m or min Minute(s ) mCPBA Meta-Chloroperoxybenzoic acid Me0H Methanol MeCNAcetonitrile mL Milliliter(s) MTBE Methyl tertiary butyl ether NaN3 Sodium azide NHS N-hydroxysuccinimide PAZAM poly(N-(5-azidoacetamidilpentyl) acrylamide-co-acrylamide) of any ratio of acrylamide to azapa PG Protecting group Ph Phenyl ppt Precipitate rt Room temperature SFA Silane-free acrylamide as defined in US Patent Publication no. 2011/0059865 Sulfo-HSAB or SHSAB N-Hydroxysulfosuccinimidyl-4-azidobenoate TEA Triethylamine TEMPO (2,2,6,6-Tetramethylpiperidin-l-yl)oxyl TCDI 1,1'-Thiocarbonyl diimidazole Tert, t tertiary TFA Acid trifluoroacetic THF Tetrahydrofuran TEMED Tetramethylethylenediamine μL Microliter(s)

[0040] As used herein, the term “array” refers to a population of different probe molecules that are attached to one or more substrates in such a way that the different probe molecules can be differentiated from each other in a according to relative location. An array may include different probe molecules, which are each located in a different addressable location on a substrate. Alternatively or additionally, an array may include separate substrates, each having a different probe molecule, wherein the different probe molecules may be identified according to the locations of the substrates on a surface to which the substrates are attached or according to with the locations of substrates in a liquid. Examples of matrices in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in US Patent no. 6,355,431 Bl, US 2002/0102578 and PCT Publication no. WO 00/63437. Exemplary formats that can be used in the present invention to distinguish beads in a liquid matrix, for example, using a microfluidic device such as a fluorescent activated cell sorter (FACS), are described, for example, in Patent no. . 6,524,793. Other examples of matrices that can be used in the present invention include, without limitation, those described in US Patent Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897.

[0041] As used herein, the term “covalently fixed” or “covalently linked” refers to the formation of a chemical bond that is characterized by the sharing of electrons between pairs of atoms. For example, a covalently fixed polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as compared to attaching to the surface through other means, for example, adhesion or electrostatic interaction. It should be noted that polymers that are covalently attached to a surface can also be attached via addition means for covalent attachment.

[0042] As used herein, the term “polymer blocking” refers to the process in which functional groups on the surface of a substrate react with the polymer coating such that the polymer coating is covalently bonded to the surface.

[0043] As used herein, any “R” group(s), such as, without limitation, R2, R3, R4, R5, R6, R7 and R8 represent substituents that can be attached to the indicated atom. An R group can be substituted or unsubstituted. If two “R” groups are described as being “taken together” the R groups and the atoms to which they are attached can form a cycloalkyl, aryl, heteroaryl or heterocycle. For example, without limitation, if R2 and R3 or R2, R3 or R4 and the atom to which they are attached are said to be “taken together” or “joined together” means that they are covalently attached to each other to form a ring, an example of which is described below:

[0044] Whenever a group is described as being “optionally substituted”, that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being “substituted or unsubstituted”, if substituted, the substituent may be selected from one or more of the indicated substituents. If no substituent is indicated, it is understood that the indicated “optionally substituted” or “substituted” group may be individually and independently substituted with one or more groups individually and independently selected from a group of functionalities including, but not limited to, limitation, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl) alkyl, hydroxyl, protected hydroxyl, alkoxyl, arylaxyl, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-starch, N-starch, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanate, thiocyanate , isothiocyanate, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanessulfonyl, trihalomethanesulfonamido, amino, monosubstituted amino group, the disubstituted amino group and their protected derivatives.

[0045] As used herein, “alkyl” refers to a linear or branched hydrocarbon chain comprising a fully saturated hydrocarbon group (there are no double or triple bonds). In some embodiments, the alkyl group may have from 1 to 20 carbon atoms (wherever appearing herein, a numerical range such as “1 to 20” refers to each whole number in the given range inclusive of endpoints; e.g. For example, “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl”, where no numerical range is designated). The alkyl group may also be a medium-sized alkyl of about 7 to about 10 carbon atoms. The alkyl group may also be a lower alkyl group having 1 to 6 carbon atoms. The alkyl group of the compounds may be designated as “C1-C4 alkyl” or similar designations. By way of example, “C1-C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, that is, the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl , isobutyl, sec-butyl and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyls. The alkyl group can be substituted or unsubstituted.

[0046] As used herein, “alkenyl” refers to an alkyl group that contains in the linear or branched hydrocarbon chain one or more double bonds. An alkenyl group may be unsubstituted or substituted.

[0047] As used herein, the term “alkynyl” refers to an alkyl group that contains in the linear or branched hydrocarbon chain one or more triple bonds. An alkynyl group may be unsubstituted or substituted.

[0048] As used herein, “cycloalkyl” refers to a fully saturated monocyclic or multicyclic hydrocarbon ring system (there are no double or triple bonds). When composed of two or more rings, the rings may be fused together. Cycloalkyl groups can contain 3 to 10 ring atoms. In some embodiments, the cycloalkyl groups may contain 3 to 8 ring atoms. A cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0049] As used herein, “cycloalkenyl” refers to a monocyclic or multicyclic hydrocarbon ring system that contains one or more double bonds in at least one ring; although, if there is more than one, the double bonds cannot form a system of fully delocalized pi electrons throughout the rings (otherwise the group would be “aryl” as defined here). When composed of two or more rings, the rings may be connected together in a fused manner. A cycloalkenyl group may be unsubstituted or substituted.

[0050] As used herein, “cycloalkynyl” refers to a monocyclic or multicyclic hydrocarbon ring system that contains one or more triple bonds in at least one ring. If there is more than one triple bond, the triple bonds cannot form a system of fully delocalized pi electrons throughout all the rings. When composed of two or more rings, the rings may be fused together. A cycloalkynyl group may be unsubstituted or substituted.

[0051] As used herein, “aryl” refers to a monocyclic carbocyclic (all carbon) ring system or multicyclic aromatic ring system (including, for example, fused, bridged or spiro ring systems in which two carbocyclic rings share a chemical bond, e.g., one or more aryl rings with one or more aryl or non-aryl rings) having a fully delocalized pi electron system along at least one of the rings. The number of carbon atoms in an aryl group can vary. For example, in some embodiments, the aryl group may be a C6-C14 aryl group, a C6-C10 aryl group, or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group can be substituted or unsubstituted.

[0052] As used herein, “heterocyclyl” refers to ring systems that include at least one heteroatom (e.g., O, N, S). Such systems may be unsaturated, may include some unsaturation, or may contain an aromatic moiety or be all aromatic. A heterocyclic group can be unsubstituted or substituted.

[0053] As used herein, “heteroaryl” refers to a monocyclic or multicyclic aromatic ring system (a ring system having at least one ring with a fully delocalized pi electron system) containing one or more heteroatoms that is, an element other than carbon, including but not limited to nitrogen, oxygen and sulfur and at least one aromatic ring. The number of atoms in the ring of a heteroaryl group can vary. For example, in some embodiments, a heteroaryl group may contain 4 to 14 ring atoms, 5 to 10 ring atoms, or 5 to 6 ring atoms. Furthermore, the term "heteroaryl" includes fused ring systems in which two rings, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2, 3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidiazole na, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinoline and triazine. A heteroaryl group may be substituted or unsubstituted.

[0054] As used herein, “heteroalicyclic” or “heteroalicyclic” refers to a monocyclic, bicyclic and tricyclic ring system with three, four, five, six, seven, eight, nine, ten, up to 18 members , wherein carbon atoms together with 1 to 5 heteroatoms constitute said ring system. A heterocycle may optionally contain one or more unsaturated bonds situated in such a way, however, that a fully delocalized pi electron system does not occur throughout all of the rings. Heteroatoms are independently selected from oxygen, sulfur and nitrogen. A heterocycle may further contain one or more carbonyl or thiocarbonyl functionalities, so making the definition include oxo systems and thiosystems such as lactams, lactones, cyclic imides and cyclic thioimides, cyclic carbamates. When composed of two or more rings, the rings may be fused together. Furthermore, any nitrogens in a heteroalicyclic can be quaternized. Heteroalicyclic or heteroalicyclic groups may be unsubstituted or substituted. Examples of such "heteroalicyclic" or "heteroalicyclic" groups include, but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1 ,4-dioxolane, 1,3-oxathione, 1,4-oxathione, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathione, tetrahydro-l, 4-thiazine , 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline , oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine-N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidine, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetra -hydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone and their benzofused analogues (e.g. benzimidazolidinone, tetrahydroquinoline, 3,4-methylenedioxyphenyl).

[0055] As used herein, “aralkyl” and “aryl (alkyl)” refers to an aryl group connected, as a substituent, through a lower alkylene group. The lower alkylene and aryl group of an aralkyl group may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl and naphthylalkyl.

[0056] As used herein, “heteroaralkyl” and “heteroaryl (alkyl)” refers to a heteroaryl group connected, as a substituent, through a lower alkylene group. The alkylene and heteroaryl group of lower heteroaralkyl may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 1,3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl and imidazolylalkyl and their benzo-fused analogues.

[0057] A “(heteroalicyclyl)alkyl” is a heterocyclic group or a heteroalicyclic group connected, as a substituent, through a lower alkylene group. The heterocyclic lower alkylene or a heterocyclyl of an (heteroalicyclyl) alkyl may be substituted or unsubstituted. Examples include, but are not limited to, tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4 -yl)methyl and (1,3-thiazinan-4-yl)methyl.

[0058] As used herein, “alkoxy” refers to the formula -OR where R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or a cycloalkynyl is defined as above. A non-limiting list of alkoxy groups is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy and tert-butoxy. An alkoxy group can be substituted or unsubstituted.

[0059] As used herein, “hydroxyalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a hydroxy group. Exemplary hydroxyalkyl groups include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkyl can be substituted or unsubstituted.

[0060] As used herein, “alkylamino” refers to an alkyl group in which one or more of the hydrogen atoms is replaced by an amino group. Exemplary alkylamino groups include, but are not limited to, aminomethyl, 2-aminoethyl, 3-aminoethyl. An amino can be substituted or unsubstituted.

[0061] As used herein, “alkylamido” refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a C-amido group or an N-amido group. A starch can be substituted or unsubstituted.

[0062] As used herein, “alkylthio” refers to RS-, where R is an alkyl. Alkylthio can be substituted or unsubstituted.

[0063] As used herein, a “C-amide” group refers to a “-C(=O)N(RaRb)” group in which Ra and Rb can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl)alkyl. A C-starch can be substituted or unsubstituted.

[0064] As used herein, an “N-amido” group refers to a “RC(=O)N(Ra)-” group in which R and Ra can independently be hydrogen, alkyl, alkenyl, alkynyl , cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl) alkyl. An N-starch can be substituted or unsubstituted.

[0065] The term “halogen atom”, “halogen” or “halo”, as used herein, means any of the radiostable atoms in column 7 of the Periodic Table of the Elements, such as fluorine, chlorine, bromine and iodine.

[0066] The term “amine” as used herein refers to a -NH2 group in which one or more hydrogen atoms can be optionally replaced by a group R. R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl)alkyl.

[0067] The term “aldehyde” as used herein refers to a group a-Rc-C(O)H, in which Rc may be absent or is independently selected from alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, arylene, heteroarylene, heteroalicyclylene, aralkylene or (heteroalicyclyl)alkylene.

[0068] The term “activated ester” as used herein refers to an ester that reacts spontaneously with a nucleophile, for example, NHS ester, pentafluorophenyl ester or nitrophenyl ester.

[0069] The term “nitrene” as used herein refers to the nitrogen analogue of a carbene, in which the nitrogen atom is not charged with six valence electrons.

[0070] The term “amino” as used herein refers to a -NH2 group.

[0071] The term “hydroxy” as used herein refers to an -OH group.

[0072] The term “cyano” group as used herein refers to a “-CN” group.

[0073] The term “azido” as used herein refers to a -N3 group.

[0074] The term “thiol” as used herein refers to a -SH group.

[0075] The term “hydrazinyl” as used herein refers to a -NHNH2 group.

[0076] The term “hydrazonyl” as used herein refers to a group.

[0077] The term “formyl” as used herein refers to a -C(O)H group.

[0078] The term “glycidyl” or “glycidyl ether” as used in This document refers to •

[0079] The term “epoxy” as used herein refers to

[0080] The term “carboxylic acid” as used herein refers to -C(O)OH.

[0081] The term “thiocyanate” as used herein refers to the -SC-N group.

[0082] The term “oxo-amine” as used herein refers to the group -O-NH2, in which one or more hydrogen atoms of the -NH2 may be optionally replaced by a group R. R may independently be hydrogen , alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl)alkyl.

[0083] As used herein, the prefixes “photo” or “foto-” mean relating to light or electromagnetic radiation. The term may encompass all or part of the electromagnetic spectrum, including, but not limited to, one or more of the bands commonly known as radio, microwave, infrared, visible, ultraviolet, parts of spectrum range. The portion of the spectrum may be one that is blocked by a region of a metal surface, such as the metals set forth herein. Alternatively or additionally, the portion of the spectrum may be one that passes through an interstitial region of a surface such as a region made of glass, plastic, silica or other materials set forth herein. In particular embodiments, radiation which is capable of passing through a metal can be used. Alternatively or additionally, radiation which is masked by glass, plastic, silica or other materials set forth herein may be used.

[0084] As used herein, the term “reactive site” means a site on the polymer coatings described herein that can be used to attach one or more molecules through a chemical reaction or molecular interaction. Such a bond may be through a covalent bond or through other bonding or interactive forces.

[0085] As used herein, the term “percent surface remaining” may refer to the intensity measured using a qc TET to stain the P5/P7 surface primers. Primers P5 and P7 are used on the surface of commercial flow cells sold by Illumina Inc. for sequencing on HiSeq, MiSeq, and Genome Analyzer platforms. Primer sequences are described in Pub. Pat. US no. 2011/0059865 Al, which is incorporated herein by reference. TET is a dye-labeled oligonucleotide with the sequence complementary to the P5/P7 primer. TET can be hybridized to the P5/P7 primer on a surface; excess TET can be washed off and the concentration of bound dye can be measured by fluorescence detection using scanning instruments such as a Typhoon scanner (General Electric).

Substrate compositions having a polymer coated surface

[0086] A first aspect of the compositions described herein relates to a substrate comprising a surface having a polymer coating covalently fixed thereto. In a preferred embodiment, the polymer comprises PAZAM. In some embodiments, the polymer coating comprises a recurring unit of Formula (I) and a recurring unit of Formula (II): wherein: each R1 and R1' is independently selected from hydrogen, halo, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or optionally substituted variants thereof; each R2 and R3 is independently selected from hydrogen, alkyl, alkylamino, alkylamido, alkylthiol, aryl or optionally substituted variants thereof; each R4, R4', R5 and R5' is independently selected from H, R6 or 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 bonded to form a heterocycle.

[0087] In some embodiments of such compositions, R2 is H and R3 is an optionally substituted alkyl group.

[0088] In still other embodiments of such compositions, R3 represents an alkyl group substituted by an N-amido group.

[0089] In some embodiments of the compositions described in this document, the recurring unit of Formula (I) is also represented by Formula (Ia): wherein R1 is H or alkyl; RA is selected from the group consisting of hydrogen, amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, halo, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester , epoxy, aziridine, triazoline and thiol; each of -(CH2)-o may be optionally substituted; o is an integer between 1-50; and since when R1 is H and RA is halo, RA cannot be a bromine group.

[0090] In some such embodiments, o is 5 and RA is azide.

[0091] According to other embodiments, R1 is hydrogen.

[0092] In still other embodiments, R4 is a -C(O)NR7R8 group, in which each R7 and R8 is independently selected from hydrogen, alkyl or hydroxyalkyl.

[0093] In some embodiments of such polymeric compositions, R4 is -C(O)NH2, -C(O)NH(CH3)2 or -C(O)N(CHe)2.

[0094] In other embodiments, R4 is a group -C(O)NH(CH2)2OH or -C(O)N(CH3)(CH2)2OH.

[0095] In still other embodiments, R4 is NR7R8, in which R7 and R8 are joined together with the atoms to which they are attached to form a five-membered heterocycle.

[0096] In preferred embodiments comprising a five-membered heterocycle, the five-membered heterocycle is an optionally substituted pyrrolidino.

[0097] In still other embodiments, R4 is -C(O)OR6, where R6 is selected from hydrogen, alkyl or hydroxyalkyl.

[0098] In a preferred embodiment, R6 is hydrogen.

[0099] In another preferred embodiment, R6 is methyl.

[0100] In yet another preferred embodiment, R6 is hydroxyethyl.

[0101] In other preferred embodiments, R4 is hydrogen.

[0102] In still other preferred embodiments, R4 is alkyl.

[0103] In still other preferred embodiments, R4 is methyl.

[0104] In some preferred embodiments, R5 is hydrogen.

[0105] In additional preferred embodiments, R5 is alkyl.

[0106] According to other preferred embodiments, R5 is methyl.

[0107] In yet another embodiment of the substrates comprising a polymer coated surface described herein, the polymer coating comprises a polymer of Formula (III) or (III'): wherein R1 is selected from H or alkyl; RA is selected from the group consisting of hydrogen, amine, optionally substituted alkene, optionally substituted alkyne, oxo amine, azido, formyl, halo, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester, epoxy, aziridine, triazoline and thiol; each of -(CH2)-o may be optionally substituted; o is an integer in the range 1-50; R5 is selected from H or alkyl; n is an integer in the range 1 to 50,000; en is an integer in the range 1 to 50,000; since when R1 and R5 are H, o is 5, then RA cannot be a bromine group. In some embodiments, the is 5.

[0108] In one embodiment, the polymer of Formula (III) or (III') is also represented by Formula (IIIa) or (Illb): where n is an integer in the range 1-20,000 and m is an integer in the range 1-100,000.

[0109] In some of these embodiments, the polymer coating is covalently attached to a series of functional groups attached to the surface, wherein the functional groups are selected from alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether , amine, maleimide or benzoyl ester with a phosphine substituent in the ortho position.

[0110] In a preferred embodiment of these compositions, the functional groups comprise alkynes and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is azide.

[0111] In another preferred embodiment of these compositions, the functional groups comprise nitrenes and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is azide.

[0112] In yet another preferred embodiment of these compositions, the functional groups comprise activated esters and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is amine.

[0113] In yet another preferred embodiment of these compositions, the functional groups comprise hydrazines and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is formyl.

[0114] In yet another preferred embodiment of these compositions, the functional groups comprise the aldehyde group and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is amine, oxo-amine or hydrozinyl.

[0115] In yet another preferred embodiment of these compositions, the functional groups comprise glycidyl ether and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is amine.

[0116] In yet another preferred embodiment of these compositions, the amine functional groups comprise and the recurring unit of Formula (I) is also represented by Formula (Ia), where RA is azide.

[0117] In yet another preferred embodiment of these compositions, the functional groups comprise maleimide, the recurring unit of Formula (I) is also represented by Formula (Ia) and where RA is thiol.

[0118] In yet another embodiment of the substrates comprising a polymer-coated surface described herein, the recurring unit of Formula (I) is also represented by Formula (Ib): wherein R2 is optionally substituted aryl.

[0119] In some such embodiments, R2 in Formula (Ib) is phenyl azide, optionally substituted with one or more halogen groups. In a particular embodiment, R2 is phenyl perfluoro azide.

[0120] In some embodiments, R1 in Formula (Ib) is hydrogen.

[0121] According to other embodiments, R4 in Formula (Ib) is - C(O)NR7R8. In a particular embodiment, R4 is a -C(O)NH2 group.

[0122] In some embodiments, R4 in Formula (Ib) is hydrogen.

[0123] In some embodiments, R5 in Formula (Ib) is alkyl.

[0124] In other embodiments, R5 in Formula (Ib) is hydrogen.

[0125] In some embodiments, R5 in Formula (Ib) is hydrogen.

[0126] In another embodiment of the substrates comprising a polymer coated surface described here, the polymer coating comprises a polymer of Formula (IV): where n is an integer in the range 1-50,000 and m is an integer in the range 1-100,000. In a particular embodiment, the phenyl group of Formula (IV) is optionally replaced by one or more fluorine groups.

[0127] The polymer coating described herein can be covalently fixed to a variety of substrates. Essentially, any substrate material that can be functionalized with Reactive groups, including but not limited to alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether, amine, maleimide can be used. Acrylamide, enone or acrylate can also be used as a substrate material. The substrates may comprise a single material or a plurality of different materials. Substrates can be composite or laminated. In some embodiments, the substrate has at least one surface comprising glass. In other embodiments, the substrate has at least one surface comprising a metal. In some such embodiments, the metal is gold. In some embodiments, the substrate has at least one surface comprising a metal oxide. In one embodiment, the surface comprises a tantalum oxide. Other substrate materials may include, but are not limited to, plastic, silicon, silicon dioxide, silicon nitride, fused silica, gallium arenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers and copolymers . The substrate can be flat, round or textured.

[0128] In some embodiments of the compositions described herein, the substrate surface comprises two polymer-coated regions and inert regions that are not polymer-coated. The polymer-coated regions can comprise reactive sites and thus can be used to attach molecules through chemical bonding or other molecular interactions. In some embodiments, the polymer-coated regions and inert regions may alternate to form a pattern or grid. Such patterns can be in one or two dimensions. In some embodiments, the inert regions may be selected from the group consisting of glass regions, metal regions, mask regions, and interstitial regions. In a preferred embodiment, the surface comprises regions of glass. In another preferred embodiment, the surface comprises metal regions. In yet another preferred embodiment, the surface comprises mask regions. In yet another preferred embodiment, the surface comprises interstitial regions. In some embodiments of the compositions described herein, the substrate may be a granule. In a preferred embodiment, the surface of the bead is functionalized. Functionalization can occur before or after introducing the bead into a well. In one embodiment, the well is predefined on the surface of a flow cell. Examples of substrate materials that can be coated with a polymer of the present invention or that can otherwise be used in a composition or method set forth herein are described in US documents nos. serial numbers 13/492,661 and 13/661,524, each of which is incorporated herein by reference.

[0129] A second aspect of the compositions described herein relates to a flow cell comprising one or more substrates comprising a surface having a polymer coating covalently fixed thereto. In some embodiments, the flow cells described herein comprise one or more of the substrates described above. In a preferred embodiment, the polymer comprises PAZAM.

[0130] In some embodiments, the flow cells further comprise polynucleotides attached to the surface of the substrate through the polymer coating. In preferred embodiments, the polynucleotides are present in the flow cells in groups of polynucleotides, wherein the polynucleotides of the polynucleotide clusters are attached to a surface of the flow cell by means of the polymer coating. In such embodiments, the surface of the flow cell body to which the polynucleotides are attached is considered the substrate. In other embodiments, a separate substrate having a polymer-coated surface is inserted into the body of the flow cell. In preferred embodiments, the flow cell is a flow chamber that is divided into a plurality of lanes or a plurality of sectors, wherein one or more of the plurality of lanes or the plurality of sectors comprises a surface that is coated with a coating. of covalently fixed polymer described herein. In some embodiments of the flow cells described herein, the fixed polynucleotides within a single polynucleotide cluster have the same or similar nucleotide sequence. In some embodiments of the flow cells described herein, fixed polynucleotides from different polynucleotide clusters have different or dissimilar nucleotide sequences. Examples of flow cells and substrates for manufacturing flow cells that may be used in the method or composition described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA) or described in document US 2010/0111768 Al or US 2012/0270305, each of which is incorporated herein by reference

Methods for coating substrates with covalently fixed polymers

[0131] A first aspect of the present methods disclosed herein relates to a process for preparing a polymer coating immobilized on a surface of a substrate. In some embodiments, the method comprises contacting a polymer with a surface of a substrate, wherein the surface comprises a plurality of functional groups, thereby forming a polymer coating layer on the surface and wherein the polymer coating it is covalently linked to functional groups on the surface. In some embodiments, the polymer is formed in situ on the surface of the substrate by polymerization of a polymerizable material on the surface of the substrate. In another embodiment, the polymer is preformed before coming into contact with the surface of the substrate. In a preferred embodiment, the polymer comprises PAZAM.

[0132] In some embodiments of methods of immobilizing a polymer coating to a substrate surface, the polymer coating comprises a recurring unit of Formula (I) and a recurring unit of Formula (II). wherein: each R1 and R1' is independently selected from hydrogen, halo, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or optionally substituted variants thereof; each R2 and R3 is independently selected from hydrogen, alkyl, alkylamino, alkylamido, alkylthiol, aryl or optionally substituted variants thereof; each R4, R4', R5 and R5' is independently selected from H, R6 or 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 bonded to form a heterocycle.

[0133] In some embodiments, R2 is H and R3 is an optionally substituted alkyl. In one embodiment, R3 is an alkyl group substituted by an N-amido group.

[0134] In other embodiments of methods of coating a substrate surface with a polymer, the recurring unit of Formula (I) is also represented by Formula (Ia): wherein R1 is H or alkyl; RA is selected from the group consisting of hydrogen, amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, halo, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester , aziridine, triazoline, epoxy and thiol; each of -(CH2)-o may be optionally substituted; o is an integer between 1-50; provided that when R1 is H and RA is halo, RA cannot be a bromine group.

[0135] In one embodiment of such a method, o is 5 and RA is azide.

[0136] In another embodiment of such a method, R1 is hydrogen.

[0137] In yet another embodiment of such a method, R4 is a group - C(O)NR7R8, in which each R7 and R8 is independently selected from hydrogen, alkyl or hydroxyalkyl. In a particular embodiment of such a method, R4 is a -C(O)NH2 group.

[0138] In another embodiment of such a method, R4 is hydrogen.

[0139] In another embodiment of such a method, both R4 and R5 are hydrogens.

[0140] In yet another embodiment of such a process, at least one of R4 and R5 is alkyl, for example, a methyl group.

[0141] In some embodiments, the polymer coating comprises a polymer of Formula (III) or (III'): wherein R1 is selected from H or alkyl; RA is selected from the group consisting of hydrogen, amine, optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, halo, hydroxy, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxylic acid, glycidyl, activated ester , aziridine, triazoline, epoxy and thiol; each of -(CH2)-o may be optionally substituted; o is an integer in the range 1-50; R5 is selected from H or alkyl; n is an integer in the range 1 to 50,000; en is an integer in the range 1 to 50,000; since when R1 and R5 are H, o is 5, then RA cannot be a bromine group. In some embodiments, the is 5.

[0142] In one embodiment, the polymer of Formula (III) or (III') is also represented by Formula (IIIa) or (Illb): where n is an integer in the range 1-20,000 and m is an integer in the range 1-100,000.

[0143] In some embodiments of such methods, the functional groups on the surface of the substrate comprise photoactivatable azides. In some embodiments, the photoactivatable azides are optionally substituted phenyl azide groups. In some of these embodiments, phenyl azide is prepared by reacting an amine group on the surface of the substrate with N-hydroxy sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB).

[0144] In some embodiments of the methods described above, phenyl azide is photoactivated before contacting the polymer with the surface of the substrate. In a preferred embodiment of these methods, photoactivated functional groups generate nitrene. In another preferred embodiment of these methods, the polymer coating is covalently linked to nitrene groups through photoactivation.

[0145] In some embodiments of the above-described methods of covalently attaching a polymer coating to the surface of a substrate, the functional groups on the surface of the substrate comprise alkyne groups.

[0146] In some embodiments of the above-described methods of covalently attaching a polymer coating to the surface of a substrate, the polymer coating is covalently linked to functional groups in the presence of a catalyst. In a particular embodiment, the catalyst is a copper catalyst. In some embodiments, the polymer coating is covalently bonded to the alkyne groups without the use of a copper catalyst.

[0147] In some embodiments of the above-described methods of covalently attaching a polymer coating to the surface of a substrate, the functional groups on the surface of the substrate comprise alkene groups. In some embodiments, alkene groups are prepared by reacting the amine-functionalized surface with acryloyl groups. In some preferred embodiments, the amine-functionalized surface is prepared by treating the surface with 3-aminopropyl trimethoxy silane (APTMS). In some other preferred embodiments, the acryloyl group may be selected from an activated acrylic ester, acrylic acid, acrylic chloride or COMU (CAS Number 1075198-30-9). In one embodiment, the activated acrylic ester is an N-hydroxysuccinimide (NHS) acrylic acid ester. In some other embodiments, alkene groups are prepared by directly contacting the surface of the substrate with functionalized silanes. In some other embodiments, the functionalized silane can be selected from 3-acrylamidotrimethoxysilane or methacryloxypropyltrimethoxysilane.

[0148] In some embodiments of the above-described covalent attachment methods of a polymer coating to the surface of a substrate, the polymer coating is covalently bonded to functional groups at an elevated temperature. In a preferred embodiment, the elevated temperature is any temperature in a range of 60°C to 90°C.

[0149] In other embodiments of methods of immobilizing a polymer coating to a substrate surface, the recurring unit of Formula (I) is also represented by formula (Ib): wherein R2 is optionally substituted aryl.

[0150] In one embodiment of such methods, R2 in Formula (Ib) is phenyl azide, optionally substituted with one or more halogen groups. In a particular embodiment, R is phenyl azide perfluoro.

[0151] In another embodiment of the methods of coating a substrate surface with a polymer, R1 in Formula (Ib) is hydrogen.

[0152] In some embodiments of such methods, R4 in Formula (Ib) is -C(O)NR7R8. In a particular embodiment, R4 is a group - C(O)NH2.

[0153] In other embodiments of such methods, R5 in Formula (Ib) is hydrogen.

[0154] In still other embodiments of such methods, both R4 and R5 in Formula (Ib) are hydrogen.

[0155] In some embodiments of such methods, the polymerizable material is applied in liquid form.

[0156] In some embodiments of methods of immobilizing a polymer coating to a substrate surface, the plurality of functional groups are disposed on the substrate surface so as to form a plurality of polymer-coated regions and a plurality of inert regions. for subsequent polymerization of the polymeric material. In some embodiments the inert regions are selected from the group consisting of glass regions, metal regions, mask regions, and interstitial regions. In a preferred embodiment, the inert regions comprise glass. In some embodiments, the plurality of polymer-coated zones and the plurality of inert regions are arranged on the surface to form a pattern or grid. Such patterns or grids may be one-dimensional or two-dimensional with respect to the surface of the substrate. Exemplary patterned surfaces that can be used are described in US documents nos. serial numbers 13/492,661 and 13/661,524, each of which is incorporated herein by reference.

[0157] In some embodiments of the above-described methods of preparing a polymer coating immobilized on a surface of a substrate, the polymer coating is dissolved in an aqueous solution before covalently bonding to functional groups on the surface. In some embodiments, the substrate is a granule.

[0158] A second aspect of the present methods disclosed herein relates to a process for preparing a polynucleotide matrix. In such embodiments, the methods may comprise the steps of (a) reacting a plurality of first oligonucleotides and a plurality of second oligonucleotides with the reactive sites in a polymer coating present on a surface of any of the substrates described herein or a polymer coating prepared by any of the methods of immobilizing a polymer coating on a surface of a substrate as described herein; (b) contacting the plurality of first oligonucleotides attached to the polymer coating with templates to be amplified; and (c) amplifying the templates using the first oligonucleotides and the second oligonucleotides, thus generating a pooled array of polynucleotides. In some embodiments, each template comprises at the 3' end a sequence capable of hybridizing with the first oligonucleotides and at the 5' end a complement sequence that is capable of hybridizing with the second oligonucleotides. In the above methods, the second oligonucleotide is optional. Thus, the second oligonucleotide need not be present in some embodiments. If present, the second oligonucleotide can be fixed to the polymer coating or can be supplied in solution surging the amplification step.

[0159] In some embodiments of the above-described preparation methods of a polynucleotide matrix, the first oligonucleotides or second oligonucleotides comprise alkyne groups to be reacted with the azido groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0160] In other embodiments of the above-described preparation methods of a polynucleotide matrix, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with cyanuric chloride of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0161] In still other embodiments of the above-described preparation methods of a polynucleotide array, the first oligonucleotides or second oligonucleotides comprise aldehyde groups to be reacted with activated amine groups, such as hydrazonyl or hydrazinyl groups of the polymer coating. . In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0162] In still other embodiments of the above-described preparation methods of a polynucleotide matrix, the first oligonucleotides or second oligonucleotides comprise amine groups to react with carboxylic acid or thiocyanate groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0163] In additional embodiments of the above-described preparation methods of a polynucleotide array, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with glycidyl groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0164] According to other embodiments of the above-described preparation methods of a polynucleotide matrix, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with amine groups of the polymer coating, through a dialdehyde linker. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0165] In still other embodiments of the above-described preparation methods of a polynucleotide matrix, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with activated ester or epoxy groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0166] In still other embodiments of the above-described preparation methods of a polynucleotide matrix, the first oligonucleotides or second oligonucleotides comprise aldehyde groups to be reacted with the oxo-amine groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0167] In a preferred embodiment of the above-described preparation methods of a polynucleotide matrix, the polymer coating on the surface of the substrate comprises a polymer of Formula (IIIa) or (IIIb).

[0168] A third aspect of the present methods disclosed herein relates to a method for preparing a surface of a substrate comprising: forming a plurality of functional groups on the surface of one or more granules; contacting a polymer coating described herein with the granules so as to form a polymer coating layer on the surface of the granules, wherein the polymer coating is covalently linked to functional groups on the surface of the granules; and attaching the polymer-coated granules to the surface of the substrate. In one embodiment, the substrate is a flow cell. In one embodiment, the polymer coating comprises PAZAM. The polymer coating layer may cover the granules fully or partially. In one embodiment, the polymer coating layer on the surface of the beads has a thickness of about 20 nm. In some embodiments, the beads have a diameter of about 1.2 microns or less. In some other embodiments, the beads have a diameter of about 0.5 microns or less. In a preferred embodiment, the surface of the substrate is patterned. However, larger granules may be used, including, but not limited to, those having a diameter of about 10 microns or less, 5 microns or less, 3 microns or less, or 2 microns or less.

[0169] In some embodiments of methods of preparing a surface of a substrate, as described herein, the functional groups on the surface of the granules comprise alkenes. In some embodiments, the alkene groups are prepared by reacting the amine-functionalized surface of the beads with acryloyl groups. In some preferred embodiments, the amine-functionalized surface is prepared by treating the surface with 3-aminopropyl trimethoxy silane (APTMS). In some other preferred embodiments, the acryloyl group may be selected from an activated acrylic ester or an acrylic chloride. In one embodiment, the activated acrylic ester is an acrylic ester of N-hydroxysuccinimide acid (NHS). Other useful binding agents include acrylic acid, COMU (CAS Number 1075198-30-9), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS). In some other embodiments, alkene groups are prepared by directly contacting the substrate surface with functionalized silanes. In some other embodiments, the functionalized silane can be selected from 3-acrylamidotrimethoxysilane or methacryloxypropyltrimethoxysilane. In some embodiments, the pretreated granules are exposed to a solution comprising a polymerization inhibitor before coming into contact with the polymer coating. In some embodiments, the polymerization inhibitor is selected from butylated hydroxyl toluene (BHT), diethylhydroxylamine, or (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO). In some preferred embodiments, the polymer coating is covalently bonded to alkene groups on the surface of the granules at an elevated temperature. In a preferred embodiment, the elevated temperature is any temperature in a range of 60°C to 90°C.

[0170] In some embodiments of the methods of preparing a surface of a substrate, as described herein, the functional groups on the surface of the granules comprise photoactivatable azides. In some embodiments, the photoactivatable azides are optionally substituted phenyl azide groups. In some of these embodiments, phenyl azide is prepared by reacting an amine group on the surface of the substrate with N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB). In some embodiments, the phenyl azide is photoactivated before coming into contact with the polymer with the surface of the substrate. In a preferred embodiment of these methods, photoactivated functional groups generate nitrene. In another preferred embodiment of these methods, the polymer coating is covalently linked to the functional groups through photoactivation.

[0171] In some embodiments of the methods of preparing a surface of a substrate, as described herein, the functional groups on the surface of the granules comprise alkyne groups. In some embodiments, the polymer coating is covalently linked to functional groups in the presence of a catalyst. In a particular embodiment, the catalyst is a copper catalyst. In some embodiments, the polymer coating is covalently bonded to the alkyne groups without the use of a copper catalyst.

[0172] In some embodiments of methods of preparing a surface of a substrate, as described herein, polymer-coated beads are fixed to the surface of the substrate by loading said beads into open wells on the surface of the substrate. The beads and wells may have dimensions that result in no more than one bead residing in each well. Alternatively, the relative dimensions may result in multiple beads per well. In some preferred embodiments, the polymer-coated beads are attached to the surface of the substrate by reaction of functional groups in the polymer coating with the functional groups on the surface of the substrate. Surface functional groups can be localized to particular features of a surface, for example, pits or blocks. Alternatively, the functional groups of the substrate can be dispersed across a flat surface. Examples of methods for preparing such surfaces, which can be coated using the methods and compositions presented here, are described in US document nos. serial numbers 13/492,661 and 13/661,524, each of which is incorporated herein by reference. In one embodiment, the functional groups of the polymer coating comprise amines and the functional groups on the surface of the substrate comprise N-hydroxysuccinimide (NHS) esters. In another embodiment, the functional groups of the polymer coating comprise azides and the functional groups on the surface of the substrate comprise alkynes. In yet another embodiment, the functional groups of the polymer coating comprise thiols and the functional groups on the surface of the substrate comprise maleimides.

[0173] In some embodiments of the methods of preparing a surface of a substrate, as described herein, the method further comprises washing the polymer-coated granules to remove excess unbound polymer coating before loading the granules to the surface of the substrate. In some embodiments, the polymer coating is dissolved in a solution before contacting the granules. In one embodiment, the solution is an aqueous solution.

[0174] In some embodiments of the methods of preparing a surface of a substrate, as described herein, the method further comprises reacting a plurality of first oligonucleotides and a plurality of second oligonucleotides with reactive sites on the polymer coating of the substrates. granules; contacting the plurality of first oligonucleotides bound 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 complement sequence which is capable of hybridizing to the first oligonucleotides. second oligonucleotides; and amplify the templates using the first oligonucleotides and the second oligonucleotides, thus generating a pooled polynucleotide matrix.

[0175] In some embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise alkyne groups to be reacted with the azido groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0176] In other embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with cyanuric chloride of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0177] In still other embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise aldehyde groups to be reacted with activated amine groups, such as hydrazonyl or hydrazinyl groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0178] In still other embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise amine groups to react with carboxylic acid or thiocyanate groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0179] In additional embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with glycidyl groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0180] According to still other embodiments of the methods described above, the first oligonucleotides or the second oligonucleotides comprise amine groups to be reacted with amine groups of the polymer coating, through a dialdehyde linker. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0181] In still other embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise amine groups to be reacted with activated ester or epoxy groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0182] In still other embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise maleimide groups to be reacted with the thiol groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0183] In still other embodiments of the methods described above, the first oligonucleotides or the second oligonucleotides comprise alkene groups to be reacted with the alkene groups of the olefin polymer coating through cross-metathesis. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0184] In still other embodiments of the methods described above, the first oligonucleotides or second oligonucleotides comprise aldehyde groups to be reacted with the oxo-amine groups of the polymer coating. In a preferred embodiment, both the first oligonucleotides and the second oligonucleotides comprise these groups.

[0185] In some embodiments of the methods of preparing a surface of a substrate, as described herein, the method further comprises the step of staining the polymer-coated granules with an optical imaging agent. In some embodiments, the optical imaging agent is selected from the group consisting of Dylight488 phosphine, DyLight 550phosphine, DyLight 650phosphine (both from ThermoFisher Scientific), and strained alkyne dyes (DBCO-based dyes from Click Chemistry Tools, Inc. ) such as: DBCO-Fluor R488, DBCO-Fluor 525, DBCO-Fluor 545, DBCO-Fluor 568, DBCO-Fluor 585 and DBCO-SETA 650. In one embodiment, the optical imaging agent is DyLight488 phosphine.

[0186] In some embodiments of the methods of preparing a surface of a substrate, as described herein, the substrate may be selected from a silicon substrate, a plastic substrate, or a plastic substrate impregnated with additives. In some other embodiments, the plastic substrate may be impregnated with SiO2, TiO2 or carbon black.

[0187] A method set forth herein may use any of a variety of amplification techniques. Examples of techniques that may be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random major amplification (RPA). In particular embodiments, one or more primers used for amplification may be attached to a polymer coating. In PCR embodiments, one or both of the primers used for amplification can be linked to a polymer coating. Formats that utilize two species of fixed primers are often referred to as bridge amplification because double-stranded amplicons form a bridge-like structure between the two fixed primers flanking the template sequence that was copied. Examples of conditions and reagents that can be used for bridged amplification are described, for example, in US Patent no. 5,641,658; US Patent Publication no. 2002/0055100; US Patent No. 7,115,400; US Patent Publication no. 2004/0096853; US Patent Publication no. 2004/0002090; US Patent Publication no. 2007/0128624; and US Patent Publication no. 2008/0009420, each of which is incorporated herein by reference. PCR amplification can also be performed with one of the amplification primers attached to a polymer coating and the second primer in solution. An exemplary format that uses a combination of a fixed primer and a soluble primer is emulsion PCR as described, for example, in Dressman et al., Proc. Natl. academic Sci. USA 100: 8817-8822 (2003), document WO 05/010145 or US Patent Publications nos. 2005/0130173 or 2005/0064460, each of which is incorporated herein by reference. Emulsion PCR is illustrative of the format and it will be understood that for the purposes of the methods set forth herein the use of an emulsion is optional and in fact, for several embodiments, an emulsion is not used. Furthermore, primers do not need to be linked directly to solid supports as set forth in the ePCR references and can, instead, be linked to a polymer coating as set forth herein.

[0188] RCA techniques can be modified for use in a method of the present disclosure. Examples of components that can be used in an RCA reaction and principles by which RCA produces amplicons are described, for example, in Lizardi et al., Nat. Genet. 19: 225-232 (1998) and US 2007/0099208 Al, each of which is incorporated herein by reference. Initiators used for RCA can be in solution or attached to a polymer coating.

[0189] MDA techniques can be modified for use in a method of the present disclosure. Some basic principles and useful conditions for MDA are described, for example, in Dean et al., Proc Natl. academic Sci. USA 99: 5261-66 (2002); Lage et al., Genome Research 13: 294307 (2003); Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20: 1691-1696 (1992); US 5,455,166; US 5,1302,38; and US 6,214,587, each of which is incorporated herein by reference. Initiators used for MDA can be in solution or attached to a polymer coating.

[0190] In particular embodiments, a combination of the amplification techniques exemplified above may be used. For example, RCA and MDA can be used in a combination where RCA is used to generate a concatameric amplicon in solution (e.g., using solution-phase primers). The amplicom can then be used as a template for MDA using primers that are attached to a polymer coating. In this example, the amplicons produced after the combined RCA and MDA steps will be fixed to the polymer coating.

[0191] A third aspect of the present methods disclosed herein relates to a process of determining a nucleotide sequence of a polynucleotide. In such embodiments, the methods may comprise the steps of (a) contacting a polynucleotide polymerase with the polynucleotide clusters attached to a surface of a substrate through any of the polymer coatings described herein; (b) delivering nucleotides to the polymer-coated surface of the substrate such that a detectable signal is generated when one or more nucleotides are used by the polynucleotide polymerase; (c) detect signals in one or more polynucleotide clusters; and (d) repeating steps (b) and (c), thereby determining a nucleotide sequence of a polynucleotide present in one or more polynucleotide clusters

[0192] In some embodiments of the methods described herein, a nucleotide sequence is determined by a polynucleotide fixed to the surface of the substrate that is present within a flow cell. In some embodiments, the polymer coated surface is an integral part of the flow cell. In other embodiments, the polymer-coated surface is a separate substrate placed within the flow cell. In other embodiments, the separate substrate may be coupled, bonded, or otherwise secured to a surface or other portion of the flow cell.

[0193] Nucleic acid sequencing can be used to determine a nucleotide sequence of a polynucleotide through various processes known in the art. In a preferred method, sequencing synthesis (SBS) is used to determine a nucleotide sequence of a polynucleotide attached to a surface of a substrate through any of the polymer coatings described herein. In such a process, one or more nucleotides are supplied to a template polynucleotide that is associated with a polynucleotide polymerase. Polynucleotide polymerase incorporates one or more nucleotides into a newly synthesized nucleic acid chain that is complementary to the template polynucleotide. Synthesis is initiated from a primer oligonucleotide that is complementary to a portion of the template polynucleotide or to a portion of a nucleic acid or universal variable that is not covalently linked to one end of the template polynucleotide. As nucleotides are incorporated against the template polynucleotide, a detectable signal is generated that allows determination of which nucleotide was incorporated during each step of the sequencing process. In this way, the sequence of a nucleic acid complementary to at least a portion of the template polynucleotide can be generated, thereby allowing determination of the nucleotide sequence of at least a portion of the template polynucleotide. Flow cells provide a convenient format for housing a matrix that is produced by the methods of the present invention and that is subjected to sequencing synthesis (SBS) or other detection technique that involves the repeated distribution of reagents in cycles. For example, to initiate a first cycle of SBS, one or more labeled nucleotides, DNA polymerase, etc., may be flowed into/through a flow cell housing a nucleic acid matrix produced by methods set forth herein. Those sites on an array where primer extension causes a labeled nucleotide to be incorporated can be detected. Optionally, the nucleotides may further include a reversible termination property that terminates beyond the primer extension once a nucleotide has been added to a primer. For example, a nucleotide analogue having a reversible terminator moiety can be added to a primer in such a way that subsequent extension cannot occur until an unblocking agent is released to remove the moiety. Thus, for embodiments utilizing reversible termination, an unblocking reagent may be released to the flow cell (before or after detection occurs). Washes can be carried out between the various release steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thus detecting a sequence of length n. Examples of SBS procedures, fluidic systems and detection platforms that can be readily adapted for use with a matrix produced by the methods of the present invention are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US 2008/0108082, each of which is incorporated herein by reference in its entirety.

[0194] Other sequencing processes that use cyclic reactions can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al., Science 281(5375), 363 (1998); US 6,210,891; US 6,258,568 and US 6,274,320, each of which which are incorporated herein by reference in their entirety). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase and the level of ATP generated can be detected through luciferase-producing photons. Thus, the sequencing reaction can be monitored using a luminescence detection system. Excitation radiation sources used for fluorescence-based detection systems are not required for pyrosequencing procedures. Useful fluidic systems, detectors and procedures that can be used to apply matrix pyrosequencing of the present invention are described, for example, in WO 12/058096 Al, US 2005/0191698 Al, US 7,595,883 and US 7,244,559 , each of which is incorporated herein by reference in its entirety.

[0195] Sequencing-by-ligation reactions are also useful, including, for example, those described in Shendure et al. Science 309: 1728-1732 (2005); US 5,599,675; and US 5,750,341, each of which is incorporated herein by reference in its entirety. Some embodiments may include sequencing-by-hybridization procedures as described, for example, in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58(1998).; Fodor et al., Science 251 (4995), 767-773 (1995); and WO 1989/10977, each of which is incorporated herein by reference in its entirety. In both ligation-sequencing and hybridization sequencing procedures, nucleic acids that are present at sites on an array are subjected to repeated cycles of oligonucleotide release and detection. Fluidic systems for SBS methods as set forth herein or in references cited herein can be easily adapted to release reagents for ligation sequencing or hybridization sequencing procedures. Typically, oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described in connection with the SBS procedures described herein or in references cited herein.

[0196] Some embodiments may use methods that involve real-time monitoring of DNA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and nucleotides labeled with Y-phosphate or zero-mode waveguides (ZMWs). Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. academic Sci. USA 105, 1176-1181 (2008), the descriptions of which are incorporated herein by reference in their entirety.

[0197] Some embodiments of SBS include the detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on proton detection may utilize an electrical detector and techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or the associated sequencing methods and systems described in US 2009 documents /0026082 Al; US 2009/0127589 Al; US 2010/0137143 Al; or US 2010/0282617 Al, each of which is incorporated herein by reference in its entirety.

[0198] Another useful application for a provision of the present disclosure, for example, after it has been produced by a method defined herein, is gene expression analysis. Gene expression can be detected or quantified using RNA sequencing techniques, such as those called digital RNA sequencing. RNA sequencing techniques can be performed using sequencing methodologies known in the art, such as those presented above. Gene expression can also be detected or quantified using hybridization techniques performed by direct hybridization to an array or using a multiplex assay, the products of which being detected on an array. A matrix of the present disclosure, for example, having been produced by a method defined herein, can also be used to determine the genotypes of a sample of genomic DNA from one or more individuals. Examples of methods for array-based expression and genotyping analysis that can be performed on an array of the present invention are described in U.S. Patent Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Patent Publication Nos. 2005/0053980 Al.; 2009/0186349 Al or US 2005/0181440 Al, each of which is incorporated herein by reference in its entirety.

[0199] In some embodiments of the method described above, which employ a flow cell, only a single type of nucleotide is present in the flow cell during a single flow step. In such embodiments, the nucleotide sequences may be selected from the group consisting of dATP, dCTP, dGTP, dTTP and analogs thereof. In other embodiments of the above-described method employing a flow cell, a plurality of different types of nucleotides are present in the flow cell during a single flow step. In such methods, nucleotides can be selected from dATP, dCTP, dGTP, dTTP and their analogues.

[0200] Determination of the nucleotide or nucleotides incorporated during each flow step for one or more of the polynucleotides attached to the polymer coating on the surface of the substrate present in the flow cell is achieved by detecting a signal produced at or near the template polynucleotide. In some embodiments of the methods described above, the detectable signal comprises optical signal. In other embodiments, the signal comprises a non-optical detectable signal. In such embodiments, the optical signal does not comprise a change in pH at or near one or more of the template polynucleotides.

EXAMPLES Example 1 Preparation of PAZAM General

[0201] Unless otherwise indicated, all reactions were conducted under a nitrogen or argon atmosphere and starting materials were obtained from commercial suppliers (Aldrich Chemical Company, Fisher Scientific, Dow) and were used as received without further purification. All recorded reaction temperatures indicate the temperature of the bath/air in contact with the reaction vessel. No anhydrous solvent was required.

[0202] 1H-NMR and 13C-NMR spectra were recorded in deuterium oxide (by LC NMR 99.9 at %D) on a Bruker Avance 400 MHz instrument. Chemical shifts are expressed in parts per million, (ppm , δ) from tetramethylsilane (TMS) and are referenced to the solvent indicated as internal standard.

[0203] GPC analyzes were performed by Smithers Rapra Technology Limited using the following chromatographic conditions: Instrument: TDA301 Malvern/Viscotek Triple Detector Array with associated pump and autosampler. Columns: Agilent lx PLaquagel-OH 40 plus 1 x PLaquagel-OH 60, 30 cm, 13 μm or Agilent PLaquagel-OH Guard plus 2 x PLaquagel-OH Mixed, at 30 cm, 8 μm Eluent: NaNO30.2 M; NaH2PO4 0.01 M adjusted to pH = 7.0 Flow: 1.0 mL/min (nominal), Temperature: 30°C (nominal) Detector: Index of refraction with differential pressure and right-angle light scattering Data were collected and analyzed using Malvern/Viscotek OminSec' software.

Photoluminescence Spectroscopy

[0204] Fluorescence measurements were conducted on a Typhoon Trio Variable Mode Imager (GE) and data processed using ImageQuant TL software.

[0205] Thin films were prepared for analysis by coating using any of the rotating coating flow methods on suitably prepared clean silicon or glass substrates. Photoluminescence spectra of dried grafted films hybridized with complementary fluorescent oligos were collected on a Typhoon Trio (GE).

Synthesis

[0206] The polymer coating described herein can be prepared in several ways. A method for synthesizing the polymer of Formula (IIIa) is shown in Scheme 1A. Scheme 1A: Synthesis of a PAZAM mixture in solution state

[0207] The synthesis of N-(5-bromoacetamidylpentyl)acrylamide (BRAPA) (1c) was described in Patent Publication no. 2011/0059865, the disclosure of which is incorporated herein by reference in its entirety.

[0208] The water-soluble acrylamide polymer (PAZAM) was prepared by a chemically initiated free radical polymerization. The crude polymer was purified by successive precipitations to obtain PAZAM with number average molecular weights of 300-400 kDa. Average molecular weights in a wider range can also be obtained, for example in a range of 30-600 kDa.

[0209] Acrylamide (5.0 g, 70.3 mmol) was dissolved in 3/40 deionized water (45 mL) and the mixture was vortexed carefully to ensure complete dissolution. In a separate flask, BRAPA (1.0 g, 3.61 mmol) was added to DMF (10 mL, 9.44 g) and the resulting mixture was carefully vortexed to facilitate dissolution. A mixture of BRAPA solution (9.25 g or 9.80 ml), aqueous acrylamide solution (47.5 ml) and water (190 ml) was combined and then stirred for 5 min. The solution was then filtered through a 0.2 μm Whatman filter.

[0210] Potassium persulfate (125 mg, 0.46 mmol) was added to water (2.5 mL) and the mixture was vortexed until dissolved. Sodium azide (11.5 g, 177 mmol) was added to deionized water (82.9 ml) and the solution was vortexed to dissolve. (The final concentration of NaN3 (the raw polymer mixture) is 0.53 M, which corresponds to a 50-fold excess of NaN3 over available BRAPA).

[0211] The BRAPA/acrylamide premix (-250 mL) was purged with argon for 20 minutes using a plastic pipette. To this mixture of monomers were added tetramethylethylenediamine (TEMED) (pure, 273 μL) and potassium persulfate (50 mg/mL; 2,370 μL). The mixture was stirred and heated at 35°C for 1.5 h and then allowed to cool to room temperature. While stirring, Chainguard I-15 (1390 μL) was added to the crude mixture, followed by 2.03 M sodium azide solution (82.9 mL). To ensure complete quenching of any remaining radicals generated during polymerization, air was bubbled through the mixture for 10 min. The quenched polymer mixture was stirred at 65°C for 2 h. The mixture was then cooled to room temperature and the temperature could be stored in this state (at 4°C) for prolonged periods.

[0212] The crude mixture (-335 mL) was added dropwise to a large excess (~1.5 L) of IPA, while maintaining gentle stirring. The polymer precipitated as a white solid which was collected and dried in the pump. The partially dried polymer (-10 g) was redissolved in H2O (100 mL) by stirring the mixture at room temperature for at least one hour to facilitate dissolution. The viscous solution was then added dropwise to 1.5 L of IPA while stirring. The solid was filtered under vacuum and then dried using high vacuum to obtain PAZAM as a white solid (~5.6 g, >95%). 1H NMR Spectra are shown in FIG. 1A.

[0213] GPC (0.2 M NaNO3; NaH2PO40.01 M adjusted to pH = 7.0; calibrated using narrow distribution Pullulan polysaccharide with a peak molecular weight of 130,000, an intrinsic viscosity of 0.511 and a differential refractive index in 0.1 M sodium nitrate of 0.147 mL/g A value for the differential refractive index (dn/dc) of 0.191 mL/g was used to calculate the sample molecular weight data): Mn = 3.15 x 105 Da, Mw = 1.0 x 106 Da, Mw/Mn = 3.2 (see FIG 1B). The polymer showed a multi-modal MW distribution. The PAZAM sample was difficult to filter before starting chromatographic analysis which contributed to the wide weight distribution and led to measurement artifacts, increasing the complexity of the analysis. The dried PAZAM can then be redissolved in water at a desired concentration, typically 0.01-12% w/v. Preparation of PAZAM Linear

[0214] Alternatively, the synthesis of a linear PAZAM (IIIb) is shown in Scheme 1B. Scheme 1B. Synthesis of a Linear PAZAM

[0215] Preparation of 1d/acrylamide premix: 1a (915 mg), sodium azide (236 mg) and DMF (9 ml) were mixed in a 25 ml round bottom flask equipped with a stir bar. The flask was placed in a drysyn bath and the solution was heated under a nitrogen atmosphere with stirring for 2 h at 35°C (bath temperature) to form 1d. Acrylamide (4.78 g) was dissolved in deionized water and the resulting 1d solution was added to the acrylamide solution and stirred to mix. The reagent mixture was filtered through a 0.2μm filter.

[0216] A 1L flange flask was placed in a 1L drysyn bath on a stirrer/hot plate with a flask lid secured over it. The flask was equipped with an anchor-shaped stirring paddle through an agitator press, and the stirring paddle was connected to a top agitator. An air condenser was attached to a quickFit gasket of the balloon lid and a tube adapter connected to a nitrogen manifold (but no gas flow at this time) was attached to the top of the condenser. The filtered reagent mixture solution was transferred to the flask setup and nitrogen was bubbled through the solution using a 5 mL pipette attached to the nitrogen manifold for 30 minutes while preheating the reagent mixture to 35°C (temperature of bath).

[0217] Preparation of raw PAZAM polymer solution: Although degassing of the acrylamide/1d premix was ongoing, potassium persulfate (119 mg) was dissolved in deionized water (2.4 ml) with stirring in vortex. The nitrogen flow over the top of the condenser was turned on to ensure a flow of nitrogen over the degassed reactant mixture. After the degassing process, TEMED (99 μL) was added to the reagent mixture while stirring at 200 rpm. Then, potassium persulfate solution was added to the stirred reaction mixture to initiate polymerization. Stirring was maintained at 200 rpm under nitrogen at 35°C (bath temperature) for 1.5 hours.

[0218] Extinguishing polymerization: Although the polymerization reaction was ongoing, the IPHA.HCl solution was prepared by dissolving IPHA.HCl (312 mg) in deionized water (2.80 ml). Once the polymerization reaction had been ongoing for 1.5 h, the nitrogen line was removed from the top of the condenser, leaving the reaction vessel open to the air. Sodium hydroxide solution (1M) was added to the stirred reaction mixture, followed by the prepared IPHA.HCl solution to quench the polymerization. The quenched reaction mixture was stirred at 200 rpm at 35°C (bath temperature) for a further 30 minutes.

[0219] Purification of PAZAM polymer: The crude mixture was slowly added by pipette to 2-propanol with stirring (750 mL) and continued stirring for another 1h. The solvent was decanted and discarded as residue containing sodium azide. The precipitated polymer was crushed to squeeze out the trapped solvent. The polymer was redissolved in deionized water (150 mL) and the polymer solution was added slowly by pipette to 2-propanol with stirring (750 mL) and stirring continued for a further 1h. The solvent was decanted again and discarded as residue containing sodium azide. The precipitated polymer was crushed to squeeze out the trapped solvent. The resulting polymer was dried in a desiccator under high vacuum for 18 h and then transferred to a labeled container, tared, and stored in the dark at room temperature.

PAZAM Preparation Lightly Crosslinked

[0220] Likewise, a lightly cross-linked PAZAM was prepared following general synthetic Scheme 1B with a modified procedure as described herein.

[0221] Preparation of 1d/acrylamide premix: 1a (915 mg), sodium azide (106 mg) and DMF (9 ml) were mixed in a 25 ml round bottom flask equipped with a stir bar. The flask was placed in a drysyn bath and the solution was heated under a nitrogen atmosphere with stirring for 2 h at 35°C (bath temperature) to form 1d. Acrylamide (4.78 g) was dissolved in deionized water and the resulting 1d solution was added to the acrylamide solution and stirred to mix. The reagent mixture was filtered through a 0.2μm filter.

[0222] A 1L flange flask was set up in a 1L drysyn bath on a shaker/hot plate with a flask lid secured over it. The flask was equipped with an anchor-shaped stirring paddle through a stirring press, and the stirring paddle was connected to a top stirrer. An air condenser was attached to a QuickFit gasket of the flask lid and a tube adapter connected to a nitrogen manifold (but no gas flow at this time) was attached to the top of the condenser. The filtered reagent mixture solution was transferred to the configured flask and nitrogen was bubbled through the solution using a 5 mL pipette attached to the nitrogen manifold for 30 minutes, while preheating the reagent mixture to 35°C (bath temperature ).

[0223] Preparation of raw PAZAM polymer solution: While degassing of the acrylamide/ld premix was in progress, potassium persulfate (119 mg) was dissolved in deionized water (2.4 ml) with stirring in vortex. The nitrogen flow over the top of the condenser was turned on to ensure a flow of nitrogen over the degassed reactant mixture. After the degassing process, TEMED (99 μL) was added to the reagent mixture while stirring at 200 rpm. Then, potassium persulfate solution was added to the stirred reaction mixture to initiate polymerization. Stirring was maintained at 200 rpm under nitrogen at 35°C (bath temperature) for 1.5 hours.

[0224] Polymerization Quenching and Azidolysis: Although the polymerization reaction was ongoing, the IPHA.HCl solution was prepared by dissolving IPHA.HCl (312 mg) in deionized water (2.80 ml). In addition, a sodium azide solution was prepared by dissolving 575 mg of sodium azide in 8 ml of deionized water. Once the polymerization reaction had been ongoing for 1.5 h, the nitrogen line was removed from the top of the condenser, leaving the reaction vessel open to the air. After removing one of the stoppers from the lid, a sodium hydroxide solution (1M) was added to the stirred reaction mixture, followed by the prepared IPHA.HCl solution to quench the polymerization. Then, sodium azide solution was added to the stirred reaction mixture. The quenched reaction mixture was heated to 65°C (bath temperature) and stirring continued at 200 rpm for 2 h maintaining the temperature.

[0225] Purification of PAZAM polymer: The crude mixture was slowly added by pipette to 2-propanol with stirring (750 mL) and stirring was continued for another 1h. The solvent was decanted and discarded as a residue containing sodium azide. The precipitated polymer was crushed to squeeze out the trapped solvent. The polymer was redissolved in deionized water (150 ml) and the polymer solution was added slowly by pipette to 2-propanol with stirring (750 ml) and stirring continued for a further 1h. The solvent was again decanted and discarded as a residue containing sodium azide. The precipitated polymer was crushed to squeeze out the trapped solvent. The resulting polymer was dried in a desiccator under high vacuum for 18 h and then transferred to a labeled container, tared, and stored in the dark at 4°C.

[0226] The 1H NMR of lightly cross-linked PAZAM was obtained from a mixture of 100 μL of D2O with a solution of 500 μL of water (3%) of the polymer (see FIG. 1C). Example 2 Preparation of a PAZAM derivative

[0227] The synthesis of a PAZAM derivative is shown in Scheme 2. First, BRAPA (lc) is reacted with a t-Boc-protected hydroxylamine (2a) to form an intermediate (2b), which is treated with dichloroacetic acid to form an oxo-amine derivative of PAZAM (2c). 2c can subsequently be grafted with aldehyde-functionalized oligonucleotides to form 2d. Example 3 Polymer Coatings PAZAM coatings on glass/flow cell substrates

[0228] Aqueous PAZAM solutions were deposited on glass, plastic or silicon substrates. The coatings were subsequently grafted (using an alkyne-functionalized oligonucleotide) and a complementary dye hybridized to the grafted surface. See FIGS 2A and 2B. Typical fluorescence scans are shown in FIG. 2B. The polymer was then grafted using primers (alkyne oligonucleotides) at different concentrations, to obtain a surface that contained four lanes at approximately the standard oligonucleotide primer density (~15,000 primers/μm) and four lanes at a higher primer density. high (~50,000 primers/μm). Primer densities can range from 2500 to 1 x 106 primers/μm2 depending on the graft primer concentration chosen (FIG 2A).

[0229] A Typhoon scan of a HiSeq glass flow cell (Illumina) coated using PAZAM is shown in FIG. 3A. The polymer surface was grafted using primers (alkyne oligonucleotides) with increasing concentrations demonstrating the range of primer densities (primers/μm2) that can be achieved using this technique (FIG 3B).

[0230] FIG. 4A illustrates a Typhoon scan of a glass substrate that has been spin-coated using PAZAM. A flow cell sized to fit inside the Genome-Analyzer (Ilumina) was then made using the wafer. As described in FIGS. 2A and 2B, the polymer surface was then grafted using primers (alkyne oligonucleotides) with increasing concentrations demonstrating the range of primer density (primers/μm2) that can be achieved on a spin-coated surface via this approach (FIG 4B). .

[0231] Flux-coated PAZAM surfaces were able to support amplification of seeded DNA templates. FIG. 5A illustrates that the DNA clusters from the templates in the standard SFA channel (lane 1) were identical to those in the PAZAM-coated channels. Clusters were grown using cBot 28-cycle cluster amplification ([PhiX] = 1pM, short template, 80 b.p.) then SYBR green dye. The results demonstrated that this approach provided a robust surface capable of supporting bridged amplification (FIG 5B and FIG 5C).

[0232] FIGS. 6A and 6B show the DNA clusters on a spin-coated PAZAM surface. This illustrates that the spin-coated PAZAM surfaces were able to support amplification of the seeded DNA template, to obtain clusters similar to those observed on the standard SFA surface. Typical images of the clusters were obtained using Mantéia (20x objective, 1 mJ exposure) and SYBR green dye (FIG. 6C). Deposition of Poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM)

Materials and equipment

[0233] (3-aminopropyl) trimethoxy-silane (97%) (APTMS) and isopropanol (Analytical grade reagent) (IPA), N,N,N',N',N''- pentamethyldiethylenetriamine (PMDETA), sulfate copper (CuSO4. 5H2O, 4% w/v) and sodium ascorbate (NaAsc) were purchased from Sigma Aldrich. A 50% aqueous solution of IPA was prepared for washes. N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB) was purchased from Webscientific. An aqueous solution of poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) at 2% w/v (PAZAM) was prepared in a laboratory. Dilution occurred in a hybridization oven overnight at 35°C. Potassium phosphate buffer (10mM aqueous solution, pH = 7) (KPi) and sodium chlorate and sodium citrate solution (SSC) were prepared in the laboratory. Flow cell cleaning was performed using an Emitiech K105OX oxygen plasma incinerator. An MJ Research PTC-200 thermal cycler (MJ) was used for solution deposition of sulfo-HSAB and PAZAM. A UVP XX-Series UV Bench Lamp was used for the photochemical reaction of sulfo-HSAB with PAZAM. An Ophir PD300- UV photodiode sensor (20pW-300mW) was used to monitor the UV source supplied to the flow cell throughout the photochemical reaction.

Method

[0234] The raw flow cell (Illumina) was placed in Asher plasma for 10 minutes at 100W. The cleaned glass substrate was then silanized by vapor deposition. The flow cell ports were placed on top of two open glass vials containing 100μI of pure APTMS in a vacuum desiccator. The desiccator was placed under vacuum and incubated at 60°C overnight. After removal from the vacuum desiccator, the flow cell was placed on an MJ and primed with KPi (10mM) for 2 minutes at 75μL.min-1. 21.1 mM sulfo-HSAB solution in 10 mM KPi was then flowed for 2 minutes at 100 μL.min-1. A static incubation was performed in the dark for 1.5 hours at room temperature. Subsequent washes were then performed with deionized water, 50% IPA solution and deionized water for 2 minutes at 100 μL.min-1, respectively, ensuring that no air gaps were introduced into the flow cell channels. The 2% PAZAM solution was then poured into the channels for 1 minute at 100 μL.min-1, ensuring that no air gaps were introduced into the flow cell channels. After removing MJ, the flow cell was UV illuminated using a UV lamp. The distance between the UV source and the flow cell was 1 cm. The exposure time was adjusted so that 15 J per square centimeter of area was delivered to the flow cell. The flow cell was then subsequently washed with deionized water, 50% IPA, deionized water and KPi (10 mM) for 2 minutes at 100 μL.min-1 respectively. The flow cell was then functionalized by reacting alkyne oligonucleotides in KPi (10 mM) with PMDETA, copper sulfate and NaAsc (500 mg.mL-1 aqueous solution) at 60°C for 30 minutes. The presence of the alkyne oligonucleotides on the surface was confirmed by staining with a sequenced complementary oligonucleotide functionalized with a fluorescent dye at the 5′ end of the alkyne primers.

Results

[0235] Fluorescence detection was performed with a Typhoon Trio fluorescent scanner (GE Healthcare). A typical image (FIG 7B) and a graph indicating the median intensities across the channels (FIG 7A) are shown.

[0236] A 2x26 cycle test was completed on a 2.6 mm HiSeq flow cell (Illumina). The first baseline report indicated that clusters were detected in all lanes (see FIG 8A to FIG 8C). Clusters were still present in the PAZAM-coated channels after 26 cycles. The results also showed that PAZAM clusters could be sequenced in the Ilumina paired-end protocol and returning data for 52 cycles (i.e., a pair of 26 cycles). See FIG. 8D to FIG. 8I.

PAZAM spin coated on a solid substrate

[0237] In a typical experiment, a patterned glass substrate was first cleaned by calcining for five minutes in a plasma calciner (Emitiech K105OX) at 100 W. After cleaning, the substrate was silanized by placing it in a vacuum desiccator containing 0.5 mL of (3-aminopropyl)triethoxy-silane (APTES) in open bottles. The desiccator was placed under reduced pressure and incubated at 60°C for one hour. After silanization, the desiccator was opened and the substrate recovered. The substrate was then placed face down in a Petri dish containing a solution of 5 mg/mL N-Hydroxysulfosuccinimidyl-4-azidobenoate (SHSAB) in 10 mM potassium phosphate buffer (KPi) at pH 7.0. After one hour at room temperature, the substrate was rinsed with water and dried by nitrogen ventilation. The substrate was placed on the spin coater and a 2% w/v PAZAM solution in water was pipetted onto the substrate. After spin coating, the substrate was immediately irradiated with 365 nm UV radiation (UVP, XX15L lamp) for 30 minutes. The surface was then extensively washed with water and dried by nitrogen ventilation. After sealing with a coverslip, the surface was then functionalized by reaction with alkyne oligonucleotides (3 x 109 mol) in 10 mM KPi at pH 7.0 (1.429 mL), together with N,N,N',N', N”- pentamethyldiethylenetriamine (PMDETA, 13.14 uL, 6.3 x 10-5 moles), copper sulfate (4% w/v CuSO4.5H2O solution, 7.49 μL, 1.2 x 10-6 moles) and sodium ascorbate (4.75 μL of a 500 mg/mL solution in water, 1.2 x 10-5 moles), at 60°C for 30 minutes. The presence of the alkyne oligonucleotides on the surface was confirmed by staining with complementary sequenced oligonucleotide functionalized with a Texas red fluorescent dye at the 5′ end. As shown in FIG. 7A, the fluorescent signal can be detected by scanning on a Trio Typhoon fluorescence scanner (GE Healthcare).

[0238] FIG. 9 shows a spin-coated substrate functionalized with alkyne oligonucleotides hybridized with complementary fluorescent oligonucleotides. Dark color indicates the presence of oligonucleotides on a spin-coated polymer layer. Example 4 Preparation of Alkyne Functionalized Surface Scheme 3. Suggested Synthetic Routes for Preparation of Alkyne Functionalized Surfaces

[0239] As described herein, the various functional groups can be used for polymer blocking. Scheme 3 illustrates several suggested synthetic routes for preparing the alkyne-functionalized surface.

[0240] An alternative approach involves the use of the photoactive coupling agent N-hydroxy-sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB). Sulfo-HSAB is a commercially available bifunctional cross-linking agent comprising a photoactive aryl azide and an activated NHS moiety. Upon exposure to UV light (~250-374 nm), aryl azide generates a nitrene with the release of nitrogen. This highly reactive species can undergo a variety of rapid insertion reactions. Example 5 Preparation of a Photoactive Surface Scheme 4. Route Used to Generate a Photoactive Surface

[0241] Scheme 4 shows a way to prepare a photoactive surface. The surface is pretreated with APTS (methoxy or ethoxy silane) and baked to form an amine group monolayer (or multilayer). The amine groups are then reacted with sulfo-HSAB to form an azido derivative. UV activation at 21°C with 1 to 30 J/cm3 of energy generates the active nitrene species, which can be easily subjected to a variety of insertion reactions with PAZAM.

[0242] Initial verifications indicate that this approach, involving the use of a photoactive surface and purified PAZAM mixtures, can be used to coat standard GA flow cells (Ilumina) and the resulting coatings can be used to grow clusters . Additionally, polymers can be deposited (e.g., spin coating, dip, dip, spray, etc.) onto a prepared photoactive wafer with similar results. A coated PAZAM mixture, derived as described in Scheme 4 above, was taken through sequencing and a summary of the run is detailed below. Alternative Crosslinking Agents

[0243] In addition to sulfo-HSAB, photostability and efficiency of three other cross-linking agents were also examined and compared with sulfo-HSAB.

[0244] Alternative crosslinking agents were tested with standard process flow with a GA flow cell (Illumina). Cross-linking agents tested include Sulfo-HSAB, diazirine, BBA, and ATFB. Coupling reactions were performed at 20°C in DMF at a concentration of about 20 mM. After APTES deposition, alternating lanes of an aminosilane-functionalized flow cell were treated with a 20 mM aqueous/DMF solution of each of the cross-linking agents. The channels were incubated for an identical time as the sulfo-HSAB control channel. After successive washing, PAZAM coating, and UV exposure to lock the polymer to the surface, the lanes were grafted with our standard oligonucleotide primers and stained with a complementary fluorescent oligonucleotide sequence. Post-hybridization results with Texas VRed are summarized in FIGS 10A and 10B.

[0245] The performance of the three alternative cross-linkers was evaluated by measuring the fluorescent signal of the hybridized complementary oligonucleotides using a Typhoon imager. The results were clear with the measured surface initiator densities being less than half of those recorded for sulfo-HSAB. For any of these photoactive crosslinkers (including sulfo-HSAB), the insertion mechanism to form a covalent bond with the coated polymer can likely be thermally triggered. Example 6 Unsaturated Surface Preparation and Thermal Crosslinking of PAZAM

[0246] Another alternative approach to polymer blocking is PAZAM thermal cross-linking, which includes functionalizing the substrate surface with unsaturated groups, such as alkene. Scheme 5 illustrates some synthetic pathways to generate alkene-functionalized surfaces. Scheme 5. Route Used to Generate an Acrylamide Functionalized Surface

[0247] The substrate surface was first treated with 3-aminopropyltrimethoxy-silane (APTMS) (5a) to form an amine-functionalized surface. Then, the amine functional groups were reacted with acryloyl chloride (5b) or activated acryloyl NHS ester (5c). Subsequently, PAZAM was introduced to the unsaturated surface either by pumping a 12% PAZAM aqueous solution into the flow cell or by spin coating on top of an open glass slide. The amount of PAZAM that is present in the aqueous solution pumped into a flow cell can be, for example, 0.1-10%. The substrate was incubated at an elevated temperature, typically 60°C. In this process, the azide groups on PAZAM reacted with the unsaturated alkene groups on the surface. Different mechanisms may be involved in this process, as suggested by Kriille et al., Tetrahedron:. Asymmetry (1997), 8: 3087-3820, which is incorporated herein by reference in its entirety.

[0248] After a wash with water or aqueous buffer to remove excess unreacted PAZAM, the surface can be grafted with nucleic acid primers. Standard technique can be used in all other downstream processes, e.g. cluster growth or QC. The surface has similar performance characteristics to blocking polymer through UV activation. For example, it can be grafted with different concentrations of graft initiators to achieve different initiator densities. It can also be dehydrated, stored dry, and rehydrated while maintaining an active surface transfer rate (see FIG 12). Example 7 Application of Polymer Coatings on Clusters and Sequencing

[0249] In FIG. 8A-I, a 2x26 cycle test was completed with PAZAM-coated HiSeq flow cell (Illumina). The first baseline report indicated that clusters were detected in all lanes. Furthermore, PAZAM clusters were still present after 26 cycles. PAZAM clusters finished paired-end reading and data returned for 52 complete cycles (i.e., a pair of 26 cycles).

[0250] The polymer-coated surface prepared by the thermal cross-linking method is also active for cluster growth and sequencing using standard methods as described herein. FIG. 13A shows clusters grown on a thermally crosslinked surface with Phix V3 as a model, imaged on a Hi-SEQ (Ilumina). FIG. 13B illustrates sequencing metrics obtained from a 2 x 26 cycle run on a Hi-Seq with conventional sequencing chemistry. Example 8 Application of Polymer Coating in Patterning

[0251] Furthermore, to coat regular flow cells, the generation of a photoactive layer can also be used to form a patterned surface. Microfabrication techniques can generally be used to create “islands” of amino functionality in a “sea” of inert or passive material.

[0252] An aryl azide, such as sulfo-HSAB, can be located on the amino nucleus to create a patterned photoactive layer. Coating this surface with a polymer blend such as PAZAM followed by exposure to UV rays and subsequent washing leaves a patterned polymer matrix. This process is demonstrated in FIG. 11 A.

[0253] The background region is free of any photocoupled polymer because the crosslinker is located only in the patch. The radical insertion reaction is confined to a monolayer on the surface minimizing uncontrolled radical propagation. Aryl azides functionalized with silane and phosphate units can also be readily produced from commercially available starting materials, allowing the anchoring of a wide variety of patch types (see FIG. 11A). Preferential polymer deposition on patches with functionalized features was observed using a slide with plain glass features and a gold interstitial region (FIG. 11B). The right side shows a fluorescence image of Mantéia and the left side shows a Typhoon image of the slide, with the darker areas indicating an increased polymer coating.

[0254] Likewise, the thermal cross-linking method also allows the formation of patterned functional polymer patches when the underlying functional layer is patterned. For example, standardized amino silane patches can be prepared using common lithographic techniques. Typically, this involves coating a substrate with a resist, then exposing the resist development to leave simple patches on the substrate. The single patches are then functionalized with the silane, followed by removing most of the resistance and leaving behind small patches of amino-functionalized substrate with an interstitial of large amounts of the non-functional substrate. These patches can then be functionalized with the unsaturated alkene groups as shown in Scheme 5 and then incubated with PAZAM. The resulting surface is composed of small patches of PAZAM in a predefined grid, which can then support cluster growth in a defined patterned matrix. In FIG. 14, a surface made as described above is shown to support bridging amplification selectively in the patterned regions. The features are 450 nm in diameter, with a spacing of 1.4 μm. Clusters were grown using an Illumina V3 PE cBotT kit with 1pM human DNA template. The surface was imaged with Sybrgree after amplification.

[0255] Other methods for making patterned surfaces that can be used include, for example, those described in US document Nos. by grade. 13/492,661 and 13/661,524, each of which is incorporated herein by reference. Example 9 PAZAM coated granules

[0256] Various methods can be used to apply a polymer coating to the granules. One approach described here includes the use of a UV-activated surface for blocking the polymer. An alternative approach described in Example 11 includes the use of a surface functionalized with alkene or acrylamide by thermally cross-linking the polymer. In the thermal-based approach, the bead is generally exposed to washing solutions containing a polymerization inhibitor (e.g., BHT, diethylhydroxylamine or TEMPO, etc.) after functionalization with alkene or acrylamide groups. This can be done to prevent the granules from aggregating due to premature polymerization of the alkene or acrylamide groups.

[0257] After coating PAZAM on the granules, the granules were washed to remove excess unbound PAZAM. The PAZAM layer was about 20 nm thick after washing. The beads were then grafted onto the primers (P5/P7 mixtures) by reacting the azide groups in PAZAM with a 5' alkyne-modified oligonucleotide. Alternative grafting chemistry can also be used, which includes, but is not limited to: reacting the amine groups on the polymer with 5'-NHS-modified oligonucleotides; reacting the oxo-amine groups in the polymer with 5'-aldehyde-modified oligonucleotides to form oxime; reacting the thiol groups in the polymer with 5'-maleimide-modified oligonucleotides; perform cross-olefin metathesis (reaction of terminal alkene groups on both the polymer and 5' modified oligonucleotides in the presence of a metathesis catalyst); reacting the amine groups in the polymer with oligonucleotides modified by 5'-cyanuric chloride; etc. Excess primers not grafted onto PAZAM on the bead can then be washed away, leaving the beads with a layer of oligo-PAZAM on them.

[0258] Various methods can be used to perform quality control on polymer-coated granules prior to application to the substrate surface. One method is to directly stain PAZAM with dyes (e.g. Dylight488 Phosphine). Another method is to label the grafted oligonucleotides with dye-labeled reverse complements of the oligonucleotides on PAZAM. Labeled granules can be measured by flow cytometry or cast onto a surface and then analyzed by light microscopy (including fluorescent light microscopy). Additionally, an aliquot of the beads can be used in a destructive test to evaluate how the beads will perform in an assay. An example is loading an aliquot onto a substrate, performing sequencing, and determining whether the majority of the granules are of good enough grain to be used in loading many matrices.

[0259] Different types of corrosible substrates can be used to load the granules, for example, silicon, plastic (Zeonor®) and plastic impregnated with additives (for example, SiO2 or carbon black). FIG. 15 illustrates the patterned PAZAM matrices prepared by loading PAZAM-coated beads onto clear Zeonor slides.

[0260] There are several advantages of using polymer-coated granules for preparing patterned matrices. First, the bead approach is cost-effective and time-saving. The bead loading approach allows the preparation of a matrix surface with high density compared to conventional flow cell coating and grafting methods. An exemplary convention approach uses a device that supplies different reactants to the flow cell and allows the temperature of the flow cell to be controlled, which results in only a fraction of the pumped initiators being grafted, resulting in considerable loss. Furthermore, the granule loading procedure described in this document is much faster than the conventional process and allows for large-scale commercial applications. Example 10 Photoactive polymers

[0261] Described here is the use of alternative water-soluble polymers that are capable of supporting bridge amplification and subsequent sequencing by chemical synthesis (SBS). Additionally, the polymer can be easily applied to surfaces with a variety of different techniques. In particular, the development of a linear polymer that can be fully characterized before being applied to the surface is beneficial in order to limit potential variation in surface coating quality. Furthermore, the approach described herein includes the ability to use alternative coating strategies, including dip, spray, and polymer spin coating.

[0262] Scheme 6 presents the preparation of a linear polymer that can be prepared using commercially available starting materials. The polymer can be used to coat, in a controllable form, glass and silicon wafers, allowing full characterization for both the polymer solution and the coated wafer prior to fabrication in flow cells. This method also allows one or both surfaces of the flow cells to be functionalized as appropriate. Scheme 6. Synthesis of a Photoactive Linear Polymer

[0263] Polymerization can be conducted on a large scale using traditional solution phase techniques. Acrylamide and a new monomer 6c (synthesized via a one-step procedure), can be polymerized using free radical polymerization analogous to the current procedure for preparing PAZAM.

[0264] One modification involves the use of perfluoroaryl azides (keana, JFW; Cai, SX New Reagents for Photoaffinity Labeling: Synthesia and Photolysis of Functionalized Perfluorophenyl Azides. J. Org. Chem. 1990, 55, 3640-3647), the disclosure of which is incorporated herein by reference in its entirety. These species have a reduced tendency to undergo ring expansion upon nitrene generation. As a result, the UV-triggered reaction is cleaned with the exclusive formation of insertion products. Scheme 7. Synthesis of an Aryl Azide Perfluoro Monomer to Prepare a Photoactive Polymer

[0265] The use of aryl azides as labeling agents was reported as early as 1969. (Fleet, G.W.J.; Porter, R.R.; Knowles, J.R. Affinity Labeling od Antibodies with Aryl Nitrene as Reactive Group 1969, 224, 511-512), the disclosure of which is incorporated herein by reference in its entirety. In the present application, the dual reactivity of these functional groups is exploited; as a partner in the 1,3-dipolar cycloaddition (for grafting of the oligos) and a photocoupling agent following UV-triggered generation of a singlet nitrene species. The advantage of using a UV-active monomer is that the reaction is “clean” (insertion products only), chemoselective and localized with the polymer environment providing a large number of bonds that can be inserted.

[0266] This approach eliminates the need to perform any post-deposition functionalization steps. Once coated with the pre-grafted polymer, the wafer can be cut, a coverslip attached and a QC step performed in order to determine/confirm the number of surface initiators available.

[0267] The purified batch is then divided with a portion (about ~80%) grafted into solution, using, for example, copper-catalyzed Huisgen 1,3-cycloaddition (FIG 16: lane 2). The grafted polymer can be recombined with the unreacted material to provide a polymer mixture that contains both paired terminal initiators as well as free photoactive groups capable of blocking both the wafer surface and adjacent polymer chains (see FIG. 16).

[0268] This photocoupling approach improves the robustness of the polymer to support the cluster growth and sequencing steps. Previous work has shown that dynamic silicon-free acrylamide mixtures are capable of supporting SBS but exhibit weak undesirable “creases” visible after cluster formation and become more pronounced during sequencing. Use of the photocoupling approach disclosed herein can improve anchoring of the polymer to the wafer, while decreasing excessive flexibility and maintaining access to the grafted initiators. Example 11 Coating Granules with PAZAM Using Thermal Crosslinking

[0269] Silica granules were obtained as a 10% solids suspension from Bang'sLabs of Fishers, IN (PN SS04N/9348, but note that many other granule sizes and compositions can be used in a similar manner ). A 1 ml aliquot (100 mg) was transferred to a 1.5 ml eppendorf tube. The tube was centrifuged in a benchtop centrifuge (5000 rfu) for 30 sec to spin the beads. The solution was removed by aspiration. Next, 1 ml of acetonitrile (Aldrich PN 34967) was added to the tube, the tube was vortexed for 30 seconds to homogenize the suspension, and then centrifuged again. This process was repeated 5 times to change the solvent. Finally, the last solvent was removed by aspiration (after centrifugation to spin the beads).

[0270] In a separate 15 ml tube, 5 ml of acetonitrile and 100 μL of 3-acrylamidopropyltrimethoxy-silane (Gelest PN SIA0146.0) were added. This solution was vortexed for 15 seconds to mix the solution. Next, a polymerization inhibitor was added to the solution to prevent premature polymerization: 2 μL of N,N-diethylhydroxylamine (Aldrich PN 471593) was added to the 2 ml of silane solution to produce an inhibitor concentration at 1000 ppm. . Then, 1 ml of the silane solution with an inhibitor solution was added to the beads. The tube was vortexed for 30 seconds to homogenize the bead suspension in the silane solution. The granules were then allowed to react with silane for 30 minutes at room temperature in a rotisserie mixer. The tube of beads was then centrifuged to spin the beads and the solution was removed by aspiration. The beads were then solvent exchanged into acetonitrile containing 1000 ppm polymerization inhibitor N,N-diethylhydroxylamine in a similar manner to that described above.

[0271] After silanization, the granules were solvent exchanged in Ethanol containing 1000 ppm of polymerization inhibitor (through 5 successive centrifuges, aspiration and new additions of solvents). Then, the granules were solvent exchanged in acetone containing 1000 ppm of polymerization inhibitor. Finally, the residual acetone was removed by aspiration (after centrifuging the granules), the tube of granules was capped with a kimwipe fixed by a rubber band to ensure that dry granules would not escape into the evacuation chamber. The granules were then placed in a vacuum oven preheated to 40°C and subjected to a vacuum chamber (approx. 27 Torr) for 1 h. The granules were then removed from the vacuum oven and 1 ml of HPLC grade water containing 1000 ppm of the polymerization inhibitor was added. The bead suspension was then homogenized by vortexing for 30 seconds.

[0272] In a separate 1.5 ml eppendorf tube, 10 ul of the aqueous suspension of granules from the previous step were added. Next, 1 ml of 1.0 wt% solution of PAZAM polymer in HPLC grade water was then added to the beads. The solution was homogenized by vortexing for 20 seconds on a bench shaker, then centrifuged at 7000 rfu for 90 seconds to obtain the granules. The PAZAM polymer solution was then removed by aspiration. Then, 1 ml of a 2.0 wt % PAZAM solution was added to the beads and the solution was homogenized by vortexing on a bench shaker for 20 seconds. Then, PAZAM was thermally grafted to the silane by reacting the tube at 60°C for 1 h. After thermal grafting of PAZAM onto the beads, the beads were washed with 5x1ml HPLC-grade water (using the solvent exchange method described above) at room temperature and then 3x1ml of pre-treated HPLC-grade water. heated (approximately 40°C). The beads were diluted in 100 μL of HPLC-grade water to produce a 10 wt% solids suspension.

[0273] Oligonucleotides P% and P7 with 5' alkynyl functional groups were synthesized separately using standard oligonucleotide synthesis procedures (or, alternatively, obtained commercially from oligonucleotide synthesis suppliers). The oligonucleotide solution was supplied as a 100 μM solution in HPLC water.

[0274] The grafting of the alkynyl-functionalized oligonucleotides to the azido-functionalized PAZAM was done as follows. To the 100 μL aliquot of 10 wt% solids PAZAM-grafted bead solution, 800 μL of 1xPBS buffer at pH 7.4 and 100 μL of the oligonucleotide solution were added. This solution was sparged with nitrogen gas for 10 minutes at a gentle nitrogen bubbling flow rate. Then, 2 μl of pure PMDETA (Aldrich PN 369497) was added. Next, 17 μl of a 160 mM aqueous copper sulfate solution (Aldrich PN C2284) was then added. The solution turned purple as it was vortexed for 20 seconds. Finally, 25 μl of 40 mg/ml sodium ascorbate solution (Aldrich A7631 PN) in HPLC water was added and the solution turned blue after vortex mixing for 20 seconds. The solution was allowed to react at 60°C for 1 h. Then, the beads were centrifuged and the reaction solution was removed by aspiration. The beads were washed in 1lxPBS buffer (pH 7.4) in a similar manner as described previously (solvent exchange using 5x1ml buffer washes).

[0275] The oligo-functionalized beads were then loaded onto an empty microarray slide that was functionalized with 1.2 micron holes. Slides were prepared as described in US 6,770,441. The beads were loaded as follows: the wells containing the slides were pre-cleaned by immersion in 0.1N NaOH for 5 min at room temperature, then vigorously washing with HPLC-grade water flow (about 100 ml) and dried under a stream of nitrogen. Then, 10 μl of 200 proof Ethanol (Aldrich PN E7023) was added to 100 μl of bead solution. The bead solution was then applied to the microscope slide via a pipette. Then, the slide was placed in a vacuum oven preheated to 40°C and the slides were allowed to evaporate for 30 min. Residual granules were removed from the surface of the microscope slide by gentle manual application of a kimwipe saturated with ethanol.

[0276] To a slide prepared in this way, a solution of 10 μM of 5' dye-labeled oligonucleotide (preferably Cy5) in 5 x SSC buffer (Aldrich PN S6639) was added with the reverse complement to the oligo that was immobilized to the bead. grafted with PAZAM. The slide was then covered with a glass coverslip. The slide was left to hybridize at room temperature for 30 minutes. Then the coverslip was removed and the slide was washed with copious amounts of flowing 5x SSC buffer (about 100 ml). Finally, 100 μl of 5xSSC buffer and then a coverslip were added to the slide and the slide was imaged on a GE Typhoon FLA 9500, scanning in the Cy5 dye channel adjusting with a setting of 500 PMT.

[0277] To another slide prepared in this way a rubber gasket was added and another microscope slide was pre-drilled with the appropriate location of holes in the glass to allow the introduction of liquid through an Ilumina HiSeq sequencing instrument. The DNA clusters were then grown on slides and the clusters were sequenced according to the manufacturer's instructions for the HiSeq 2000 (Illumina Inc., San Diego, CA). This slide has been shown to be capable of sequencing up to 150 bp of sample DNA per cluster.

Claims (15)

1. Substrate, having a surface comprising a polymer coating covalently bonded thereto, characterized in that the polymer coating comprises a recurring unit of Formula (I) 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 azide; 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 bonded to form a heterocycle; each of -(CH2)-o is optionally substituted; and o is an integer between 1-50. 2. Substrate according to Claim 1, characterized in that R1 is H or alkyl. 3. Substrate according to Claim 1, characterized in that the polymer coating comprises a polymer of Formula (III): wherein R1 is H or alkyl; R5 is H or alkyl; n is an integer in the range 1 to 50,000; em is an integer in the range 1 to 100,000. 4. Substrate according to Claim 3, characterized in that the polymer of Formula (III) is also represented by Formula (IIIa): m (Illa) where n is an integer in the range 1-20,000 and m is an integer in the range 1-100,000. 5. Substrate according to any one of Claims 1 to 4, characterized in that the polymer coating is covalently linked to a series of functional groups linked to said surface, wherein the functional groups are selected from the group consisting in alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether, amine, maleimide and benzoyl ester with a phosphine substituent in the ortho position for Staudinger bond. 6. Substrate according to any one of Claims 1 to 5, characterized in that the surface comprises both polymer-coated regions and inert regions; optionally, the inert regions are selected from the group consisting of glass regions, metal regions, mask regions and interstitial regions; and, optionally, the inert regions comprise glass. 7. Substrate according to any one of Claims 1 to 6, characterized in that the surface of the substrate comprises open wells. 8. Substrate according to any one of Claims 1 to 6, characterized in that the substrate is a granule. 9. Method for Preparing Immobilized Polymer Coating on a Substrate Surface, characterized in that said method comprises: contacting a polymer coating as defined in any one of Claims 1 to 4, with a surface of a substrate, wherein the surface comprises a plurality of functional groups, thereby forming a polymer coating layer on the surface and wherein the polymer coating is covalently linked to the functional groups on the surface. 10. Method for preparing polymer coating immobilized on a substrate surface, according to Claim 9, characterized in that the functional groups on the surface of the substrate comprise photoactivatable azides, optionally, the phenyl azide is prepared by reacting an amine group on the substrate surface with N-hydroxysulfosuccinimidyl-4-azidabenzoate (sulfo-HSAB) and, optionally, the polymer is covalently linked to the surface functional groups through photoactivation. 11. Method for preparing polymer coating immobilized on a substrate surface, according to Claim 9, characterized in that the functional groups on the surface of the substrate comprise alkyne, alkene or acrylamide groups, optionally, the acrylamide groups are prepared by making react functionalized surface amine with acryloyl groups selected from activated acrylic esters or acrylic chlorides and, optionally, the polymer is covalently linked to the surface functional groups via thermal activation or in the presence of a copper catalyst. 12. Method for preparing substrate surface-immobilized polymer coating according to any one of claims 9 to 11, characterized in that the plurality of functional groups are arranged on the surface of the substrate so as to form a plurality of coated regions of polymer and a plurality of inert regions, after the polymer layer is coated onto the surface. 13. Method for Preparing Granule Matrix, characterized in that it comprises: forming a plurality of functional groups on the surface of one or more granules; and contacting a polymer coating as defined in any one of Claims 1 to 4, with the granules so as to form a polymer coating layer on the surface of the granules, wherein the polymer coating is covalently bonded to the functional groups on the the surface of the granules. 14. Method for preparing granule matrix, according to Claim 13, characterized in that the functional groups on the surface of the granules comprise alkenes or acrylamides, optionally, the alkene or acrylamide groups are prepared by directly contacting the surface of the granules with silane functionalized and, optionally, the polymer is covalently linked to functional groups on the surface of the beads at an elevated temperature. 15. Method for preparing granule matrix according to Claim 13, characterized in that the functional groups on the surface of the granules comprise optionally substituted phenyl azide groups, optionally, the phenyl azide is prepared by reacting an amine group on the surface of the granules with N-hydroxysulfosuccinimidyl-4-azidabenzoate (sulfo-HSAB) and, optionally, the polymer is covalently linked to the functional groups on the surface of the granules through photoactivation.