阅读说明：本技术 具有稳定的聚合物涂层的流动池及其用于基因测序的用途 (Flow cell with stable polymer coating and its use for gene sequencing ) 是由 方晔 J·G·林 孙伟 魏莹 于 2019-08-05 设计创作，主要内容包括：提供了流动池制品,其中,所述流动池制品包括：具有一个或多个层的基材；设置在基材中的流体通道,其中,所述流体通道包括至少一个反应性表面,所述反应性表面包括：偶联剂,所述偶联剂具有共价附接于流体通道的基材的第一官能团和共价附接于式(I)的聚合物的第二酰亚胺官能团,其中,R-1是已经与马来酐共聚的不饱和单体的残基；R-2是H、烷基、低聚(乙二醇)和/或二烷基胺；m、n和o各自为1至10,000；X是二价NH、O和/或S；并且Z是第一官能团。(A flow cell article is provided, wherein the flow cell article comprises: a substrate having one or more layers; a fluid channel disposed in a substrate, wherein the fluid channel comprises at least one reactive surface comprising: a coupling agent having a first functional group covalently attached to a substrate of a fluid channel and a second imide functional group covalently attached to a polymer of formula (I), wherein R 1 Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r 2 Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; m, n and o are each 1 to 10,000; x is divalent NH, O and/or S; and Z is a first functional group.)

1. A flow cell article, comprising:

a substrate comprising one or more layers;

a fluidic channel disposed in or on a substrate and comprising at least one reactive surface comprising:

a coupling agent comprising a first functional group covalently attached to a substrate and a second functional group covalently attached to a polymer of formula (I) using at least one imide linkage:

wherein:

R₁is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride;

R₂is H, alkyl, oligo (ethylene glycol) and/or dialkylamine;

m, n and o are each 1 to 10,000;

x is divalent NH, O and/or S; and is

Z is a first functional group.

2. The flow cell article of claim 1, wherein the substrate comprises glass, glass-ceramic, silicon, fused silica, quartz, thermoplastic, or thermoset.

3. The flow cell article of claim 1 or 2, wherein the coupling agent comprises an aminosilane and/or an aminoorganophosphate.

4. The flow cell article of claim 3, wherein the aminosilane is selected from the group consisting of: 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3- (2-aminoethyl) -aminopropyltrimethoxysilane, aminopropylmethyldialkoxysilane, 4-aminobutyltriethoxysilane, 4-amino-3, 3-dimethylbutyltrimethoxysilane and N- (6-aminohexyl) aminomethyltriethoxysilane.

5. The flow cell article of claim 3 wherein the aminoorganophosphate is selected from the group consisting of: 3-aminopropyl dihydrogen phosphate, 4-aminophenyl phosphate, 2-aminoethyl dihydrogen phosphate, and 2- (3-aminopropyl) aminoethyl phosphorothioate.

6. The flow cell system of any of claims 1-5, wherein the relative ratio of m to n (m: n) is from about 0.5 to about 10.

7. The flow cell article of any one of claims 1-6, further comprising a nucleic acid primer molecule covalently attached to a polymer.

8. The flow cell article of claim 7, wherein the nucleic acid primer molecule comprises an amine-terminated nucleic acid, or a mixture of amine-terminated nucleic acids.

9. The flow cell article of any one of claims 7 and 8, wherein the density of nucleic acid primer molecules is from 1 to 500,000 probe molecules per square micron of surface area.

10. A flow cell system, comprising:

a substrate comprising one or more layers;

a fluidic channel disposed in or on a substrate and comprising at least one reactive surface comprising:

a coupling agent comprising a first functional group covalently attached to a substrate of the fluid channel and a second functional group located away from the substrate;

a polymer of formula (II) covalently attached to a second functional group of the coupling agent using at least one imide linkage:

wherein:

R₁is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride;

R₂is H, alkyl, oligo (ethylene glycol) and/or dialkylamine;

n and o are each an integer of 1 to 10,000; and is

X is divalent NH, O and/or S; and

a nucleic acid primer molecule covalently attached to a polymer.

11. The flow cell system of claim 10, wherein the nucleic acid primer molecule comprises an amine-terminated nucleic acid, or a mixture of amine-terminated nucleic acids.

12. The flow cell system of any of claims 10 and 11, wherein the density of nucleic acid primer molecules is from 1 to 500,000 probe molecules per square micron of surface area.

13. The flow cell system of any of claims 10-12, wherein the substrate comprises glass, glass-ceramic, silicon, fused silica, quartz, thermoplastic, or thermoset.

14. The flow cell system of any of claims 10-13, wherein the coupling agent comprises an aminosilane and/or an aminoorganophosphate.

15. The flow cell system of claim 14, wherein the aminosilane is selected from the group consisting of: 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3- (2-aminoethyl) -aminopropyltrimethoxysilane, aminopropylmethyldialkoxysilane, 4-aminobutyltriethoxysilane, 4-amino-3, 3-dimethylbutyltrimethoxysilane and N- (6-aminohexyl) aminomethyltriethoxysilane.

16. The flow cell system of claim 14, wherein the amino organophosphate is selected from the group consisting of: 3-aminopropyl dihydrogen phosphate, 4-aminophenyl phosphate, 2-aminoethyl dihydrogen phosphate, and 2- (3-aminopropyl) aminoethyl phosphorothioate.

17. The flow cell system of any of claims 10-16, wherein the relative ratio of m to n (m: n) is from about 0.5 to about 10.

18. A method of making a flow cell article, the method comprising:

contacting a fluid channel disposed in or on a substrate with a coupling agent to covalently attach a first functional group to the fluid channel;

contacting the polymer of formula (II) with a coupling agent to covalently attach the polymer to a second functional group, thereby forming an imide linkage on the tethered polymer;

wherein:

R₁is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride;

R₂is H, alkyl, oligo (ethylene glycol) and/or dialkylamine;

n and o are each an integer of 1 to 10,000; and is

X is divalent NH, O and/or S; and

contacting the nucleic acid primer molecule with the tethered polymer to covalently attach the nucleic acid primer molecule to the tethered polymer.

19. The method of claim 18, wherein the nucleic acid primer molecule comprises an amine-terminated nucleic acid, or a mixture of amine-terminated nucleic acids.

20. The method of any one of claims 18 and 19, wherein the density of nucleic acid primer molecules is 1 to 500,000 probe molecules per square micron of surface area.

21. The method of any of claims 18-20, wherein the substrate comprises glass, glass-ceramic, silicon, fused silica, quartz, a thermoplastic material, or a thermoset material.

22. The method of any of claims 18-21, wherein the coupling agent comprises an aminosilane and/or an aminoorganophosphate.

23. The method of claim 22, wherein the aminosilane is selected from the group consisting of: 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3- (2-aminoethyl) -aminopropyltrimethoxysilane, aminopropylmethyldialkoxysilane, 4-aminobutyltriethoxysilane, 4-amino-3, 3-dimethylbutyltrimethoxysilane and N- (6-aminohexyl) aminomethyltriethoxysilane.

24. The method of claim 22, wherein the amino organophosphate is selected from the group consisting of: 3-aminopropyl dihydrogen phosphate, 4-aminophenyl phosphate, 2-aminoethyl dihydrogen phosphate, and 2- (3-aminopropyl) aminoethyl phosphorothioate.

25. A method for sequencing a nucleic acid, the method comprising:

providing a flow cell article comprising:

a substrate comprising one or more layers;

a fluidic channel disposed in or on a substrate and comprising at least one reactive surface comprising a coupling agent comprising a first functional group covalently attached to the substrate and a second functional group covalently attached to a polymer of formula (I) using imide bonds:

wherein:

R₁is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride;

R₂is H, alkyl, oligo (ethylene glycol) and/or dialkylamine;

m, n and o are each 1 to 10,000;

x is divalent NH, O and/or S; and

z is a second functional group, and Z is a second functional group,

contacting a nucleic acid primer molecule with a polymer of formula (I) to covalently attach the nucleic acid primer molecule to the polymer of formula (I);

capturing DNA fragments using nucleic acid primer molecules, wherein each DNA fragment comprises a sequence complementary to a nucleic acid primer molecule; and

adding nucleotides to the ends of the nucleic acid primer molecules to synthesize complementary DNA sequences for each captured DNA fragment, wherein the complementary DNA sequences are covalently linked to the polymer of formula (I) through the nucleic acid primer molecules.

26. The method of claim 25, wherein the nucleic acid probe comprises an amine-terminated nucleic acid, or a mixture of amine-terminated nucleic acids.

27. The method of any one of claims 25 and 26, wherein the density of nucleic acid probe molecules is from 1 to 500,000 probe molecules per square micron of surface area.

28. The method of any one of claims 25-27, wherein the substrate comprises glass, glass-ceramic, silicon, fused silica, quartz, thermoplastic, or thermoset.

29. The method of any of claims 25-28, wherein the coupling agent comprises an amine-containing silane and/or an amine-containing organophosphate ester.

30. The method of claim 29, wherein the amine-containing silane is selected from the group consisting of: 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3- (2-aminoethyl) -aminopropyltrimethoxysilane, aminopropylmethyldialkoxysilane, 4-aminobutyltriethoxysilane, 4-amino-3, 3-dimethylbutyltrimethoxysilane and N- (6-aminohexyl) aminomethyltriethoxysilane.

31. The method of claim 29, wherein the amine-containing organophosphate is selected from the group consisting of: 3-aminopropyl dihydrogen phosphate, 4-aminophenyl phosphate, 2-aminoethyl dihydrogen phosphate, and 2- (3-aminopropyl) aminoethyl phosphorothioate.

32. The method of any one of claims 25-31, further comprising:

denaturing the complementary DNA sequences from the DNA fragments; and

complementary DNA sequences and/or DNA fragments are collected from the flow cell preparation.

33. The method of any one of claims 25-31, further comprising:

denaturing the complementary DNA sequences from the DNA fragments;

collecting DNA fragments from the flow cell preparation;

synthesizing a target DNA fragment using a complementary DNA sequence covalently linked to a polymer of formula (I) by a nucleic acid primer molecule; and

target DNA fragments are collected from the flow cell preparation.

Technical Field

The present disclosure relates to microfluidic flow cell devices, and methods of making and using microfluidic devices for biomolecule analysis, particularly gene sequencing.

Background

Various biomolecule analysis techniques, such as DNA microarrays, Next Generation Sequencing (NGS), or DNA-based biosensors employ synthetic DNA probe molecules as the most critical detection elements. These DNA probe molecules often need to be covalently attached to a solid support, including flat two-dimensional surfaces and three-dimensional surfaces, e.g., microbeads and micro/nanoparticles. Covalent attachment of these DNA probe molecules to the surface can be achieved using a variety of different methods. Regardless of the method used, these gene sequencing techniques are accompanied by many sequencing cycles, each of which involves multiple harsh washing steps, which in turn often results in loss of DNA molecules over time, thus limiting the length of reads to be achieved.

NGS techniques based on optical detection often utilize high density DNA primers (also known as primer lawn) to capture DNA fragments from biological samples, all of which have adaptor sequences complementary to the DNA primer sequences. Once the DNA fragments are captured, all DNA fragments are amplified locally (locally) to form the desired monoclonal cluster. After clustering, the sequence of the DNA fragments in each cluster is determined by synthesis consisting of several hundred cycles, each cycle consisting of nucleotide addition using DNA polymerase, followed by washing, imaging, terminator cleavage and washing. Thus, DNA sequencing by synthesis faces a drastic multi-step process in a microfluidic device (e.g., a flow cell), such that attaching DNA to a reaction substrate in a microfluidic device can be problematic.

Accordingly, there is a need for improved techniques and corresponding chemistries for more efficiently linking DNA fragments to a variety of different substrates used in microfluidic flow cell devices that can withstand cycling conditions used in sequencing-by-synthesis of DNA strands. The development of emerging chemistry that can provide more stable linkages between DNA fragments and the substrate of a flow cell device can translate into improved reliability, improved accuracy, and improved manufacturing costs that result in longer DNA sequences.

Disclosure of Invention

According to some aspects of the present disclosure, a flow cell article comprises: a substrate having one or more layers; a fluid channel disposed in a substrate, wherein the fluid channel comprises at least one reactive surface having: a coupling agent having a first functional group covalently attached to a substrate of a fluid channel and a second imide functional group covalently attached to a polymer of formula (I):

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; m, n and o are each 1 to 10,000; x is divalent NH, O and/or S; and Z isA first functional group.

According to some aspects of the present disclosure, a flow cell system comprises: a substrate having one or more layers; a fluid channel disposed in a substrate, wherein the fluid channel comprises at least one reactive surface having: a coupling agent having a first functional group covalently attached to a substrate of the fluid channel and a second functional group located away from the substrate; a polymer of formula (II) to be covalently attached to a second functional group of a coupling agent:

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; n and o are each an integer of 1 to 10,000; and X is divalent NH, O and/or S; a nucleic acid primer molecule covalently attached to a polymer.

According to other aspects of the present disclosure, methods of making a flow cell article are provided. The method comprises the following steps: contacting a fluidic channel disposed in a substrate with a coupling agent to covalently attach a first functional group to the fluidic channel; contacting the polymer of formula (II) with a coupling agent to covalently attach the polymer to a second functional group, thereby forming an imide linkage on the tethered polymer;

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; n and o are each an integer of 1 to 10,000; and X is divalent NH, O and/or S; contacting the nucleic acid primer molecule with the tethered polymer to covalently attach the nucleic acid primer molecule to the tethered polymer.

According to other aspects of the present disclosure, methods for sequencing nucleic acids are provided. The method comprises the following steps: providing a flow cell article having: a substrate comprising one or more layers; a fluid channel disposed in a substrate, wherein the fluid channel comprises at least one reactive surface comprising a coupling agent having a first functional group covalently attached to the substrate of the fluid channel and a second imide functional group covalently attached to a polymer of formula (I):

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; m, n and o are each 1 to 10,000; x is divalent NH, O and/or S; and Z is a second functional group; contacting a nucleotide primer molecule with a polymer of formula (I) to covalently attach the nucleotide primer molecule to the polymer of formula (I); capturing DNA fragments using the nucleic acid primer molecules, wherein each DNA fragment comprises a sequence complementary to the nucleic acid primer molecule; and adding nucleotides to the ends of the nucleic acid primer molecules to synthesize complementary DNA sequences for each captured DNA fragment, wherein the complementary DNA sequences are covalently linked to the polymer of formula (I) through the nucleic acid primer molecules.

Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the various embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide an overview or framework for understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments and, together with the detailed description, serve to explain the principles and operations of the embodiments.

Drawings

The following is a description of the figures in the drawings. The figures are not necessarily to scale and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

FIG. 1 is a schematic illustration of a flow cell preparation covalently linked to a DNA probe molecule according to some aspects of the present disclosure;

FIG. 2 is a schematic representation of a polymeric coating covalently bound to a coupling agent attached to a substrate forming a fluid channel, according to some aspects of the present disclosure;

FIG. 3 is a schematic representation of a polymer coating covalently bonded to a coupling agent attached to a metal oxide layer coated on a substrate forming a fluid channel, according to some aspects of the present disclosure;

4A-4C are scanning electron microscope images of a surface of a fluid channel having a polymer coating, according to some aspects of the present disclosure;

fig. 5A and 5B show a photographic view (5A) and a fluorescence view (5B), respectively, of a flow cell article having 8 channels, each channel consisting of an amine-terminated dA30 attached to a polymer coating, after hybridization of Cy3 dye-labeled dT30, according to some aspects of the present disclosure; and

figure 6 is a graph showing comparative differences in sequencing read lengths for amide and imide linkages used to covalently bind a coupling agent to a polymer coating for attaching DNA.

Detailed Description

Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described in the following description, taken in conjunction with the claims and the appended drawings.

As used herein, the term "and/or," when used in a list of two or more items, means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition is described as containing components A, B and/or C, the composition may contain only a; only contains B; only contains C; a combination comprising A and B; a combination comprising A and C; a combination comprising B and C; or a combination comprising A, B and C.

In this document, relative terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.

Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is to be understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and are not intended to limit the scope of the present disclosure, which is defined by the following claims as interpreted according to the principles of patent law, including the doctrine of equivalents.

For the purposes of this disclosure, the term "connected" (in all forms: connected, and the like) generally means that two elements are joined to each other either directly or indirectly. Such a combination may be stationary in nature or movable in nature. Such joining may be achieved by the two components being integrally formed as a single unitary body with one another, with any additional intermediate members, or by both components. Unless otherwise specified, such joining may be permanent in nature, or may be removable or releasable in nature.

As used herein, the term "about" means that quantities, dimensions, formulas, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller as desired, such as to reflect tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. When the term "about" is used to describe a value or an endpoint of a range, it is to be understood that the disclosure includes the particular value or endpoint referenced. Whether or not the numerical values or endpoints of ranges in the specification are listed as "about," the numerical values or endpoints of ranges are intended to include both embodiments: one modified with "about" and the other not modified with "about". It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

As used herein, the terms "substantially", "essentially" and variations thereof are intended to mean that the recited feature is equal or approximately equal to a numerical value or description. For example, a "substantially planar" surface is intended to mean that the surface is a planar or substantially planar surface. Further, "substantially" is intended to mean that two numerical values are equal or approximately equal. In some embodiments, "substantially" may mean values within about 10% of each other, such as values within about 5% of each other, or values within about 2% of each other.

Directional terminology used herein, such as upper, lower, right, left, front, rear, top, bottom, is for reference only to the accompanying drawings and is not intended to be absolute.

The terms "the", "a" or "an" as used herein mean "at least one" and should not be limited to "only one" unless explicitly stated to the contrary. Thus, for example, reference to "a component" includes embodiments having two or more such components, unless the context clearly indicates otherwise.

The term "next generation sequencing" or "NGS" as used herein is defined to include a type of DNA sequencing technology that utilizes parallel sequencing of many small fragments of DNA from a biological sample to determine gene sequence. NGS can be used to sequence every nucleotide in a genome, or a small portion of a genome (e.g., an exon or a preselected subset of genes).

Over the past few decades, remarkable progress has been made in classifying human genetic variations and correlating these variations with disease susceptibility, responsiveness to specific therapies, susceptibility to dangerous drug side effects and other medically operable characteristics. Advances in NGS have reduced the cost per megabase and increased the number and diversity of genomes sequenced. The key to the progress of whole genome sequencing is the use of flow cells to distribute millions of DNA fragments generated from a biological DNA sample onto the surface of the flow cell so that nearly all immobilized fragments can be sequenced simultaneously. Some NGS techniques, particularly short read sequencing techniques, involve covalently immobilizing DNA fragments onto the surface of a flow cell for sequencing. For sequencing efficiency and quality, it is important to achieve the ability to stably, reproducibly, and optimally attach DNA molecules to the flow cell surface.

Embodiments of the present disclosure relate generally to nucleic acid analysis and, more particularly, to methods of making and using microfluidic flow cell devices for, e.g., large-scale parallel genomic analysis (e.g., next generation sequencing, NGS). Many important molecular applications, such as DNA microarrays, NGS or DNA-based biosensors, can use synthetic DNA probe molecules attached to solid supports comprising flat two-dimensional surfaces, such as glass, silica or silicon slides, and to three-dimensional surfaces, such as microbeads and microparticles/nanoparticles. Immobilization of DNA probe molecules on a microfluidic surface can generally be achieved using a variety of methods, e.g., electrostatic interaction, covalent coupling, embedding, etc. One technique for covalently attaching DNA probes to surfaces involves the use of bifunctional linker molecules that link the DNA probe molecules to the surface in the presence of amines using amide bonds. For example, many gene sequencing methods based on optical detection have in common the use of a surface containing a high density of oligonucleotide primers or probes, to which DNA primers are covalently attached. However, covalent attachment based on amide bonds is generally accompanied by relatively low stability relative to vigorous sequencing cycles, and therefore only short read lengths can be achieved.

The present disclosure provides materials and methods for covalently attaching amine-terminated DNA primer molecules to solid supports for nucleic acid analysis, particularly gene sequencing. The present disclosure also provides reactive surfaces for covalently capturing amine-terminated DNA probe fragments. Fragment density and position can be precisely controlled so that the synthesis and growth of polyclonal clusters can be spatially controlled, and sequencing is efficient with improved quality and accuracy.

According to various aspects of the present disclosure, a flow cell article 10 is provided. The flow cell article 10 includes a substrate 14 having one or more layers 16; a fluid channel 18 disposed in the substrate 14, wherein the fluid channel 18 comprises at least one reactive surface 22 having a coupling agent 26, the coupling agent 26 having a second functional group 34 (e.g., a primary amine) covalently attached to a polymer 38 using an imide bond and a first functional group 30 of the substrate 14 covalently attached to the fluid channel 18, the covalently attached polymer 38 being represented by formula (I):

R₁is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine. m, n and o are each 1 to 10,000. X is a divalent NH, O and/or S. Z is a first functional group 30.

Referring now to fig. 1, a schematic illustration of a flow cell article 10 covalently attached to a plurality of nucleic acid primer molecules 42 is provided according to some aspects of the present disclosure. The solid support or substrate 14 is operatively attached to a coupling agent 26, wherein the coupling agent 26 includes a first functional group 30 covalently attached to the substrate 14 and a second functional group 34 covalently attached to a polymer 38. In some aspects, the first functional group 30 of the coupling agent 26 includes an aminosilane and/or an aminoorganophosphate. The polymer 38 includes a linker group 36 that can be used to covalently link the second functional group 34 to form a polymer link 35. In some aspects, the same type of linker group 36 can be used to covalently link the plurality of nucleic acid primer molecules 42 and the second functional group 34 using the same or different types of polymer linkages 35. As an alternative to the more reactive amide or ester linkages traditionally used, in some aspects, the polymer linkages 35 used in embodiments disclosed herein may include an imide functional group, which may be formed by a condensation reaction between a primary amine and an anhydride functional group. In some aspects, the linker group 36 is a succinic anhydride group, the second functional group 34 is a primary amine, and the corresponding polymer linker group 35 is an imide. The fully synthetic flow cell article 10 includes a substrate 14 covalently bonded to a polymer 38 using a coupling agent 26 in addition to at least one nucleic acid primer molecule 42 covalently attached to the substrate 14 by the polymer 38. The DNA-bound flow cell article 10 will be positioned facing or extending into the fluid channel 18 to provide a reactive surface 22 for capturing or binding the desired DNA fragments.

In some aspects, the polymer 38 may comprise a polymer containing anhydride functionality. In some aspects, the polymer 38 may comprise poly (ethylene-co-maleic anhydride) (EMA), polyethylene-graft-maleic anhydride, polypropylene-graft-maleic anhydride, poly (ethylene/maleic anhydride), poly (ethylene-co-ethyl acrylate-co-maleic anhydride), poly (isobutylene-co-maleic anhydride), poly (maleic anhydride-co-1-octadecene), poly (styrene-co-maleic anhydride), poly (styrene-co-maleic acid), or a combination thereof. Each of the copolymers listed above may include alternating copolymer (e.g., ABABAB), block copolymer (e.g., aaabbba aabbb) and/or random copolymer (e.g., abbababab) structures. For example, in some aspects, the polymer 38 may comprise poly (ethylene-co-maleic anhydride), wherein the ethylene and maleic anhydride monomers are combined together in an alternating, random, or block structure. In some aspects, the relative ratio of m to n (m: n) is from about 0.5 to about 10.

In some aspects, the polymer 38 used to covalently bind and attach the coupling agent 26 is represented by the following formula (II):

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; n and o are each an integer of 1 to 10,000; and X is divalent NH, O and/or S. When ethylene is copolymerized with maleic anhydride, the residue R of the unsaturated monomer₁Will be, for example, a dirylethyl group. Once the polymer 38 having the formula (II) is reacted with the second functional group 34, the modified structure of the polymer 38 is represented by the following formula (I):

wherein: r₁、R₂And X are all as set forth above in formula (II); m, n and o are each an integer of 1 to 10,000; and Z is a first functional group 30. In some aspects, for example, for a flow cell system, the nucleic acid primer molecules 42 may be covalently bound, linked and attached to the polymer 38 having formula (I) by forming imide linkages with amine groups of the nucleic acid primer molecules 42 using a reactive succinic anhydride function incorporated into the polymer backbone.

In some aspects, the present disclosure may also provide flow cells having surfaces modified with DNA probe molecules, wherein the density of the DNA probe molecules may be precisely controlled, thereby enabling excellent DNA hybridization, subsequent polyclonal cluster formation and sequencing. By incubating the polymer-modified flow cell surface with an amine-terminated DNA probe molecule in the presence of a specific concentration of a second regulatory small molecule (e.g., surface-modifying molecule 50), the DNA probe molecule can be covalently attached to the flow cell surface coated with the amine-reactive polymer.

Still referring to fig. 1, the surface-modifying molecules 50 may be used to control the extent (e.g., amount or breadth) of the coupling reaction of the coupling agent 26 and/or the nucleic acid primer molecules 42 to the polymer 38, which may control or determine the density of attached nucleic acid primer molecules. The ratio of nucleic acid primer molecules 42 to surface-modifying molecules 50 can determine the amount or density of nucleic acid primer molecules 42 located at the reactive surface 22. In some aspects, depending on the density desired for a given application, the molar ratio (P: S) of nucleic acid primer molecules 42 to surface-modifying molecules 50 can be, for example, 0.01:1, 0.1:1, 1:2, 1:5, 1: 10; ratios of 1:20, 1:50, 1:100, 1:1000, 1:10,000, and the like, including numbers and ranges therebetween. For example, in some aspects where clustering can be achieved using bridge amplification methods, the density of nucleic acid primer molecules 42 can be relatively low (e.g., 10,000 or 100,000 nucleic acid primer molecules 42 per square micron of surface area of the fluid channel 18). When clustering is achieved using template walking, the density of nucleic acid primer molecules 42 can be relatively high (e.g., 250,000 or 500,000 nucleic acid primer molecules 42 per square micron of surface area of the fluid channel 18).

In some aspects, the surface modifying molecules 50 can be small molecules containing amines. In some aspects, R₂May be a surface modifying molecule 50. The surface-modifying molecules 50 may include, for example, ethanolamine, N-dimethylethylenediamine, N-diethylethylenediamine, (2-aminoethyl) -trimethylammonium, amine-terminated polyethylene glycol, and/or oligoethylene glycol. The surface-modified molecules 50 may also prevent non-specific binding of biomolecules to the surface of the polymer 38 during sequencing and reduce background signal during sequencing cycles. In other aspects, the surface-modifying molecules 50 may be introduced during the polymer coating, or may be introduced during the nucleic acid primer molecule 42 functionalization step. In some aspects, the surface-modifying molecules 50 can include, for example, but are not necessarily limited to, monoamino, diamino, and triaminosilanes, such as, for example, y-aminopropylsilane, 3- (aminopropyl) triethoxysilane (APTES), 3-aminopropyl) trimethoxysilane, 3-aminopropyldimethylmethoxysilane, 3-aminopropyl (diethoxy) methylsilane, Aminopropylsilsesquioxane (APS), N- [3- (trimethoxysilyl) propyl ] silane]Ethylenediamine, N1- (3-trimethoxysilylpropyl) diethylenetriamine, ethylenediamine, propylamine, allylamine, ethanolamine, (2-aminoethyl) trimethylammonium, or combinations thereof.

In some aspects, the nucleic acid primer molecule 42 can be, for example, an amine-terminated nucleic acid or nucleic acid fragment. In some aspects, the nucleic acid primer molecules 42 can have a surface density, e.g., 1 to 10,000 nucleic acid primer molecules 42 per strand of the polymer 38. In other aspects, the nucleic acid primer molecules 42 can have a density, e.g., 1 to 500,000 nucleic acid primer molecules 42 per square micron of surface area of the fluid channel 18. In other aspects, where polyclonal clustering is desired for sequencing, the density of nucleic acid primer molecules 42 can be, for example, 1,000 to 500,000 nucleic acid primer molecules 42 per square micron (μm2) of surface area of the fluid channel 18. Alternatively, when single molecule analysis is required for sequencing, the density of nucleic acid primer molecules 42 may be, for example, 1 to about 1000 nucleic acid primer molecules 42 per square micron of surface area of the fluid channel 18. In some aspects, the nucleic acid primer molecule 42 can be, for example, 5 '-amine terminated dA30, 3' -amine terminated dA30, amine terminated dT30, and similar probe molecules. In other aspects, the nucleic acid primer molecule 42 may be replaced with an RNA probe molecule for sequencing RNA.

In some aspects, the flow cell article 10 can, for example, comprise at least two solid substrates 14 bonded together using, for example, techniques known in the art, including, for example, laser assisted processes, adhesive tape, polyimide adhesive, pressure sensitive adhesive tape, or combinations thereof. In some aspects, the two substrates 14 can be made of the same material, or in other aspects, the two substrates can be made of different materials. Depending on the application, substrate 14 may be, for example, plastic, glass, silicon, fused silica, or quartz. The flow cell article 10 defines a chamber, cavity, and/or liquid channel 18. The flow cell product 10 may comprise, for example, ports for media flow that direct liquid into and out of the chamber [ see illumina.com; hamming Sequencing Technology, Inc. (Illumina Sequencing Technology), focus:sequencing]. In some aspects, the substrate 14 may comprise glass, glass-ceramic, silicon, fused silica, quartz, thermoplastic, or thermoset.

Referring now to fig. 2, a schematic illustration of an intermediate microfluidic device 44 including a polymer 38 covalently bound to a coupling agent 26 attached to a substrate 14 is provided according to some aspects of the present disclosure. The intermediate microfluidic device 46 shown in fig. 2 includes a polymer 38 coated on the surface of the substrate to be located within the fluid channel 18. The surface of the substrate 14 located in the fluid channel 18 of the intermediate microfluidic device 44 may be first coated with the coupling agent 26. As exemplified, in some aspects, the coupling agent 26 can include an aminosilane. The use of an aminosilane as coupling agent 26 provides the silane to be used as first functional group 30 and the amine to be used as second functional group 34. Once the silane as the first functional group 30 is covalently attached (e.g., forms a covalent bond) to the surface of the substrate 14, the amine serving as the second functional group 34 can be covalently attached to the polymer 38. In some aspects, the succinic anhydride moieties or linker groups 36 (see fig. 1) on the polymer 38 for linking amines can condense to form imide linkages or imide bonds, wherein water molecules are generated in the condensation reaction. The resulting polymer 38 is covalently attached to the coupling agent 26 to form a coating on the substrate 14 comprising three different types of functional groups: 1) an imide group covalently linking coupling agent 26 to polymer 38; 2) amine-reactive anhydride groups (e.g., succinic anhydride groups) incorporated into the polymer backbone that support the attachment of the coupling agent 26 and the DNA segment 42; and 3) modifier functional groups (e.g., esters, amines, carboxylic acids, carboxylic acid esters, and/or thioesters) for facilitating the modulation of DNA density and surface properties.

Still referring to fig. 2, the coupling agent 26 may include an aminosilane, while the solid substrate may be glass, glass-ceramic, silicon, fused silica, a thermoplastic, a thermoset, and/or quartz. Aminosilanes may include molecules that contain both primary amine and silane functional groups, typically linked to each other using, for example, alkyl, alkoxy, and/or diol groups. In some aspects, the aminosilane may include 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3- (2-aminoethyl) -aminopropyltrimethoxysilane, aminopropylmethyldialkoxysilane, 4-aminobutyltriethoxysilane, 4-amino-3, 3-dimethylbutyltrimethoxysilane, N- (6-aminohexyl) aminomethyltriethoxysilane, or a combination thereof. In some aspects, the silane of the aminosilane may include, for example, a silsesquioxane or a mixture thereof. In other aspects, the aminosilane, for example, can include 3- (aminopropyl) triethoxysilane, and the silsesquioxane, for example, can be aminopropyl silsesquioxane.

Referring now to fig. 3, a schematic illustration of an intermediate microfluidic device 44a including a polymer 38 covalently structured with a coupling agent 26 is provided, the coupling agent 26 being attached to a metal oxide layer 46 on a substrate 14, according to some aspects of the present disclosure. The intermediate microfluidic device 44a includes a polymer 38 coated on a surface of a substrate. In some aspects, the surface of the substrate 14 of the intermediate microfluidic device 44a may be first coated with the metal oxide layer 46, wherein the metal oxide layer 46 may then be coated with the coupling agent 26. As exemplified, in some aspects, the coupling agent 26 can include an aminoorganophosphate. The use of an aminoorganophosphate as the coupling agent 26 provides an organophosphate to be used as the first functional group 30 and an amine to be used as the second functional group 34. Once the first functional group 30 or organophosphate is covalently attached (e.g., forms a covalent bond) to the surface of the metal oxide layer 46 of the substrate 14, the second functional group 34 or amine may be covalently attached to the polymer 38. In some aspects, the succinic anhydride moieties or linker groups 36 (see fig. 1) on the polymer 38 for linking amines can condense to form imide linkages or imide bonds, wherein water molecules are generated in the condensation reaction.

Still referring to fig. 3, the coupling agent 26 may include an aminoorganophosphate in which the solid substrate 14 is attached or coated with a metal oxide layer 46. The metal oxide for the metal oxide layer 46 may include, for example, but is not limited to, Al₂O₃、ZnO₂、Ta₂O₅、Nb₂O₅、SnO₂MgO, indium tin oxide, CeO₂、CoO、Co₃O₄、Cr₂O₃、Fe₂O₃、Fe₃O₄、In₂O₃、Mn₂O₃、NiO、a-TiO₂(anatase), r-TiO₂(rutile), WO₃、Y₂O₃、ZrO₂. In some aspects, the metal oxide layer 46 is transparent in the visible wavelength range (e.g., 400nm to 750 nm). For example, the metal oxide layer 46 may have a transmittance of at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the visible wavelength range.

In some aspects, the amino organophosphate ester may include molecules having at least one or both of a primary amine and an organophosphate functionality. The ammonia and organophosphate functional groups may be linked to each other using, for example, alkyl, alkoxy, and/or diol groups. The aminoorganophosphate can include 3-aminopropyl dihydrogen phosphate, 4-aminophenyl phosphate, 2-aminoethyl dihydrogen phosphate, 2- (3-aminopropyl) aminoethyl phosphorothioate, or a combination thereof.

Referring now to fig. 4A-4C, scanning electron microscope images of fluid channel surfaces having polymer coatings are provided, according to some aspects of the present disclosure. The surface of the microfluidic channel 18 has a stable polymer coating that has been treated with an amine-terminated dA30 primer DNA moiety, presenting three scanning electron microscopy images of the surface including: after reaction with dA30, (a) low resolution SEM image of the surface showing two areas: left (a) and right (b); (B) a high resolution SEM image of the left portion of surface a showing the presence of the polymer as nanoparticles; and (C) high resolution SEM images of the right portion of surface a, these figures show that most of the polymer nanoparticles are open to form a uniform dA 30-polymer coating on the surface.

Still referring to fig. 4A-4C, once covalently attached to a surface using an initial imide bond, polymer 38 may be in nanoparticle form, particularly single-stranded nanoparticle form, rather than an extended polymer chain form. Nanoparticle coating schemes can be achieved by incubating the amine-bearing surface with a polymer in a mixture of organic solvents, where each individual polymer self-assembles into a nanoparticle. The mixture of organic solvents comprises, consists essentially of, or consists of: polymer-soluble solvents, such as N-methyl-2-pyrrolidone (NMP); and polymer insoluble solvents, for example, isopropyl alcohol (IPA). As shown in fig. 4A and 4B, the polymer covalently attached to the surface primarily by imide bonds through the coupling agent comprises, consists essentially of, or consists of individual nanoparticles. However, once functionalized by the DNA primer (here 5' -amino-C6-dA 30), the polymeric nanoparticles become open and cover the entire surface to form a uniform coating, as shown in fig. 4A and 4C. Since DNA is negatively charged, self-repulsion between DNA molecules will cause the polymer molecules to expand to cover the entire surface, even if there are spaces between the attached polymer nanoparticles. It is also noteworthy that the polymer nanoparticles are well distributed after attachment to the substrate. Without being bound by any theory, it is believed that this is due to the well-known crowding effect of the polymer particles in the solvent. Sequencing by functionalized surface synthesis using dA30, together with template walking based clustering and reversible terminator DNA synthesis chemistry, showed that the surface coated with EMA nanoparticles was able to achieve an average of 75 reads, while the surface coated with extended EMA polymer chains only allowed an average of a few reads (<10 bp). By controlling the concentration of the polymer 38 and the duration of the reaction for coating, the density of the polymer nanoparticles covalently attached to the substrate can be controlled. The low concentration of polymer 38 used in the coating process results in an attached lower density of polymer nanoparticles. Based on the polymer crowding effect and the limited surface coverage of each polymer after induced extension by the attached DNA primer molecules, the polymer coated substrate can be viewed as a patterned surface, even though the polymer nanoparticles may have different sizes (as shown in fig. 4B). Thus, when the density of attached polymer nanoparticles is low (e.g., 1 polymer nanoparticle/10,000 nm2, or 1 polymer nanoparticle/20,000 nm2, or 1 polymer nanoparticle/40,000 nm2, or 1 polymer nanoparticle/100,000 nm2, or 1 polymer nanoparticle/250,000 nm2, or 1 polymer nanoparticle/1000,000 nm2), individual clusters can be created within the region covered by a single polymer nanoparticle, thus effectively forming a random but well-separated cluster array. This is particularly useful for high density patterned sequencing applications.

Referring now to fig. 5A-5B, which provide a photographic image (5A) and a fluorescence image (5B) of a flow cell article having 8 channels, each channel comprising, consisting essentially of, or consisting of an amine-terminated dA30 attached to a polymer coating, according to some aspects of the present disclosure. Fig. 5A shows a photographic image of the entire flow cell, and fig. 5B shows a confocal fluorescence image of the entire flow cell with 8 channels, each channel comprising, consisting essentially of, or consisting of an amine-terminated dA30 attached to a reactive polymer coating, after hybridization with Cy 3-labeled dT 30.

Specifically, all channels were first coated with y (gamma) -aminopropylsilane followed by covalent attachment of propylamine derivatized poly (ethylene-alt-maleic anhydride).

Thereafter, all channels were incubated with 50. mu.M of 5' -amine-terminated dA30 in the presence of 100. mu.M ethanolamine, followed by further processing as described below. From top to bottom, channels 1 to 8 are: channels 1, 2, 7 and 8: washing three times with Phosphate Buffered Saline (PBS); channels 3 and 6: incubate 5 minutes with 0.05M NaOH, wash three times with PBS; and channels 4 and 5: the washing was carried out five times for 1 minute each time with water at 60 ℃. Finally, all channels were incubated with 1 μ M Cy3 labeled dT30 for 45 minutes. After washing three times with PBS and drying, the entire flow cell was scanned using confocal microscopy. All the acquired fluorescence images are assembled together to form a fluorescence image of the entire flow cell as shown.

Referring now to fig. 6, a graph showing comparative differences in sequencing read lengths for amide and imide linkages used to covalently bind a coupling agent to a polymer coating for attaching DNA is provided. The figure illustrates the comparative difference in sequencing read length for two polymer coatings: the attached one polymer is linked using an amide bond; the other polymer attached is linked using an imide linkage.

In some aspects of the present disclosure, a two-step sequential reaction scheme is used to form imide linkages between the anhydride-containing polymer 38 and the amine terminal groups of the coupling agent 26. First, the anhydride group of the polymer reacts with the amine group on the surface to form an amide bond, and then cyclized by heat treatment (120 to 150 ℃ for several hours) to form an imide bond. As shown in fig. 6, sequencing by functionalized surface synthesis using dA30, along with template-walking based clustering and reversible terminator DNA synthesis chemistry, showed that polymers covalently attached to amine-bearing surfaces via imide linkages allowed an average of 75 reads, while polymers covalently attached to amine-bearing surfaces via amide linkages only allowed an average of 25 reads. Further optimization of DNA attachment (e.g., density and distribution) can further significantly increase read length (e.g., up to 200 reads for single-end sequencing) given the high stability of the imide linkages against harsh treatments (e.g., NaOH, vigorous washing, room temperature to high temperature cycling). Here, the microfluidic channel surfaces were treated with the same coating and functionalization protocol, but with a3 hour heat treatment at 120 ℃ to drive imide bond formation.

In some aspects of the present disclosure, a method of making a flow cell article 10 is provided. The method comprises the following steps: contacting the fluid channel 18 disposed in the substrate 14 with a coupling agent 26 to covalently attach a first functional group to the fluid channel 18; contacting the polymer 38 of formula (II) with a coupling agent 26 to covalently attach the polymer 38 to the second functional group 34, thereby forming an imide linkage on the tethered polymer;

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; n and o are each an integer of 1 to 10,000; and X is divalent NH, O and/or S. The method may further comprise: the nucleic acid primer molecules 42 are contacted with the tethered polymer 38 (see formula (II)) to covalently attach the nucleic acid primer molecules 42 to the tethered polymer 38. In some aspects, contacting the nucleic acid primer molecules 42 with the tethered polymer 38 can optionally include the presence of a surface modification molecule 50 to covalently attach the nucleic acid primer molecules 42 to the polymer 38 attached to the substrate 14 using the coupling agent 26 to form the flow cell article 10. In some aspects, controlling the density of nucleic acid primer molecules 42 attached to the polymer 38 can be achieved by using various ratios of surface modifying molecules 50 relative to the nucleic acid primer molecules 42.

It should be understood that the descriptions set forth and taught for the flow cell article 10 described above may be used in any combination, and that the descriptions are equally well applicable to the method of making and using the flow cell article 10.

In some aspects, the method of making a flow cell article can further comprise: the density of the nucleic acid primer molecules 42 is controlled by determining and selecting (e.g., by stoichiometry) the ratio of polymer to nucleic acid primer molecules 42 in advance.

In a further aspect of the present disclosure, the present disclosure provides a method for sequencing a nucleic acid, the method comprising: providing a flow cell article 10, the flow cell article 10 having: a substrate 14 having one or more layers; a fluid channel 18 disposed in the substrate 14, wherein the fluid channel 18 comprises at least one reactive surface 22, the reactive surface 22 comprising a coupling agent 26, the coupling agent 26 having a first functional group 30 covalently attached to the substrate 14 of the fluid channel 18 and a second functional group 34 covalently attached to a polymer of formula (I) having an imide functional linkage:

wherein: r₁Is the residue of an unsaturated monomer which has been copolymerized with maleic anhydride; r₂Is H, alkyl, oligo (ethylene glycol) and/or dialkylamine; m, n and o are each 1 to 10,000; x is divalent NH, O and/or S; and Z is a first functional group 30. The method for sequencing nucleic acids further comprises: the nucleic acid primer molecules 42 are contacted with the tethered polymer 38 to covalently attach the nucleic acid primer molecules 42 to the tethered polymer 38.

In some aspects, once the flow cell article 10 is produced, methods for sequencing nucleic acid fragments can be performed. The method further comprises the following steps: contacting the nucleic acid primer molecule 42 with the polymer 38 of formula (I) to covalently attach the nucleic acid primer molecule 42 to the polymer 38 of formula (I); capturing DNA fragments using the nucleic acid primer molecules 42, wherein each DNA fragment comprises a sequence complementary to the nucleic acid primer molecules 42; and adding nucleotides to the ends of the nucleic acid primer molecules 42 to synthesize a complementary DNA sequence for each DNA fragment captured, wherein the complementary DNA sequence is covalently linked to the polymer 38 of formula (I) through the nucleic acid primer molecules 42. In some aspects, the method for sequencing a nucleic acid fragment may further comprise: denaturing the complementary DNA sequences from the DNA fragments; collecting DNA fragments from the flow cell preparation; synthesizing a target DNA fragment using a complementary DNA sequence covalently linked to the polymer of formula (I) by a nucleic acid primer molecule 42; and collecting the target DNA fragments from the flow cell preparation 10.

In some aspects, the steps of capturing DNA fragments from the sample, each DNA fragment comprising a sequence complementary to a nucleic acid primer molecule 42, and covalently adding nucleotides from the ends of the primer molecules 42 can be used to synthesize complementary DNA sequences for each captured fragment. These complementary DNA fragments may be covalently linked to the tethered polymer, either individually or separately, as desired for the application. The double stranded DNA, complementary DNA sequence and captured DNA fragments may then be denatured and the corresponding strands may be collected and removed. In some aspects, after the DNA fragments are removed, each complementary DNA or DNA fragment can be locally amplified using polymerase chain reaction to form a monoclonal cluster. Depending on the resolution of the optical system used for fluorescence imaging, the resulting clusters may have dimensions of 50nm to 1000nm in diameter. The synthetic methods provide techniques that may be particularly useful for sequencing-by-synthesis (NGS) techniques in which a polyclonal cluster is typically formed prior to sequencing. After the nucleic acid primer molecules 42 and DNA molecules are attached to the fluid channel 18 of the substrate 14, cluster generation can be achieved using, for example, bridge amplification, exclusive amplification, or template walking methods.

It is to be understood that the descriptions set forth and taught for the flow cell article 10 described above may be used in any combination, and that the descriptions are equally well applicable to methods for sequencing nucleic acid fragments.

The present disclosure enables rapid covalent attachment of amine-terminated nucleic acid primer molecules 42 (e.g., 5 '-amine-dA 30 or 5' -amine-dT 30) to a substrate 14. The connection can be completed within 1 hour, for example.

The present disclosure more stably attaches the nucleic acid primer molecules 42 to the substrate than ligation using bifunctional or other means. This is partly due to the multivalent, strong anchoring of the polymer layer to the charged amine surface (e.g. APTES) and partly due to the formation of imide bonds. This stable linkage attachment is important because DNA sequencing often requires running many reaction cycles and involves some harsh treatment, such as NaOH washes prior to sequencing reads to denature any double stranded DNA. The use of imide linkages formed by the reaction of the succinic anhydride functional groups with primary amines built into the coupling agent 26 and/or the nucleic acid primer molecules 42 provides chemically resistant and stable covalent chemical linkages that resist degradation losses or surface separation due to these harsh chemical treatments used in sequencing chemistry.

The present disclosure also provides methods for precisely controlling hybridization (and ultimately more efficient hybridization) between a nucleic acid primer molecule 42 and a target DNA fragment containing an adaptor sequence complementary to the probe. This is mainly due to the flexibility of the polymer chains, even after coating. In contrast, for DNA attachment based on bifunctional ligation, these DNA molecules are very close to the surface, thus preventing efficient hybridization.

The present disclosure also provides precise control of the density of the surface-attached nucleic acid primer molecules 42, which allows for user-determined adjustment of DNA hybridization efficiency and subsequent clustering efficiency. Such control may be achieved in either of two different chemical reactions. First, the amine reactive polymer is partially modified and derivatized with a surface modifying molecule 50 (e.g., propylamine, etc.). Such functionalization can help control the solubility of the polymer 38 in the solution used for coating and the reactive sites once attached to the coupling agent 26 of the substrate 14. Second, the surface modified with the amine-reactive polymer 38 can be incubated with the amine-terminated nucleic acid primer molecules 42 in the presence of a desired concentration of surface-modifying molecules 50 (e.g., ethanolamine or similar reagents). This step controls the covalent ligation reaction and, in turn, the density of attached nucleic acid primer molecules 42. The combination of these two different reactions allows for excellent density control of the nucleic acid primer molecules 42 or DNA/RNA primer molecules attached to the fluidic channels 18 of the substrate 14 and ultimately the density of the clusters formed and has excellent sequencing efficiency.

The present disclosure may also be applicable to NGS using nanopatterned flow cells. Nanopatterning can be achieved using state-of-the-art photolithography or nanoimprint methods. In some aspects, the flow cell article 10 can include an array of discrete spots having an amine-reactive polymer coating and covalently bound nucleic acid primer molecules 42. Nano-patterning can be achieved, for example, using state-of-the-art photolithography or nano-imprint techniques. In an alternative embodiment, a low density polymer nanoparticle coating may be used as a randomly patterned surface, such that the clusters formed may be confined by each polymer nanoparticle and well separated from each other.

The present disclosure may also be applicable to different biomolecule analysis techniques using nucleic acid-based biosensors, where the density of attached nucleic acid primer molecules 42 may be important to contribute to the success of the bioassay.

Those skilled in the art will appreciate that the configuration of the device and other components may not be limited to any particular material. Other exemplary embodiments of the devices disclosed herein may be formed from a variety of materials, unless otherwise specified herein.

For the purposes of this disclosure, the term "couple" (in all its forms: connected, etc.) generally means that two components are joined to each other (electrically or mechanically) either directly or indirectly. Such a combination may be stationary in nature or movable in nature. Such joining may be achieved by the two components (electrical or mechanical) being integrally formed as a single unitary body with one another with any additional intermediate member or by both components. Unless otherwise specified, such bonding may be permanent in nature, or may be removable or releasable in nature.

It is also important to note that the construction and arrangement of the elements of the device as shown in the exemplary embodiments is illustrative only. Although only a few embodiments of the present inventions have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter recited herein. For example, elements shown as integrally formed may be constructed of multiple parts or elements shown as multiple parts may be integrally formed, the operation of the interfaces may be reversed or otherwise varied, the length or width of the systems structures and/or members or connectors or other elements may be varied, the nature or number of adjustment positions between the various elements may be varied. It should be noted that the elements and/or assemblies of the system may be constructed from any of a variety of materials that provide sufficient strength or durability in any of a variety of colors, textures, and combinations. Accordingly, all such modifications are intended to be included within the scope of this invention. Other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the desired exemplary embodiments and other exemplary embodiments without departing from the spirit of the present inventions.

It is understood that any described process or step in the method may be combined with other disclosed processes or steps to arrive at a structure within the scope of the present apparatus. The exemplary structures and methods disclosed herein are for purposes of illustration and are not to be construed as limitations.

It should also be understood that various changes and modifications can be made to the above-described arrangements without departing from the concepts of the present disclosure, and further it should be understood that such concepts are intended to be covered by the appended claims unless these claims by their language expressly state otherwise.

The above description is considered that of the illustrated embodiments only. Modifications of the device will occur to those skilled in the art and to those who make or use the device. It is understood, therefore, that the embodiments shown in the drawings and described above are merely for illustrative purposes and are not intended to limit the scope of the device, which is defined by the following claims as interpreted according to the principles of patent law, including the doctrine of equivalents.