阅读说明：本技术 包含杂聚物的流动池 (Flow cell comprising heteropolymers ) 是由 韦恩·N·乔治 卢多维克·文森特 安德鲁·A·布朗 马蒂厄·莱萨德-瓦伊格 于 2019-12-19 设计创作，主要内容包括：流动池包括支撑物和附接至支撑物的杂聚物。杂聚物包括包含与附接至引物的官能团反应的附接基团的丙烯酰胺单体和包含刺激响应性官能团的单体。包含刺激响应性官能团的单体可以是pH响应性的、温度响应性的、糖响应性的、亲核体响应性的和/或盐响应性的。(The flow cell includes a support and a heteropolymer attached to the support. The heteropolymer includes an acrylamide monomer including an attachment group that reacts with a functional group attached to the primer and a monomer including a stimulus-responsive functional group. The monomers comprising stimulus-responsive functional groups can be pH-responsive, temperature-responsive, sugar-responsive, nucleophile-responsive, and/or salt-responsive.)

1. A heteropolymer comprising:

an acrylamide monomer comprising an attachment group that reacts with a functional group attached to a primer; and

a monomer comprising a stimulus-responsive functional group, wherein the monomer comprising the stimulus-responsive functional group is selected from the group consisting of:

an acrylamide monomer comprising a terminal pH-responsive functional group;

a vinyl or acrylate monomer comprising a terminal pH-responsive functional group selected from the group consisting of: a hydroxyl group having an acid-labile protecting group, a hydroxyl group having a base-labile protecting group, an amino group having an acid-labile protecting group, an amino group having a base-labile protecting group, a sulfonate group and a sulfonic acid group;

a temperature-responsive N-substituted acrylamide;

an acrylamide, acrylate or vinyl monomer comprising a terminal sugar-responsive functional group;

an acrylamide, acrylate or vinyl monomer comprising a terminal nucleophile-responsive functional group; and

an acrylamide, acrylate, or vinyl monomer comprising a terminal salt-responsive functional group.

2. The heteropolymer of claim 1, wherein the monomer comprising the stimulus-responsive functional group will undergo a modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the heteropolymer.

3. The heteropolymer of claim 1 or claim 2, further comprising the primer grafted to the attachment group.

4. The heteropolymer of any of claims 1-3, wherein the attachment group is selected from the group consisting of: azido groups, alkenyl groups, alkynyl groups, aldehyde groups, hydrazone groups, hydrazine groups, tetrazole groups, tetrazine groups, and thiol groups.

5. The heteropolymer of any of claims 1-4, wherein the attachment group of the acrylamide monomer comprises an azido group.

6. The heteropolymer according to claim 5, wherein the acrylamide monomer is an azidoacetamidopentyl acrylamide monomer.

7. The heteropolymer of claim 5, further comprising a second acrylamide monomer.

8. The heteropolymer of any of claims 1-7, wherein:

the monomer comprising the stimulus-responsive functional group is the acrylamide monomer comprising the terminal pH-responsive functional group; and is

The terminal pH-responsive functional group is selected from the group consisting of: hydroxyl, 1, 2-diol, 1, 3-diol, t-butoxycarbonylamino group, 9H-fluoren-9-ylmethoxycarbonylamino group, amino group, carboxylate group, carboxylic acid group, sulfonate group and sulfonic acid group protected as acetal, hemiacetal or ketal.

9. The heteropolymer of any of claims 1-7, wherein:

the monomer comprising the stimulus-responsive functional group is the acrylamide, acrylate, or vinyl monomer comprising the terminal saccharide-responsive functional group; and is

The terminal sugar-responsive functional group includes a boronic acid group.

10. The heteropolymer of claim 9, wherein the monomer comprising the stimulus-responsive functional group is 3- (acrylamido) phenylboronic acid.

11. The heteropolymer of any of claims 1-7, wherein:

the monomer comprising the stimulus-responsive functional group is the acrylamide, acrylate or vinyl monomer comprising the terminal nucleophile-responsive functional group; and is

The terminal nucleophile-responsive functional group has the following structure:

wherein: (a) y being SO₂And Y' is CH₂(ii) a Or (b) both Y and Y' are C (O).

12. The heteropolymer of any of claims 1-7, wherein:

the monomer comprising the stimulus-responsive functional group is the acrylamide, acrylate or vinyl monomer comprising the terminal salt-responsive functional group; and is

The salt-responsive functional group is a zwitterionic functional group that exhibits a reverse polyelectrolyte behavior.

13. The heteropolymer of claim 12, wherein the monomer comprising the stimulus-responsive functional group has one of the following structures:

(i)wherein A is O or NH, and R^zIs H or C_1-4An alkyl group; or

(ii)Wherein R is^zIs H or C_1-4An alkyl group.

14. The heteropolymer of any of claims 1-7, wherein:

the monomer comprising the stimulus-responsive functional group is the temperature-responsive N-substituted acrylamide; and is

The temperature-responsive N-substituted acrylamide includes a thermosensitive hydroxyl or amino protecting group.

15. The heteropolymer of any of claims 1-7, wherein:

the monomer comprising the stimulus-responsive functional group is the temperature-responsive N-substituted acrylamide; and is

The temperature-responsive N-substituted acrylamide is N-isopropylacrylamide.

16. A heteropolymer having the structure:

wherein n ranges from 10 to 500.

17. A method of making a switchable heteropolymer comprising:

selecting a monomer comprising a stimulus-responsive functional group from the group consisting of:

an acrylamide monomer comprising a terminal pH-responsive functional group;

a vinyl or acrylate monomer comprising a terminal pH-responsive functional group selected from the group consisting of: a hydroxyl group having an acid-labile protecting group, a hydroxyl group having a base-labile protecting group, an amino group having an acid-labile protecting group, an amino group having a base-labile protecting group, a sulfonate group and a sulfonic acid group;

a temperature-responsive N-substituted acrylamide;

an acrylamide, acrylate or vinyl monomer comprising a terminal sugar-responsive functional group;

an acrylamide, acrylate or vinyl monomer comprising a terminal nucleophile-responsive functional group; and

an acrylamide, acrylate or vinyl monomer comprising a terminal salt-responsive functional group; and

copolymerizing the monomer comprising the stimulus-responsive functional group with an acrylamide monomer comprising an attachment group that reacts with a functional group attached to a primer.

18. The method of claim 17, wherein the acrylamide monomer comprising the attachment group is an azidoacetamidopentyl acrylamide monomer.

19. The method of claim 17 or claim 18, further comprising copolymerizing the monomer comprising the stimulus-responsive functional group and the acrylamide monomer comprising the attachment group with a second acrylamide monomer.

20. A flow cell, comprising:

a support; and

the heteropolymer of any of claims 1-16 attached to the support.

21. A method of making a flow cell, comprising contacting the heteropolymer of any of claims 1,2, or 4-16 with at least a portion of a flow cell support, thereby attaching the heteropolymer to the flow cell support.

22. The method of claim 21, further comprising grafting a primer to the attachment group of the heteropolymer attached to the support.

23. The method of claim 21 or claim 22, further comprising exposing the heteropolymer attached to the flow cell support to a predetermined stimulus.

24. The method of claim 23, further comprising, after the exposing, performing a sequencing operation on the flow cell.

25. A sequencing method, comprising:

grafting a primer to a switchable heteropolymer on a flow cell support;

exposing the switchable heteropolymer on the flow cell support to a predetermined stimulus, thereby causing a change in polarity and/or conformation of the switchable heteropolymer;

hybridizing a nucleic acid template to the primers on the flow cell support;

amplifying the nucleic acid template on the flow cell support to produce an amplified template; and

detecting a signal when the labeled nucleotide associates with a complementary nucleotide in the amplified template.

Background

Bioarrays are one of the tools used to detect and analyze molecules, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In these applications, arrays are designed to include probes for nucleotide sequences present in genes of humans and other organisms. For example, in certain applications, individual DNA probes and RNA probes may be attached at geometrically meshed (or random) locations on an array support. For example, a test sample from a human or organism may be exposed to the grid such that complementary fragments hybridize to the probes at individual sites in the array. The array can then be examined by scanning light at a particular frequency over the sites to identify which fragments are present in the sample by fluorescence from the sites to which the fragments hybridize.

The bioarray can be used for gene sequencing. Generally, gene sequencing involves determining the order of nucleotides or nucleic acids in a piece of genetic material (such as a fragment of DNA or RNA). Increasingly long base pair sequences are being analyzed and the resulting sequence information can be used in a variety of bioinformatic approaches to logically fit fragments together in order to reliably determine the sequence of a wide range of lengths of genetic material from which the fragments are derived. Automated computer-based inspection of feature fragments has been developed and used for genome mapping, identification of genes and their functions, risk assessment of certain conditions and disease states, and the like. In addition to these applications, biological arrays can be used to detect and evaluate a wide range of molecules, families of molecules, gene expression levels, single nucleotide polymorphisms, and genotyping.

SUMMARY

One aspect disclosed herein is a switchable heteropolymer (switchable heteropolymer) comprising: more than one monomer comprising a stimuli-responsive functional group (stimuli-responsive functional group), wherein the stimuli-responsive functional group is selected from the group consisting of: a pH-responsive functional group, a temperature-responsive functional group, a saccharide-responsive functional group, a nucleophile-responsive functional group, and a salt-responsive functional group.

The stimulus-responsive functional group is capable of undergoing a modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the switchable heteropolymer.

In some aspects, the switchable heteropolymer further comprises a primer grafted thereto.

In some aspects, the stimulus-responsive functional group is not an azido group.

In some aspects, the switchable heteropolymer includes two or more different stimuli-responsive monomers that respond to the same or different stimuli.

In some aspects, the switchable heteropolymer is a copolymer comprising more than one acrylamide monomer optionally containing an azido group. In one of these aspects, the acrylamide monomer is an azidoacetamidopentylglycolacrylamide monomer or is a combination of an azidoacetamidopentylglycolacrylamide monomer and a second acrylamide monomer.

In some aspects, the switchable heteropolymer includes a sugar monomer (sugar monomer) optionally including an azido group.

In some aspects, the pH-responsive functional group is selected from the group consisting of: hydroxyl, 1, 2-diol, 1, 3-diol, t-butoxycarbonylamino group, 9H-fluoren-9-ylmethoxycarbonylamino group, amino group, carboxylate group, carboxylic acid group, sulfonate group and sulfonic acid group protected as acetal, hemiacetal or ketal.

In other aspects, the saccharide-responsive functional group comprises a boronic acid group.

In still other aspects, the nucleophile-responsive functional group has the following structure:

wherein: (a) y being SO₂And Y' is CH₂(ii) a Or (b) both Y and Y' are C (O).

In yet further aspects, the salt-responsive functional group is a zwitterionic functional group that exhibits inverse polyelectrolyte (antipolyeletrolyte) behavior.

In some aspects, the temperature-responsive group comprises a thermosensitive hydroxyl or amino protecting group.

It is to be understood that any of the features of the switchable heteropolymers disclosed herein may be combined together in any desired manner and/or configuration.

Another aspect disclosed herein is a method of making a switchable heteropolymer, the method comprising copolymerizing more than one monomer comprising a stimulus-responsive functional group with more than one second monomer.

In some aspects, the second monomer is a sugar monomer or an acrylamide monomer comprising an azido group. In some aspects, the acrylamide monomer comprises an azido group. In some aspects, the acrylamide monomer is an azidoacetamidopentylacrylamide monomer or is a combination of an azidoacetamidopentylacrylamide monomer and a second acrylamide monomer.

It should be understood that any of the features of the method may be combined in any desired manner. Further, it is to be understood that any combination of features of the methods and/or switchable heteropolymers can be used together and/or in combination with any of the examples disclosed herein.

Another aspect disclosed herein is a flow cell comprising a support and a switchable heteropolymer attached to the support. Any of the switchable heteropolymers disclosed herein may be used. In some aspects, the flow cell further comprises a primer grafted to the switchable heteropolymer.

It will be appreciated that any of the features of the flow cell may be combined together in any desired manner. Further, it is to be understood that any combination of features of the flow cell and/or the method and/or switchable heteropolymer can be used together and/or combined with any of the examples disclosed herein.

Yet another aspect disclosed herein is a method of making a flow cell, the method comprising contacting a switchable heteropolymer with at least a portion of a flow cell support, thereby attaching the switchable heteropolymer to the flow cell support.

In some aspects, the method comprises grafting a primer to a switchable heteropolymer attached to a support.

In other aspects, the method comprises exposing a switchable heteropolymer attached to a flow cell support to a predetermined stimulus. In some aspects, the method further comprises performing a sequencing operation on the flow cell after the exposing.

It should be understood that any of the features of the method may be combined in any desired manner. Further, it is to be understood that any combination of features of the flow cell and/or any method and/or switchable heteropolymer can be used together and/or in combination with any of the examples disclosed herein.

Yet another aspect is a method of sequencing, comprising: grafting a primer to a switchable heteropolymer on a flow cell support; exposing the switchable heteropolymer on the flow cell support to a predetermined stimulus, thereby causing a change in polarity and/or conformation of the switchable heteropolymer; hybridizing a nucleic acid template to a primer on the flow cell support; amplifying the nucleic acid template on the flow cell support to produce an amplified template; and detecting a signal when the labeled nucleotide associates with a complementary nucleotide in the amplified template.

It should be understood that any of the features of the method may be combined in any desired manner. Further, it is to be understood that any combination of features of the flow cell and/or any method and/or switchable heteropolymer can be used together and/or in combination with any of the examples disclosed herein in any desired manner.

Yet another aspect disclosed herein is a heteropolymer comprising a monomer comprising an attachment group (attachment group) that reacts with a functional group attached to a primer and a monomer comprising a stimulus-responsive functional group, wherein the monomer comprising a stimulus-responsive functional group is selected from the group consisting of: an acrylamide monomer comprising a terminal pH-responsive functional group; a vinyl or acrylate monomer (acrylate monomer) comprising a terminal pH-responsive functional group selected from the group consisting of a hydroxyl group having an acid-labile protecting group, a hydroxyl group having a base-labile protecting group, an amino group having an acid-labile protecting group, an amino group having a base-labile protecting group, a sulfonate group, and a sulfonic acid group; a temperature-responsive N-substituted acrylamide; an acrylamide, acrylate or vinyl monomer comprising a terminal sugar-responsive functional group; an acrylamide, acrylate or vinyl monomer comprising a terminal nucleophile-responsive functional group; and an acrylamide, acrylate, or vinyl monomer comprising a terminal salt-responsive functional group.

In some aspects, the monomers comprising stimulus-responsive functional groups will undergo a modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the switchable heteropolymer.

Some aspects of the heteropolymer further include a primer grafted to the attachment group.

In some aspects, the attachment group is selected from the group consisting of: azido groups, alkenyl groups, alkynyl groups, aldehyde groups, hydrazone groups, hydrazine groups, tetrazole groups, tetrazine groups, and thiol groups.

In other aspects, the attachment group of the acrylamide monomer comprises an azido group. In some examples, the acrylamide monomer is an azidoacetamidopentylgramide monomer. In some examples, the heteropolymer further includes a second acrylamide monomer.

In some aspects, the monomer comprising a stimulus-responsive functional group is an acrylamide monomer comprising a terminal pH-responsive functional group; and the terminal pH-responsive functional group is selected from the group consisting of: hydroxyl, 1, 2-diol, 1, 3-diol, t-butoxycarbonylamino group, 9H-fluoren-9-ylmethoxycarbonylamino group, amino group, carboxylate group, carboxylic acid group, sulfonate group and sulfonic acid group protected as acetal, hemiacetal or ketal.

In other aspects, the stimulus-responsive functional group-containing monomer is an acrylamide, acrylate, or vinyl monomer containing a terminal sugar-responsive functional group; and the terminal sugar-responsive functional group comprises a boronic acid group. In one example, the monomer comprising a stimulus-responsive functional group is 3- (acrylamido) phenylboronic acid.

In some aspects, the monomer comprising a stimulus-responsive functional group is an acrylamide, acrylate, or vinyl monomer comprising a terminal nucleophile-responsive functional group; and the terminal nucleophile-responsive functional group has the following structure:

wherein: (a) y being SO₂And Y' is CH₂(ii) a Or (b) both Y and Y' are C (O).

In other aspects, the monomer comprising a stimulus-responsive functional group is an acrylamide, acrylate, or vinyl monomer comprising a terminal salt-responsive functional group; and the salt-responsive functional group is a zwitterionic functional group that exhibits a reverse polyelectrolyte behavior. In an example, the stimulus-responsive functional group-containing monomer has one of the following structures:

(i)wherein A is O or NH, and R^zIs H or C_1-4An alkyl group; or

(ii)Wherein R is^zIs H or C_1-4An alkyl group.

In some aspects, the monomer comprising a stimulus-responsive functional group is a temperature-responsive N-substituted acrylamide; and the temperature-responsive N-substituted acrylamide includes a thermosensitive hydroxyl or amino protecting group.

In some aspects, the monomer comprising a stimulus-responsive functional group is a temperature-responsive N-substituted acrylamide; and the temperature responsive N-substituted acrylamide is N-isopropylacrylamide.

It is to be understood that any of the features of the heteropolymers may be combined together in any desired manner. Further, it is to be understood that any combination of the features of the heteropolymers and/or flow cells and/or any method can be used together and/or in combination with any of the examples disclosed herein in any desired manner.

Another aspect disclosed herein is a heteropolymer having the structure:

wherein n ranges from 10 to 500.

It is to be understood that any combination of features of the heteropolymers and/or flow cells and/or any method can be used together and/or combined with any example disclosed herein in any desired manner.

Yet another aspect disclosed herein is a method of making a heteropolymer, the method comprising selecting a monomer comprising a stimulus-responsive functional group from the group consisting of: an acrylamide monomer comprising a terminal pH-responsive functional group; a vinyl or acrylate monomer comprising a terminal pH-responsive functional group selected from the group consisting of a hydroxyl group having an acid-labile protecting group, a hydroxyl group having a base-labile protecting group, an amino group having an acid-labile protecting group, an amino group having a base-labile protecting group, a sulfonate group, and a sulfonic acid group; a temperature-responsive N-substituted acrylamide; an acrylamide, acrylate or vinyl monomer comprising a terminal sugar-responsive functional group; an acrylamide, acrylate or vinyl monomer comprising a terminal nucleophile-responsive functional group; and an acrylamide, acrylate or vinyl monomer comprising a terminal salt-responsive functional group; and copolymerizing a monomer comprising a stimulus-responsive functional group with an acrylamide monomer comprising an attachment group that reacts with the functional group attached to the primer.

In some aspects, the acrylamide monomer comprising an attachment group is an azidoacetamidopentyl acrylamide monomer.

Some aspects of the method further include copolymerizing a monomer comprising a stimulus-responsive functional group and an acrylamide monomer comprising an attachment group with the second acrylamide monomer.

It is to be understood that any combination of features of the method and/or flow cell and/or any heteropolymer may be used together and/or combined with any of the examples disclosed herein in any desired manner.

Brief Description of Drawings

Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

Fig. 1A-1D, along with fig. 1E and 1F, or along with fig. 1G and 1H, are schematic cross-sectional views depicting various embodiments of the methods disclosed herein;

fig. 2A-2D are schematic cross-sectional views depicting another embodiment of the methods disclosed herein; and

fig. 3A and 3B are graphs depicting the percent error rate (fig. 3A) and the percent mass metric (fig. 3B) for flow cells formed from comparative polymers and embodiments of the polymers disclosed herein.

Detailed Description

The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The terms comprising, including, containing and containing are synonymous with one another and are intended to be broadly equivalent.

The terms top (top), bottom (bottom), lower (lower), upper (upper), upper (on) and related terms are used herein to describe the flow cell and/or various components of the flow cell. It should be understood that these directional terms are not intended to imply a particular direction, but are used to indicate relative directions between components. The use of directional terms should not be construed to limit the examples disclosed herein to any particular direction.

As used herein, "acetal group" refers to a group having the following linkage R₂C(OR’)₂Wherein the R group and R' group are each organic moieties. Acetal groups include acetals, ketals, hemiacetals, and hemiketals. In acetals, one R group is H. In some aspects, R' is C_1-4Alkyl, or two R' groups together form C_2-4An alkylene group. Acetal protecting groups may be used to protect hydroxyl groups, 1, 2-diols or 1, 3-diols.

"acrylate group" includes salts, esters and conjugate bases of acrylic acid and its derivatives (e.g., methacrylic acid). The acrylate ion has the formula CH₂＝CHCOO^-。

The "acrylamide monomer" is of the structureOr a substituted analog thereof (e.g., methacrylamide or N-isopropylacrylamide). An example of a monomer comprising an acrylamide group and an azido group is azidoacetamidopentylgrylamide:

as used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double or triple bonds). The alkyl group may have 1 to 20 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the like. By way of example, the name "C1-4 alkyl" indicates the presence of one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

As used herein, "alkyne" or "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups may have 2 to 20 carbon atoms.

As used herein, "aryl" refers to an aromatic ring or aromatic ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) that contains only carbon in the ring backbone. When the aryl group is a ring system, each ring in the system is aromatic. The aryl group can have 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

As used herein, the term "attached" refers to a state in which two things are joined, fastened, adhered, connected, or bound to each other, either covalently or noncovalently (e.g., through hydrogen bonds, ionic bonds, van der waals forces, hydrophilic interactions, and hydrophobic interactions). For example, the nucleic acid may be attached to the functionalized polymer by covalent or non-covalent bonds.

"Azide" or "azido" functional group means-N₃。

As used herein, "bonding region" refers to a region on a substrate that is to be bonded with another material, which may be, for example, a spacer layer, a cap, another substrate, or the like, or a combination thereof (e.g., a spacer layer and a cap). The bond formed at the bonding region may be a chemical bond (as described above), or a mechanical bond (e.g., using fasteners, etc.).

"Tert-butoxycarbonyl group" (Boc) refers toA group. "Butoxycarbonyloxy group" means-OCO₂the tBu group.

As used herein, "carbocyclyl" means a non-aromatic ring or non-aromatic ring system that contains only carbon atoms in the backbone of the ring system. When a carbocyclyl group is a ring system, two or more rings may be joined together in a fused, bridged or spiro-linked manner. The carbocyclyl group may have any degree of saturation provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl, and cycloalkynyl. Carbocyclyl groups may have 3 to 20 carbon atoms. Examples of carbocyclic rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2, 3-dihydro-indene, bicyclo [2.2.2] octyl, adamantyl, and spiro [4.4] nonyl.

As used herein, the term "carboxylic acid" or "carboxyl group" refers to-COOH.

As used herein, "cycloalkylene" means a fully saturated carbocyclic ring or ring system of carbocyclic rings attached to the remainder of the molecule via two attachment points.

As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclic ring or ring system having at least one double bond, wherein none of the rings in the ring system are aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene. As also used herein, "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclic ring or ring system having at least one double bond, having at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic.

As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclic ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclononylyne. As also used herein, "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclic ring or ring system having at least one triple bond with at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic.

As used herein, the term "deposition" refers to any suitable application technique, which may be manual or automated, and results in a change in surface properties. Generally, deposition can be performed using vapor deposition techniques, coating techniques, grafting techniques, or the like. Some specific examples include Chemical Vapor Deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dip coating or dip coating, doctor blade coating, puddle dispensing (puddle dispensing), flow through coating (flow through coating), aerosol printing, screen printing, micro-contact printing, ink jet printing, or similar techniques.

As used herein, the term "recess" refers to a discrete concave feature in a patterned support that has a surface opening that is completely surrounded by the interstitial regions of the patterned support surface. The depressions can have any of a variety of shapes at the openings in their surfaces, including, by way of example, circles, ovals, squares, polygons, stars (with any number of vertices), and the like. The cross-section of the depression taken normal to the surface may be curved, square, polygonal, hyperbolic, tapered, angular, etc. As an example, the recess may be a well. As also used herein, "functionalized recesses" refer to discrete concave features in which the polymers and primers disclosed herein are attached.

When used in reference to a collection of items, the term "each" is intended to identify a single item in the collection, but does not necessarily refer to each (every) item in the collection. Exceptions may occur if explicitly disclosed or the context clearly dictates otherwise.

As used herein, the term "flow cell" is intended to mean a container having a chamber (i.e., a flow channel) in which a reaction can be performed, an inlet for delivering a reagent to the chamber, and an outlet for removing the reagent from the chamber. In some examples, the chamber is capable of detecting a reaction or signal occurring in the chamber. For example, the chamber may include one or more transparent surfaces allowing optical detection of arrays, optically labeled molecules, or the like in the chamber.

As used herein, a "flow channel" or "flow channel region" can be a region defined between two joined components that can selectively receive a liquid sample. In some examples, a flow channel may be defined between the patterned support and the lid, and thus may be in fluid communication with one or more recesses defined in the patterned support. In other examples, a flow channel may be defined between the unpatterned support and the cover.

The Fmoc group is a base labile protecting group having the structure

As used herein, "heteroaryl" refers to an aromatic ring or aromatic ring system (i.e., two or more fused rings that share two adjacent atoms) that includes one or more heteroatoms (i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur) in the ring backbone. When the heteroaryl group is a ring system, each ring in the system is aromatic. Heteroaryl groups may have from 5 to 18 ring members.

As used herein, "heterocyclyl" means a non-aromatic ring or non-aromatic ring system containing at least one heteroatom in the ring backbone. The heterocyclic groups may be joined together in a fused, bridged or spiro-linked manner. The heterocyclyl group may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. In the ring system, the heteroatoms may be present in non-aromatic or aromatic rings. The heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon and heteroatoms). In some examples, the heteroatom is O, N or S.

As used herein, the term "hydrazine" or "hydrazino" refers to optionally substituted-NHNH₂A group.

As used herein, the term "hydrazone" or "hydrazone group" refers toGroup, wherein R_aAnd R_bEach independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

As used herein, "hydroxyl" or "hydroxy" refers to an — OH group.

As used herein, the term "interstitial regions" refers to regions in the support or on the surface that separate the depressions. For example, a gap region may separate one feature of an array from another feature of the array. Two features that are separated from each other may be discrete, i.e., lacking physical contact with each other. In another example, a gap region may separate a first portion of a feature from a second portion of the feature. In many instances, the interstitial regions are continuous, while the features are discrete, e.g., as is the case for more than one well defined in an otherwise continuous surface. The separation provided by the gap region may be a partial or complete separation. The gap region may have a surface material that is different from the surface material of the features defined in the surface. For example, a feature of an array can have an amount or concentration of coating and primers that exceeds the amount or concentration present at the interstitial regions. In some examples, the coating and primers may not be present at the interstitial regions.

As used herein, "nucleotide" includes nitrogen-containing heterocyclic bases, sugars, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e., a sugar lacking a hydroxyl group present at the 2' position in ribose. The nitrogen-containing heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of the deoxyribose is bonded to the N-1 of the pyrimidine or the N-9 of the purine.

As used herein, "plasma ashing" refers to a process of removing organic substances from a support by oxygen plasma. Products resulting from plasma ashing can be removed with a vacuum pump/system. Plasma ashing can activate the support by introducing reactive hydroxyl groups.

Reference herein to a "heteropolymer" or "heteropolymer coating" is intended to mean a macromolecule of at least two different repeating subunits (monomers), wherein one of the repeating subunits (monomers) comprises a stimulus-responsive functional group.

As used herein, a "primer" is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA or single-stranded RNA) that serves as an origin for DNA or RNA synthesis. The 5' end of the primer may be modified to allow a coupling reaction with the functionalized polymer layer. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. In an example, the primer is a short chain ranging from 20 to 40 bases.

As used herein, the terms "silane" and "silane derivative" refer to an organic or inorganic compound that includes one or more silicon atoms. An example of an inorganic silane compound is SiH₄Or halogenated SiH₄Wherein hydrogen is replaced by one or more halogen atoms. Examples of organosilane compounds are X-R^B-Si(OR^C)₃Wherein R-Si is an organic linking group, and wherein X is a functional group such as amino, vinyl, methacrylate, epoxy, sulfur, alkyl, alkenyl, or alkynyl; r^BIs a spacer, e.g. - (CH)₂)_n-, where n is 0 to 1000; r^CSelected from hydrogen, optionally substituted alkyl, optionally substitutedOptionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted 5-10 membered heteroaryl, and optionally substituted 5-10 membered heterocyclyl as defined herein. As used herein, the terms "silane" and "silane derivative" may include mixtures of different silanes and/or silane derivative compounds.

As used herein, a "spacer layer" refers to a material that binds two components together. In some examples, the spacer layer may be, or may be in contact with, a radiation absorbing material that facilitates bonding.

As used herein, "stimulus-responsive functional group" refers to a portion of atoms and/or bonds in a polymer that can change its state in response to a stimulus. The stimulus-responsive functional group can be pH-responsive, temperature-responsive, sugar-responsive, nucleophile-responsive, or salt-responsive. Specific examples of each stimulus-responsive functional group are described further below.

The term flow cell "support" or "substrate" refers to a support or substrate on which a surface chemistry can be added. The term "patterned substrate" refers to a support in or on which the recesses are defined. The term "unpatterned substrate" refers to a generally planar support. The substrate may also be referred to herein as a "support," patterned support, "or" non-patterned support. The support may be a wafer, a panel (panel), a rectangular sheet, a die (die), or any other suitable configuration. The support is generally rigid and insoluble in aqueous liquids. The support may be inert to the chemicals used to modify the depressions. For example, the support may be inert to chemicals used to form the polymer coating, to attach primers to the polymer coating, and the like. Examples of suitable supports include epoxysiloxanes, glass and modified or functionalized glass, polyhedral oligomeric silsesquioxanes (POSS) and derivatives thereof, plastics (including acrylics, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethyleneAlkenes (such as from Chemours)) Cycloolefin/cycloolefin polymers (COP) (such as from Zeon)) Polyimide, etc.), nylon, ceramic/ceramic oxide, silicon dioxide, fused silica or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p + silicon), silicon nitride (Si)₃N₄) Silicon oxide (SiO)₂) Tantalum pentoxide (TaO)₅) Or other tantalum oxides (TaO)_x) Hafnium oxide (HaO)₂) Carbon, metal, inorganic glass, or the like. The support may also be glass or silicon or a silicon-based polymer, such as a POSS material, optionally with a coating of tantalum oxide or another ceramic oxide at the surface.

The term "surface chemistry" as used herein refers to a chemical and/or bioactive component incorporated into a channel of a flow cell. Examples of surface chemistries disclosed herein include a polymeric coating attached to at least a portion of the surface of the support and a primer attached to at least a portion of the polymeric coating.

The "thiol" functional group refers to-SH.

As used herein, the terms "tetrazine" and "tetrazine group" refer to six-membered heteroaryl groups containing four nitrogen atoms. The tetrazine may be optionally substituted.

"tetrazole", as used herein, refers to a five-membered heterocyclic group containing four nitrogen atoms. The tetrazole may be optionally substituted.

Examples of flow cells disclosed herein include a support, a polymer attached to the support, and a primer grafted to the polymer. Examples of flow cells are shown in fig. 1F, 1H, and 3D, and will be described further herein. Various examples of polymers attached to the flow cell support will now be described.

Reference throughout the specification to "one example," "another example," "an example," and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that elements described in connection with any example may be combined in any suitable manner in multiple examples unless the context clearly dictates otherwise.

It is to be understood that the ranges provided herein include the stated ranges and any values or subranges within the stated ranges as if such ranges, values or subranges were expressly enumerated herein. For example, a range from about 400nm to about 1 μm (1000nm) should be interpreted to include not only the explicitly recited limits of from about 400nm to about 1 μm, but also include individual values, such as about 708nm, about 945.5nm, and the like, and sub-ranges, such as from about 425nm to about 825nm, from about 550nm to about 940nm, and the like. Further, when "about" and/or "substantially" are used to describe a value, they are meant to encompass minor variations (up to +/-10%) in the stated value.

The heteropolymers described herein comprise stimulus-responsive functional groups that are capable of undergoing modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the heteropolymer. Thus, examples of heteropolymers disclosed herein may be switchable. The switchable heteropolymer can transition from an initial state to a second switched state when exposed to a particular stimulus. Examples of switching include changes in polarity, such as hydrophobic to hydrophilic, increasing hydrophilicity; hydrophilic to hydrophobic, increasing hydrophobicity; neutral to charged; charged to neutral; anion to neutral; cationic to neutral; neutral to anionic; neutral to cationic; and neutral to neutral with increased hydrophilicity. Examples of switching also include conformational changes such as swelling, collapsed state (collapsed state) to extended state (extended state), extended state to collapsed state (e.g., with reverse polyelectrolyte behavior), and coil-sphere (coil-sphere) formation. A given stimuli-responsive group may confer more than one switching effect. In some aspects, the switching is irreversible, and in other examples, the switching is reversible under different chemical or thermal conditions.

The stimuli-responsive functional group exhibits an initial state when the switchable heteropolymer is applied to the flow cell support. In some aspects, the starting state is compatible with the hydrophobic nature of the flow cell support, and this compatibility simplifies the fabrication and handling of the flow cell. For example, the initial state (e.g., hydrophobic) can improve heteropolymer adhesion to hydrophobic flow cell supports and coating uniformity, while the switched state can be more compatible with flow cell applications such as sequencing operations.

Thus, in some aspects, the switched state may be a solution conformation that provides improved performance in some applications including sequencing operations. Prior to a sequencing operation, the stimulus responsive polymers disclosed herein can be exposed to a predetermined stimulus. The switchable heteropolymer changes polarity and/or conformation when exposed to a stimulus due to the effect of a predetermined stimulus on the stimulus-responsive functional group. Heteropolymers in the switched state can provide a low contamination surface that can reduce non-specific adsorption of proteins and can improve sequencing metrics (e.g., base or first cycle intensity, quality score, error rate, etc.).

One of ordinary skill will recognize that any of the heteropolymers described herein can be a random, block, linear, and/or branched copolymer comprising two or more repeating monomer units in any order or configuration, and can be linear, crosslinked, or branched, or a combination thereof.

In some examples, the stimulus-responsive functional group is a pH-responsive functional group. In such aspects, the switchable heteropolymer is a copolymer comprising more than one monomer containing a pH-responsive functional group. More than one monomer may each have the same pH-responsive functional group or different pH-responsive functional groups that respond to the same pH conditions. In some aspects, the heteropolymer is a copolymer having more than one acrylamide monomer. A single type of acrylamide monomer may be used, or two or more different acrylamide monomers may be used.

In some aspects, upon exposure to acidic or basic pH conditions, the pH-responsive functional group is converted to a substituent group having increased or decreased polarity (e.g., increased hydrophilicity or increased hydrophobicity). In some aspects, the pH-responsive functional group is neutral and becomes charged upon exposure to a stimulus. In some aspects, the pH-responsive functional group is charged and becomes neutral upon exposure to a stimulus. In some aspects, the pH-responsive functional group is neutral and converts to a different neutral but more polar group upon exposure to a stimulus.

In some aspects, the pH-responsive functional group is a hydroxyl group with an acid-labile protecting group (switched to a more hydrophilic free hydroxyl group upon exposure to acidic/low pH conditions), a hydroxyl group with a base-labile protecting group (switched to a more hydrophilic free hydroxyl group upon exposure to basic/high pH conditions), an amino group with an acid-labile protecting group (switched to a more hydrophilic free amino group upon exposure to acidic/low pH conditions), an amino group with a base-labile protecting group (switched to a more hydrophilic free amino group upon exposure to basic/high pH conditions), an amino group (switched to an ammonium ion under acidic/low pH conditions), a carboxylate ester/salt (-CO)₂ ^-) Groups (switching to neutral carboxylic acids upon exposure to acidic/low pH conditions), carboxylic acid groups (switching to charged and more hydrophilic carboxylic acid esters/salts upon exposure to basic/high pH conditions), sulfonate esters (-SO)₃ ^-) A group (switching to neutral sulfonic acid upon exposure to acidic/low pH conditions) or a sulfonic acid group (switching to a charged and more hydrophilic sulfonate upon exposure to basic/high pH conditions).

Exemplary switchable heteropolymers include monomers of the following structure:

wherein:

x is a pH-responsive functional group selected from the group consisting of: -O-PG, -NH-PG, -NR^aR^b、-SO₃H、-SO₃ ^-、-CO₂H and-CO₂ ^-；

PG is an acid-labile or base-labile protecting group (e.g., Boc, Fmoc, or acetal);

R^aand R^bEach independently is H or C_1-4An alkyl group; and is

Each R^zIndependently is H or C_1-4An alkyl group.

In some aspects, X is-O-Boc, -NHBoc, -NHFmoc, -NH₂、-NHCH₃or-N (CH)₃)₂. In some aspects, X is SO₃H、-SO₃ ^-、-CO₂H or-CO₂ ^-. In some aspects, R^aAnd R^bBoth are methyl. In some aspects, R^zIs H or methyl. In some aspects, the monomer has the structure:

thus, in some aspects, the pH-responsive functional group is a hydroxyl, 1, 2-diol or 1, 3-diol (switched to a more polar/hydrophilic diol upon exposure to acidic/low pH conditions) protected as an acetal, hemiacetal or ketal, a tert-butoxycarbonylamino group, a 9H-fluoren-9-ylmethoxycarbonylamino group, an amino group, a carboxylate ester/salt (-CO)₂ ^-) Group, carboxylic acid group, sulfonate (-SO)₃ ^-) A group or a sulfonic acid group.

The tert-butoxycarbonylamino group may be in a hydrophobic (or less hydrophilic) state and may be converted to a hydrophilic state (e.g., an amino group) when exposed to a low (acidic) pH (e.g., having a pH of less than 7). The t-butoxycarbonylamino group may be attached to an acrylamide monomer or an acrylate monomer. Examples of the t-butoxycarbonylamino group-containing monomer include N- (t-butoxycarbonyl-aminoethyl) methacrylamide, N- (t-butoxycarbonyl-aminopropyl) methacrylamide and (2-t-butoxycarbonyl-amino) ethyl methacrylate.

The 9H-fluoren-9-ylmethoxycarbonyl amino group can be in a hydrophobic (or less hydrophilic) state and can be converted to a hydrophilic state (e.g., an amino group) when exposed to a low (acidic) pH (e.g., having a pH of less than 7). The 9H-fluoren-9-ylmethoxycarbonyl amino group can be attached to an acrylamide monomer, or an acrylate monomer or a vinyl monomer. Examples of the monomer containing a 9H-fluoren-9-ylmethoxycarbonyl group include:

the amino group may be in a neutral state and may transition to a charged (and more hydrophilic) state (cationic) when exposed to a low (acidic) pH (e.g., having a pH of less than 7). For example, amino groups in the synthesized polymer may be protonated, which results in a cationic charge around the polymer backbone. In the synthesized polymer, the amino group may be attached to an acrylamide monomer, or an acrylate monomer or a vinyl monomer. Examples of amino group-containing monomers include 2- (dimethylamino) ethyl methacrylate, 2- (N, N-dimethylamino) ethyl acrylate, N- [3- (N, N-dimethylamino) propyl ] acrylamide, N- [2- (N, N-dimethylamino) ethyl ] methacrylamide, and N- [3- (N, N-dimethylamino) propyl ] methacrylamide.

When exposed to low (acidic) pH (e.g., pH less than 7), the acetal group can be converted to a more hydrophilic state than its starting state (hydroxyl or diol). The acetal group can be attached to an azide-functionalized hyaluronic acid (HA-N)₃)。HA-N₃Have limited solubility in organic solvents and can therefore be converted to its tetrabutylammonium salt using acidic ion exchange resins prior to the acetalization reaction. The acetalization reaction can be carried out by reacting 2-methoxypropene and pyridinium p-toluenesulfonate (pyridinium p-toluenesulfonate) with HA-N dissolved in dimethyl sulfoxide (DMSO) at room temperature₃Salt reaction.

In some aspects, the switchable heteropolymer further includes an azide-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:

and optionally

In some aspects, the switchable heteropolymer includes the structure:

wherein each R^zIndependently is H or C_1-4An alkyl group. In some examples, X is-O-Boc, -NHBoc, -NHFmoc, -NH₂、-NHCH₃or-N (CH)₃)₂. In some examples, X is-NHBoc. In some aspects, X is SO₃H、-SO₃ ^-、-CO₂H or-CO₂ ^-. In some aspects, R^aAnd R^bBoth are methyl. In some aspects, each R is^zIndependently is H or methyl.

In some examples, the switchable heteropolymer includes two pH-responsive acetal functional groups (such as one ketal group and one hemiketal group) and one carboxylic acid group. Exemplary heteropolymers have the following structure:

wherein n ranges from 10 to 500. Protection of azido-modified hyaluronic acid (HA-N) by acid-labile acetal groups₃) And changes the properties of the heteropolymer from hydrophilic to hydrophobic. Heteropolymers in their more hydrophobic starting state may be easier to handle and process, allowing the heteropolymers to more effectively coat or adhere to the hydrophobic supports disclosed herein (e.g., norbornene-functionalized glass or POSS substrates).

In some examples, the stimulus-responsive functional group is a temperature-responsive functional group. In such aspects, the switchable heteropolymer is a copolymer comprising more than one monomer containing a temperature-responsive functional group. More than one monomer may each have the same temperature-responsive functional group or different temperature-responsive functional groups that respond to the same temperature conditions. In some aspects, the heteropolymer is a copolymer having more than one acrylamide monomer. A single type of acrylamide monomer may be used, or two or more different acrylamide monomers may be used.

A temperature-responsive functional group is one that can be converted to a more or less polar functional group or that causes a conformational change in the polymer due to a change in temperature. For example, the temperature responsive group includes a thermosensitive hydroxyl or amino protecting group (such as a Boc or Fmoc group) that is removed upon exposure of the heteropolymer to heat (switching from a neutral starting state to a neutral state with increased hydrophilicity). A variety of Boc and Fmoc protected monomers can be used, including acrylic monomers. In another example, the temperature-responsive functional group can cause the polymer to exist in a neutral and relatively hydrophilic extended starting state at room temperature, and then switch the heteropolymer to a neutral and relatively hydrophobic collapsed state at elevated temperatures (such as above 32 ℃). This other example is a coil-sphere switch, which may be displayed, for example, by poly (N-isopropylacrylamide). It will be appreciated that primers grafted to the switchable heteropolymer may alter this behavior somewhat. In some aspects, the polymeric material undergoes a hot coil-to-sphere transition. In some aspects, the temperature-responsive functional group is part of a polymer of the ionizable thermosensitive gel. In some aspects, the monomer comprising a temperature-responsive functional group is an N-substituted acrylamide, such as H₂C ═ C (H or methyl) -C (O) NR^cR^dWherein R is^cIs H, and R^dIs a branched chain C_3-6An alkyl group. In some aspects, the monomer comprising a temperature-responsive functional group is N-isopropylacrylamide, optionally in blocks of poly (N-isopropylacrylamide).

In an example, the heteropolymer includes a temperature responsive functional group monomer and an acrylamide monomer. In some examples, the acrylamide monomer is selected from the group consisting of: azidoacetamidopentylacrylamide monomer and a combination of acrylamide monomer and azidoacetamidopentylacrylamide monomer, as indicated above. In some aspects, the switchable heteropolymer further includes an azide-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:

and optionally

In some aspects, the switchable heteropolymer includes the structure:

wherein each R^zIndependently is H or C_1-4An alkyl group.

In another aspect, the stimulus-responsive functional group is a saccharide-responsive functional group, which is a hydrophilic substituent group that reacts with the diol reagent to form an anionic functional group. In some aspects, the diol is an organic diol, or a sugar, or glucose. In some aspects, the saccharide-responsive functional group comprises a boronic acid, such as an alkyl boronic acid or an aryl boronic acid. The boronic acid functional group can be in an initial state of electrical neutrality (and can also be relatively hydrophobic) and can transition to a negatively charged (anionic) state (which can also be more hydrophilic than the electrical neutral state) when exposed to the sugar solution. Boronic acids have the ability to react with sugars to form boronates that undergo reversible swelling due to influx of water, which may be desirable during sequencing operations.

The boronic acid functional group may be attached to an acrylamide monomer, or an acrylate monomer or a vinyl monomer. In an example, the monomer comprising a saccharide-responsive functional group has the structure:

examples of the boronic acid group-containing monomer include 3- (acrylamido) phenylboronic acid. In some aspects, the switchable heteropolymer further includes an acrylamide monomer. In some aspects, the acrylamide monomer is an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:

and optionally

In some aspects, the heteropolymer has the structure:

wherein each R^zIndependently is H or C_1-4An alkyl group.

In some examples, the stimulus-responsive functional group is a nucleophile-responsive functional group. A nucleophile-responsive functional group is a group that is susceptible to attack by a nucleophile to effect a structural change that imparts a change in polarity and/or conformation as described herein. In some aspects, the switchable heteropolymer is a copolymer comprising more than one monomer containing a nucleophile-responsive functional group. More than one monomer may each have the same nucleophile-responsive functional group or different nucleophile-responsive functional groups that are responsive to the same nucleophile. In some aspects, the heteropolymer is a copolymer of a monomer comprising a nucleophile-responsive functional group and one or more acrylamide monomers. A single type of acrylamide monomer may be used, or two or more different acrylamide monomers may be used. In some examples, the nucleophile-responsive functional group is a cyclic sulfonate ester (such as a sultone ring) or a cyclic anhydride (such as succinic anhydride) which can undergo a ring-opening reaction upon exposure to a nucleophile, in some cases under basic (high pH) conditions (such as pH 9 or higher).

In some aspects, the nucleophile-responsive functional group has the following structure:

wherein (a) Y is SO₂And Y' is CH₂(ii) a Or (b) both Y and Y' are C (O). In other aspects, the nucleophile-responsive functional group is:suitable nucleophiles include primary alkylamines and alkyl alcohols. Examples of sultone ring-opening groups and their ring-opening reactions are as follows:

wherein M is H or a monovalent cation (sodium or potassium cation). The sultone ring-opening group may be in a hydrophobic (or less hydrophilic) state and may undergo a ring-opening reaction and transition to a (more) hydrophilic state when exposed to a high (basic) pH. The functional groups after the ring-opening reaction may also be anionic and thus in a charged state.

In some aspects, the monomer comprising a nucleophile-responsive functional group is:

in a particular example, the monomers are:

in some aspects, the nucleophile-responsive functional group may be attached to an acrylamide monomer, or an acrylate monomer or a vinyl monomer. In some aspects, the switchable heteropolymer further includes an acrylamide monomer. In some examples, the acrylamide monomer is an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:

and optionally

In some aspects, the heteropolymer has the structure:

wherein each R^zIndependently is H or C_1-4An alkyl group.

In some aspects, the stimulus-responsive functional group is a salt-responsive functional group. In some examples, the salt-responsive functional group is a zwitterionic functional group that exhibits reverse polyelectrolyte behavior, wherein the zwitterionic functional group switches from a collapsed state to an extended state upon exposure to a salt. The salt-responsive functional group is a zwitterionic functional group with a reverse polyelectrolyte behavior. As used herein, "reverse polyelectrolyte behavior" means that a monomer comprising a zwitterionic functional group switches from a collapsed state to an extended state (i.e., the monomer has greater solubility in saline than in pure water) when exposed to salt. As such, the salt-responsive functional groups can be in a collapsed state (e.g., where the polymer chains are in spheres) and can transition to an extended state (i.e., where the polymer chains are extended) when exposed to a salt solution. The influence of the local salt counter ion changes the conformation of the polymer chains comprising the salt-responsive functional groups. In one example, the monomer comprising a zwitterionic functional group is selected from the group consisting of: n- (2-methacryloyloxy) ethyl-N, N-dimethylammoniopropanesulfonate and N- (3-methacryloyiimino) propyl-N, N-dimethylammoniopropanesulfonate.

In some examples, the monomer comprising a salt-responsive functional group has the structure:

wherein A is O or NH, and R^zIs H or C_1-4An alkyl group.

In other examples, the salt-responsive functional group is a quaternary ammonium group, such as-NMe₃ ⁺. Exemplary monomers are:

wherein R is^zIs H or C_1-4An alkyl group.

These charged species may exhibit reverse polyelectrolyte behavior when combined with the anionic counterpart (present in the salt solution). Examples of suitable anionic counterparts include carboxylates, sulfonates, citrates, phosphates, and the like.

The salt-responsive functional group may be attached to an acrylamide monomer, or an acrylate monomer or a vinyl monomer. In some aspects, the switchable heteropolymer further includes an acrylamide monomer. In some examples, the acrylamide monomer is an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:

and optionally

In some aspects, the heteropolymer has the structure:

wherein each R^zIndependently is H or C_1-4An alkyl group.

In one example, a flow cell includes a support and a switchable heteropolymer attached to the support, wherein the stimulus-responsive functional group is selected from the group consisting of: a pH-responsive functional group, a temperature-responsive functional group, a saccharide-responsive functional group, a nucleophile-responsive functional group, and a salt-responsive functional group. The stimulus-responsive functional group is capable of undergoing a modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the switchable heteropolymer.

In some aspects, the flow cell support is a patterned substrate comprising recesses separated by interstitial regions, and wherein the heteropolymer is present within the recesses. In other aspects, the support is a non-patterned substrate having a flow channel region and a binding region, and wherein the heteropolymer is attached to the flow channel region.

In some aspects, the flow cell further comprises a primer grafted to the switchable heteropolymer.

In an example of this aspect of the flow cell, the surface of the support is functionalized with a silane or silane derivative, and the heteropolymer is attached to the silane or silane derivative. In some examples, the silane or silane derivative includes an unsaturated moiety capable of reacting with a functional group of the functionalized polymer layer. As used herein, the term "unsaturated moiety" refers to a chemical group comprising at least one double bond or one triple bond, including cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants thereof. The unsaturated moiety may be monovalent or divalent. When the unsaturated moiety is monovalent, cycloalkene, cycloalkyne, heterocycloalkene, and heterocycloalkyne are used interchangeably with cycloalkenyl, cycloalkynyl, heterocycloalkenyl, and heterocycloalkynyl, respectively. When the unsaturated moiety is divalent, cycloalkene, cycloalkyne, heterocyclic alkene, and heterocyclic alkyne are used interchangeably with cycloalkenylene, cycloalkynylene, heterocyclic alkenylene, and heterocyclic alkynylene, respectively.

The unsaturated moiety may be covalently attached directly to the silicon atom of the silane or silane derivative, or indirectly via a linker. Examples of suitable linkers include optionally substituted alkylene groups (e.g., divalent saturated aliphatic groups such as ethylene, believed to be derived from alkenes by opening a double bond, or from alkanes by removing two hydrogen atoms from different carbon atoms), substituted polyethylene glycols, or the like.

The heteropolymers disclosed herein are comprised of at least two different monomers. One of the monomers contains a stimulus-responsive functional group. In some aspects, another of the monomers comprises an attachment group that can react with the flow cell support and/or the primer to attach the heteropolymer thereto. The further attachment group may also be capable of attaching to the support, or the further monomer may comprise a second (different) attachment group capable of attaching to the support. It is to be understood that the polymers disclosed herein may also include one or more other monomers that do not interfere with the respective functions of the stimulus-responsive functional group and the attachment group.

In any example of the polymer disclosed herein, the attachment group is selected from the group consisting of: azido, amino, alkenyl (including cycloalkenyl or heterocycloalkenyl groups), alkynyl (including cycloalkynyl or heterocycloalkynyl groups), aldehyde, hydrazone, hydrazine, carboxyl, hydroxyl, tetrazole, tetrazine, and thiol.

The attachment group may be capable of reacting with a functional group attached to the 5' end of the primer. For example, a bicyclo [6.1.0] non-4-yne (BCN) -terminated primer can be captured by the azide attachment group of the polymer via strain-promoted catalyst-free click chemistry. For another example, an alkyne-terminated primer can be captured by the azide attachment group of the polymer via copper-catalyzed click chemistry. For yet another example, a norbornene-terminated primer can undergo a catalyst-free ring strain-promoted click reaction with the tetrazine attachment group of the polymer. It will be appreciated that other coupling chemistries may be used to attach the primers to the attachment groups including, for example, Staudinger ligation, strain-promoted reactions, and light-click cycloadditions.

Other examples of blocked primers that can be used include tetrazine blocked primers, azido blocked primers, amino blocked primers, epoxy or glycidyl blocked primers, phosphorothioate blocked primers, thiol blocked primers, aldehyde blocked primers, hydrazine blocked primers, and triazolinedione blocked primers.

In an example, the attachment group is attached to an acrylamide monomer. One example of a monomer comprising an attachment group is azidoacetamidopentyl acrylamide.

In examples of methods, applying the polymer coating to the flow cell involves flow-through deposition, chemical vapor deposition, dip coating, spin coating, spray coating, puddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, or inkjet printing.

In an example of the method, the flow cell support is a patterned flow cell support comprising recesses separated by gap regions, and the method further comprises: attaching a silane or silane derivative to the surface of the patterned flow cell support prior to applying the polymeric coating, thereby forming silanized depressions and silanized interstitial regions; applying a polymer coating in the silanized depressions and on the silanized interstitial regions; and removing (e.g., polishing) the polymer coating from the silanized interstitial regions.

In an example of the method, exposing the polymer coating to the predetermined stimulus involves one of: heating the polymer coating; exposing the polymer coating to a solution of a predetermined pH; exposing the polymeric coating to a nucleophile; exposing the polymeric coating to a solution comprising a sugar; or exposing the polymer coating to a salt solution.

In yet a further aspect, a method includes exposing a polymeric coating on at least a portion of a flow cell support to a predetermined stimulus, thereby causing stimulus-responsive functional groups of the polymeric coating to i) switch from a current state to a more hydrophilic state than the current state, or ii) switch from a neutral state to a charged state, or iii) switch from a collapsed state to an extended state; and performing a sequencing operation using the flow cell support when the polymer coating is in a more hydrophilic state, a charged state, or an extended state.

In some aspects, a method for manufacturing a flow cell. The method comprises applying a switchable heteropolymer to at least a portion of the flow cell support.

The addition of polymers (polymer coatings) and primers (i.e., surface chemistries) to the patterned substrate will be described with reference to fig. 1A-1F, and fig. 1A-1D in conjunction with fig. 1G and 1H, and the addition of surface chemistries to the unpatterned substrate will be described with reference to fig. 2A-2D.

Fig. 1A is a cross-sectional view of an example of a patterned support 12. The patterned support 12 may be a patterned wafer or a patterned die or any other patterned support (e.g., a panel, a rectangular sheet, etc.). Any of the examples of supports 12 described herein may be used. The patterned wafer may be used to form several flow cells, and the patterned die may be used to form a single flow cell. In examples, the support may have a diameter in the range of from about 2mm to about 300mm, or a rectangular sheet or panel having a maximum dimension of up to about 10 feet (-3 meters). In an example, the support wafer has a diameter in a range from about 200mm to about 300 mm. In another example, the support die has a width in a range from about 0.1mm to about 10 mm. While exemplary dimensions have been provided, it should be understood that supports/substrates having any suitable dimensions may be used. For another example, a panel that is a rectangular support with a larger surface area than a 300mm circular wafer may be used.

Patterning the support 12 includes recesses 14 defined on or in an exposed layer or surface of the support 12, and gap regions 16 separating adjacent recesses 14. In the examples disclosed herein, the recesses 14 are functionalized by surface chemistry (e.g., 20, 22), while the gap regions 16 may be available for binding, but no primers will be present thereon (shown in fig. 1E, 1F, and 1H).

The recesses 14 may be fabricated in or on the support 12 using a variety of techniques including, for example, photolithography, nanoimprint lithography, stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, and the like. As will be appreciated by those skilled in the art, the technique used will depend on the composition and shape of the support 12.

Many different layouts of the recesses 14 are envisaged, including regular, repeating and irregular patterns. In the example, the depressions 14 are arranged in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectilinear (i.e., rectangular) layouts, triangular layouts, and the like. In some examples, the layout or pattern may be an x-y format of the recesses 14 in rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of recesses 14 and/or interstitial regions 16. In still other examples, the layout or pattern may be a random arrangement of the recesses 14 and/or interstitial regions 16. The pattern may include spots, pads, wells, columns, stripes, swirls, lines, triangles, rectangles, circles, arcs, checkerboards, squares, diagonals, arrows, squares, and/or cross-hatching.

The layout or pattern may be characterized with respect to the density of the recesses 14 (i.e., the number of recesses 14) in the defined region. For example, the depressions 14 may be provided at a rate of per mm²A density of about 200 million exists. The density may be adjusted to different densities, including, for example, per mm²At least about 100, per mm²About 1,000, per mm²About 10 ten thousand per mm²About 100 million, per mm²About 200 ten thousand per mm²About 500 million per mm²About 1000 million, per mm²A density of about 5000 ten thousand or more. Alternatively or additionally, the density may be adjusted to be per mm²Not greater than about 5000 ten thousand per mm²About 1000 million, per mm²About 500 million per mm²About 200 ten thousand per mm²About 100 million, per mm²About 10 ten thousand per mm²About 1,000, per mm²About 100 or less. It is also understood that the density of the depressions 14 on the support 12 can be between one of the lower and one of the upper values selected from the ranges above. By way of example, a high density array may be characterized as having depressions 14 separated by less than about 100nm, a medium density array may be characterized as having depressions 14 separated by about 400nm to about 1 μm, and a low density array may be characterized as having depressions 14 separated by about 400nm to about 1 μmWith recesses 14 larger than about 1 μm. While exemplary densities have been provided, it should be understood that substrates having any suitable density may be used.

The layout or pattern may also or alternatively be characterized in terms of average pitch, i.e., the spacing from the center of a depression 14 to the center of an adjacent interstitial region 16 (center-to-center spacing). The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch can be, for example, at least about 10nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or greater. Alternatively or additionally, the average pitch may be, for example, up to about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. The average spacing of the particular pattern of sites 16 may be between one of the lower values and one of the upper values selected from the ranges above. In an example, the depressions 14 have a pitch (center-to-center spacing) of about 1.5 μm. While an exemplary average pitch value has been provided, it should be understood that other average pitch values may also be used.

In the example shown in fig. 1A-1H, the recesses 14 are wells 14 ', and thus the patterned support 12 includes an array of wells 14' in its surface. The wells 14' may be micro-wells or nano-wells. The dimensions of each well 14' may be characterized by its volume, well opening area, depth, and/or diameter.

Each well 14' may have any volume capable of confining a liquid. The minimum volume or the maximum volume may be selected, for example, to accommodate throughput (e.g., multiplicity), resolution, analyte composition, or analyte reactivity expected for downstream use of the flow cell. For example, the volume may be at least about 1 x 10^-3μm³About 1X 10^-2μm³About 0.1 μm³About 1 μm³About 10 μm³About 100 μm³Or larger. Alternatively or additionally, the volume may be up to about 1 × 10⁴μm³About 1X 10³μm³About 100 μm³About 10 μm³About 1 μm³About 0.1 μm³Or smaller. It is understood that the functionalized coating may fill all or part of the volume of the well 14'. The volume of coating in an individual well 14' may be greater than, less than, or between the values specified above.

The area occupied by each well opening on the surface may be selected based on criteria similar to those set forth above with respect to well volume. For example, the area for each well opening on the surface may be at least about 1 x 10^-3μm²About 1X 10^-2μm²About 0.1 μm²About 1 μm²About 10 μm²About 100 μm²Or larger. Alternatively or additionally, the area may be up to about 1 × 10³μm²About 100 μm³About 10 μm²About 1 μm²About 0.1 μm²About 1X 10^-2μm²Or smaller. The area occupied by each well opening may be greater than, less than, or between the values specified above.

The depth of each well 14' may be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or greater. Alternatively or additionally, the depth may be up to about 1 × 10³μ m, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm, or less. The depth of each well 14' may be greater than, less than, or between the values specified above.

In some cases, the diameter of each well 14' can be at least about 50nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the diameter may be up to about 1 × 10³μ m, about 100 μm, about 10 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less (e.g., about 50 nm). The diameter of each well 14' may be greater than, less than, or between the values specified above.

The patterned support 12 may be exposed to a series of processes to add the surface chemistry 20, 22 in the recesses 14.

Although not shown, it is understood that patterned support 12 may be exposed to plasma ashing in order to clean and activate the surface. For example, the plasma ashing process can remove organic materials and introduce surface hydroxyl groups. Other suitable cleaning processes may be used to clean the support 12, depending in part on the type of support 12. For example, chemical cleaning may be performed with an oxidizing agent or a caustic solution.

The patterned support 12 (shown in fig. 1A) may then be exposed to a process that will prepare the surface for deposition of the stimuli-responsive polymer disclosed herein to form the polymer coating 20 (fig. 1C). In an example, patterned support 12 may be exposed to silanization, which attaches silane or silane derivative 18 (fig. 1B) to the patterned support surface. Silanization introduces silane or silane derivative 18 across the surface, including in the recesses 14, 14' (e.g., on the bottom surface and along the sidewalls) and on the gap regions 16. In some aspects, the silane or silane derivative is selectively introduced only to the recesses of the patterned substrate or to the micro-locations of the non-patterned substrate (which are isolated from each other).

Silanization can be accomplished using any silane or silane derivative 18. The choice of silane or silane derivative 18 may depend in part on the polymer used to form the polymeric coating 20 (shown in fig. 1C), as it may be desirable to form a covalent bond between the silane or silane derivative 18 and the polymeric coating 20. The method used to attach the silane or silane derivative 18 to the support 12 may vary depending on the silane or silane derivative 18 used. Several examples are set forth herein.

In the examples, the silane or silane derivative 18 is (3-aminopropyl) triethoxysilane (APTES) or (3-aminopropyl) trimethoxysilane (APTMS) (i.e., X-R)^B-Si(OR^C)₃Wherein X is amino, R^BIs- (CH)₂)₃-, and R^CIs ethyl or methyl). In this example, the surface of support 12 may be pretreated with (3-aminopropyl) triethoxysilane (APTES) or (3-aminopropyl) trimethoxysilane (APTMS) to covalently attach silicon to one or more oxygen atoms on the surface (without intending to be held by a mechanism, each of which is each held bySilicon may be bonded to one, two or three oxygen atoms). The chemically treated surface is baked to form a monolayer of amine groups. The amine group then reacts with the sulfo HSAB to form the azido derivative. At 21 ℃ with a density of 1J/cm²To 30J/cm²UV activation of energy of (a) produces a reactive nitrene species that can readily undergo a variety of insertion reactions with the polymers disclosed herein.

Other silanization methods may also be used. Examples of suitable silylation methods include vapor deposition, YES methods, spin coating, or other deposition methods. Some examples of methods and materials that may be used to silanize support 12 are described herein, but it should be understood that other methods and materials may also be used.

In the example using a YES CVD oven, the patterned support 12 is placed in a CVD oven. The chamber may be vented and then the silylation cycle started. During the circulation, the silane or silane derivative container may be maintained at a suitable temperature (e.g., about 120 ℃ for norbornene silane), the silane or silane derivative vapor line is maintained at a suitable temperature (e.g., about 125 ℃ for norbornene silane), and the vacuum line is maintained at a suitable temperature (e.g., about 145 ℃).

In another example, silane or silane derivative 18 (e.g., liquid norbornene silane) may be deposited in a glass bottle and placed in a glass vacuum dryer with patterned support 12. The dryer may then be evacuated to a pressure in the range of from about 15mTorr to about 30mTorr and placed in an oven at a temperature in the range of from about 60 ℃ to about 125 ℃. The silanization was allowed to proceed and the dryer was then removed from the oven, cooled and vented in air.

The vapor deposition, YES process, and/or vacuum dryer may be used with a variety of silanes or silane derivatives 18, such as those silanes or silane derivatives 18 that include examples of the unsaturated moieties disclosed herein. By way of example, these methods may be used when the silane or silane derivative 18 includes a cyclic olefin unsaturated moiety such as norbornene, norbornene derivatives (e.g., (hetero) norbornene containing oxygen or nitrogen in place of one of the carbon atoms), trans-cyclooctene derivatives, trans-cyclopentene, trans-cycloheptene, trans-cyclononene, bicyclo [3.3.1] non-1-ene, bicyclo [4.3.1] dec-1 (9) -ene, bicyclo [4.2.1] non-1 (8) -ene, and bicyclo [4.2.1] non-1-ene. Any of these cycloalkenes may be substituted, for example, with an R group such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl) alkyl. Examples of norbornene derivatives include [ (5-bicyclo [2.2.1] hept-2-enyl) ethyl ] trimethoxysilane. As other examples, these methods may be used when the silane or silane derivative 18 includes an alkyne or cycloalkyne unsaturated moiety such as cyclooctyne, a cyclooctyne derivative, or bicyclononylene (e.g., bicyclo [6.1.0] non-4-yne or derivatives thereof, bicyclo [6.1.0] non-2-yne, or bicyclo [6.1.0] non-3-yne). These cycloalkynes may be substituted with any of the R groups described herein.

As shown in fig. 1B, the attachment of the silane or silane derivative 18 forms a silanized patterned support, including silanized recesses and silanized gap regions (which are one example of processed recesses and processed gap regions).

The silanized patterned support may then be exposed to a process that will form a polymer coating 20 on the silanized depressions and silanized interstitial regions.

Prior to applying the polymeric coating 20, some examples of methods may involve synthesizing heteropolymers to be deposited to form the polymeric coating 20. The synthesis may involve copolymerizing a stimulus-responsive functional group-containing monomer with a monomer selected from the group consisting of: azidoacetamidopentylgrylamide monomers and combinations of acrylamide monomers and azidoacetamidopentylgrylamide monomers. Any of the pH-responsive monomers, temperature-responsive monomers, sugar-responsive monomers, nucleophile-responsive monomers, or salt-responsive monomers described herein can be used to form the heteropolymer. Several methods may be used to prepare the polymeric materials disclosed herein. As a few examples, the polymerization method used may be free radical polymerization, controlled radical polymerization, a non-free radical method, or another suitable method.

As described herein, examples of the polymeric coating 20 include any stimulus-responsive polymer disclosed herein, including any example of a stimulus-responsive functional group and any example of an attachment group. The stimuli-responsive polymer may be present in the mixture or incorporated into the mixture. In an example, the mixture includes a stimulus responsive polymer in a mixture of ethanol and water. The polymer coating 20 can be formed on the surface of the silanized patterned support 12 (i.e., on the silanized depressions and silanized gap regions) using any suitable technique. The stimuli-responsive polymer may be deposited on the surface of patterned support 12 using Chemical Vapor Deposition (CVD), or dipping, dip coating, spin coating, spray coating or ultrasonic spray coating, puddle dispensing, doctor blade coating, aerosol printing, screen printing, micro-contact printing, or inkjet printing, or via other suitable techniques. The polymer coating 20 is shown in fig. 1C.

Dip coating may involve immersing the patterned and silanized support in a series of temperature controlled baths. The bath may also be flow controlled and/or covered with a nitrogen blanket (nitrogen blanket). The bath may comprise a polymer mixture. Throughout the multiple baths, the stimuli-responsive polymer will attach to form the polymer coating 20 in the silanized depressions and on the interstitial regions. In an example, the patterned and silanized support would be introduced into a first bath containing a polymer mixture where a reaction occurs to attach the polymer, and then the patterned, silanized, and polymer coated support would be moved to another bath for washing. The patterned support may be moved from bath to bath by a robotic arm or manually. The drying system may also be used in dip coating.

Spraying can be accomplished by spraying the polymer mixture directly onto the patterned and silanized support. The sprayed support may be incubated for a time sufficient to attach the polymer. After incubation, any unattached polymer mixture can be diluted and removed using, for example, a spin coater or by sonication in a bath or dip tank as described herein.

Puddle dispensing can be done according to the pool and spin-out method, and can therefore be done with a spin coater. The polymer mixture may be applied (manually or via an automated process) to the patterned and silanized support. The applied polymer mixture may be applied to the entire surface of the patterned and silanized support or spread across the entire surface. The polymer-coated patterned support can be incubated for a time sufficient to attach the polymer. After incubation, any unattached polymer mixture can be diluted and removed using, for example, a spin coater or by sonication in a bath or dip tank as described herein.

The attachment of the polymer coating 20 to the silanized depression and silanized interstitial regions (i.e., 18) may be by covalent bonding. Covalently attaching the polymer coating 20 to the silanized depressions is helpful in retaining the polymer coating 20 in the depressions 14, 14' throughout the life of the ultimately formed flow cell during various uses. The following are some examples of reactions that may occur between the silane or silane derivative 18 and the polymer coating 20.

When the silane or silane derivative 18 includes norbornene or norbornene derivative as the unsaturated moiety, the norbornene or norbornene derivative may: i) (ii) undergoes a 1, 3-dipolar cycloaddition reaction with the azide/azido groups of the stimulus-responsive polymer; ii) undergoes a coupling reaction with a tetrazine group attached to a stimuli-responsive polymer; iii) undergoes a cycloaddition reaction with a hydrazone group attached to the stimulus-responsive polymer; iv) undergoes a light click reaction with a tetrazolyl group attached to the stimulus-responsive polymer; or v) undergoes cycloaddition with a nitrile oxide group attached to the stimulus responsive polymer.

When silane or silane derivative 18 includes cyclooctyne or a cyclooctyne derivative as the unsaturated moiety, the cyclooctyne or cyclooctyne derivative may: i) undergo a strain-promoted azide-alkyne 1, 3-cycloaddition (SPAAC) reaction with the azide/azide groups of the stimulus-responsive polymer, or ii) undergo a strain-promoted alkyne-nitrile oxide cycloaddition reaction with a nitrile oxide group attached to the stimulus-responsive polymer.

When silane or silane derivative 18 includes bicyclononene as the unsaturated moiety, the bicyclononene may undergo a similar SPAAC alkyne cycloaddition to an azide or nitrile oxide attached to the stimulus-responsive polymer due to strain in the bicyclic ring system.

Although not shown, it is understood that in some examples of the method, patterned support 12 may not be exposed to silylation. Rather, patterned support 12 may be exposed to plasma ashing, and then polymer coating 20 may be spin coated (or otherwise deposited) directly on plasma ashed patterned support 12. In this example, plasma ashing can generate a surfactant (e.g., -OH groups, such as hydroxyl or carboxyl groups) that can adhere the polymeric coating 20 to the patterned support 12. In these examples, other functional groups of the polymer coating 20 can be selected such that it reacts with surface groups generated by plasma ashing. For example, the other functional group of the polymer coating 20 may be N-hydroxysuccinimide ester (NHS ester).

After being coated, the stimulus-responsive polymer may also be exposed to a curing process to form a polymer coating 20 across the entire patterned substrate (i.e., on the recesses 14 and interstitial regions 16). In an example, curing the stimulus-responsive polymer can be performed at a temperature in a range from room temperature (e.g., about 25 ℃) to about 60 ℃ for a time in a range from about 5 minutes to about 2 hours.

The silanized and coated patterned substrate (shown in fig. 1C) may be exposed to a cleaning process. The process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature in the range of from about 22 ℃ to about 45 ℃. In another example, the water bath temperature is in a range from about 25 ℃ to about 30 ℃.

The silanized and coated patterned support is then exposed to polishing, if necessary, to remove a portion of the polymer coating 20 from the silanized interstitial regions. The silanized, coated and polished patterned substrate is shown in fig. 1D. The portion of the silane or silane derivative 18 adjacent to the gap region 16 may or may not be removed as a result of polishing. Thus, in fig. 1D-1H, portions of the silane or silane derivative 18 adjacent to the gap regions 16 are shown in dashed lines because they may at least partially remain after polishing, or they may be removed after polishing. When these silanized portions are completely removed, it is understood that the underlying support 12 is exposed.

The polishing process may be performed with a mild chemical slurry (including, for example, abrasives, buffers, chelating agents, surfactants, and/or dispersants) that can remove the thin polymer coating 20 and, in some cases, at least a portion of the silane or silane derivative 18 from the interstitial regions 16 without detrimentally affecting the underlying support 12 at these regions. Alternatively, polishing may be performed with a solution that does not contain abrasive particles.

The chemical slurry may be used in a chemical mechanical polishing system to polish the surface of the silanized and coated patterned support shown in fig. 1C. A polishing head/pad or other polishing tool is capable of polishing the polymer coating 20 from the gap region 16 while leaving the polymer coating 20 in the recesses 14, 14' and at least substantially intact with the underlying support 12. As an example, the polishing head may be a Strasbaugh ViPRR II polishing head.

As mentioned above, polishing can be carried out with a polishing pad and a solution that does not contain any abrasives. For example, the polishing pad can be used with a solution that does not contain abrasive particles (i.e., a solution that does not contain abrasive particles).

The polishing removes portions of the polymer coating 20 (and in some cases at least a portion of the silane or silane derivative 18) from the interstitial regions 16 and leaves portions of the polymer coating 20 in the silanized recesses, as shown in fig. 1D. As also mentioned above, the gap region 16 may remain silanized after polishing is complete. In other words, the silanized gap region may remain intact after polishing. Alternatively (as indicated by the dashed portions of 18), the silane or silane derivative 18 may be removed from the gap region 16 as a result of polishing.

Although not shown, it is understood that the silanized, coated, and polished patterned support (shown in fig. 1D) may be exposed to a cleaning process. The process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature in the range of from about 22 ℃ to about 30 ℃. The silanized, coated, and polished patterned substrate may also be spin dried, or dried via another suitable technique.

The silanized, coated and polished patterned support shown in fig. 1D may then be exposed to the process shown in fig. 1E and 1F, which produces flow cell 10, or to the process shown in fig. 1G and 1H, which produces flow cell 10'. In fig. 1E and 1F, the primers 22 are grafted before the lid 26 is bonded to the patterned flow cell support 12. In fig. 1G and 1H, a cap 26 is bonded to the patterned flow cell support 12 before the primer 22 is grafted.

In fig. 1E, a grafting process is performed to graft the primer 22 to the polymer coating 20 in the recess 14, 14'. The primer 22 may be any forward amplification primer or reverse amplification primer that contains an alkyne functional group. Specific examples of suitable primers include the P5 primer and/or the P7 primer used on the surface of a commercial flow cell sold by Illumina inc^TM、HISEQX^TM、MISEQ^TM、MISEQDX^TM、MINISEQ^TM、NEXTSEQ^TM、NEXTSEQDX^TM、NOVASEQ^TM、GENOME ANALYZER^TM、ISEQ^TMAnd sequencing on other instrument platforms.

In this example, grafting can be accomplished by flow-through deposition (e.g., using a temporarily bonded lid), dip coating, spray coating, puddle dispensing, or by another suitable method of attaching primers 20 to the functionalized polymer layer 20 in at least some of the recesses 14, 14'. Each of these exemplary techniques may utilize a primer solution or mixture, which may comprise a primer, water, a buffer, and a catalyst, and may be performed as described herein.

The dip coating may involve immersing the patterned support (with the polymer coating 20 in its recesses 14) in a series of temperature-controlled baths. The bath may also be flow controlled and/or covered with a nitrogen blanket. The bath may contain a primer solution or mixture. Throughout the plurality of baths, the primers 22 will attach to the attachment groups of the polymer coating 20 in at least some of the recesses 14. In an example, the coated and polished patterned support will be introduced into a first bath containing a primer solution or mixture where a reaction occurs to attach the primer, and then the patterned substrate will be moved to another bath for washing. The patterned substrate may be moved from bath to bath by a robotic arm or manually. The drying system may also be used in dip coating.

Spraying can be accomplished by spraying the primer solution or mixture directly onto the coated and polished patterned support. The sprayed wafer may be incubated at a temperature ranging from about 0 ℃ to about 70 ℃ for a time ranging from about 4 minutes to about 60 minutes. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

Puddle dispensing can be done according to the pool and spin-out method, and can therefore be done with a spin coater. The primer solution or mixture may be applied (manually or via an automated process) to the coated and polished patterned support. The applied primer solution or mixture may be applied to or spread across the entire surface of the coated and polished patterned support. The primer coated patterned substrate can be incubated at a temperature in the range from about 0 ℃ to about 80 ℃ for a time in the range from about 2 minutes to about 60 minutes. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

As depicted in fig. 1F, the cover 26 may then be bonded to the bonding area 25 of the support 12. When the patterned flow cell support 12 is a wafer, different regions of the cover 26 may at least partially define respective flow channels 30 formed using the wafer. When the patterned flow cell support 12 is a die, the cover 26 may define one or more flow channels 30 formed.

The cover 26 may be any material that is transparent to the excitation light directed to the surface chemistry 20, 22 in the recess 14. By way of example, the cover 26 may be glass (e.g., borosilicate, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D available from Schott North America, IncA commercially available example of a suitable plastic material, i.e. a cyclic olefin polymer, is available from Zeon Chemicals l.pAnd (5) producing the product.

In some examples, the cover 26 may be integrally formed with the sidewall 29, the sidewall 29 corresponding to the shape of the bonding area 25 and to be bonded to the bonding area 25. For example, a recess (stress) may be etched into the transparent block to form a substantially planar (e.g., top) portion 27 and sidewalls 29 extending from the substantially planar portion 27. The grooves may become flow channels 30 when the etched blocks are mounted to the bonding areas of the patterned substrate 12.

In other examples, the sidewall 29 and the cover 26 may be separate components coupled to one another. For example, the lid 26 may be a generally rectangular block having an at least generally planar outer surface and an at least generally planar inner surface that defines a portion (e.g., a top portion) of the flow channel 30 (once bonded to the patterned support 12). The block may be mounted to (e.g., bonded to) a sidewall 29, the sidewall 29 bonded to the bonding region 25 of the patterned flow cell substrate 12 and forming a sidewall of the flow channel 30. In this example, the sidewalls 29 can comprise any of the materials set forth herein with respect to the spacer layer (described below).

The cover 26 may be bonded to the bonding region 25 of the patterned flow cell support 12 using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, frit bonding, or other methods known in the art. In an example, a spacer layer 28 may be used to bond the cover 26 to the bonding region 25. Spacer layer 28 may be any material that seals at least some of the gap regions 16 (e.g., bonding regions 25) of patterned substrate 12 and cap 26 together.

In one example, spacer layer 28 may be a radiation absorbing material that absorbs radiation at wavelengths transmitted by cover 26 and/or patterned support 12. The absorbed energy in turn forms bonds between the spacer layer 28 and the cap 26 and between the spacer layer 28 and the patterned substrate 12. An example of such a radiation absorbing material is black from DuPont (USA) absorbing at about 1064nm(polyimide containing carbon black). It is understood that the polyimide can be used without the addition of carbon black, except that the wavelength must be changed to a wavelength that is significantly absorbed by the natural polyimide material (e.g., 480 nm). As another example, polyimide CEN JP may be incorporated when irradiated with light at 532 nm. When the spacer layer 28 is a radiation absorbing material, the spacer layer 28 may be positioned at the interface between the cover 26 and the patterned support 12 such that the spacer layer 28 contacts the desired bonding region 25. Compression (e.g., pressure of about 100 PSI) may be applied while laser energy of a suitable wavelength is applied to the interface (i.e., the radiation absorbing material is irradiated). Laser energy may be applied to the interface from both the top and bottom in order to achieve a suitable bond.

In another example, the spacer layer 28 may include a radiation absorbing material in contact therewith. Radiation absorbing material may be applied at the interface between spacer layer 28 and cover 26 and at the interface between spacer layer 28 and patterned flow cell support 12. By way of example, the spacer layer 28 may be polyimide and the separate radiation absorbing material may be carbon black. In this example, the separate radiation absorbing material absorbs the laser energy that forms the bond between spacer layer 28 and cover 26 and between spacer layer 28 and patterned support 12. In this example, compression may be applied at the respective interfaces while laser energy at a suitable wavelength is applied to the interfaces (i.e., the radiation absorbing material is irradiated).

When the patterned flow cell support 12 is a wafer, the spacer layer 28 and the sidewall 29 (of the lid 26 or connected to the lid 26) may physically separate one flow channel 30 from an adjacent flow channel 30, and may be located at the periphery of the wafer. When the patterned support 12 is a die and the flow cell 10 being formed will include a single flow channel 30 or lane (lane), the spacer layer 28 and the sidewall 29 (of the cover 26 or connected to the cover 26) may be located at the perimeter of the die to define the flow channel 30 and seal the flow cell 10. When patterned support 12 is a die and the flow cell 10 being formed will include a plurality of isolated flow channels 30 (e.g., eight flow channels/lanes or four flow channels/lanes), spacer layer 28 and sidewall 29 (of cover 26 or connected to cover 26) may physically separate one flow channel/lane 30 from an adjacent flow channel/lane 30 and may be located at the perimeter of the die. However, it should be understood that the spacer layer 28 and sidewalls 29 may be located in any desired area depending on the implementation.

When the patterned support 12 is a die, assembling the flow cell 10 may involve the incorporation of a lid 26. When patterned support 12 is a wafer, assembling flow cell 10 may involve additional processing, such as dicing, after cover 26 is bonded. In one example, the cover 26 may be bonded to a patterned wafer and diced to form individual flow cells 10. As mentioned herein, the sidewalls 29 may physically separate one flow channel 30 from an adjacent flow channel 30 on the wafer, and thus dicing may occur through at least some of the sidewalls 29, such that each individual flow cell 10 includes a desired number of flow channels 30, each flow channel 30 having a portion of the original sidewall 29 around its perimeter. In another example, the patterned wafer may be diced to form lidless dies that may have respective covers 26 bonded thereto to form individual flow cells 10.

In the exemplary flow cell 10 shown in fig. 1F, the lid 26 includes a top portion 27 integrally formed with a sidewall 29. The sidewalls 29 are bonded to the bonding regions 25 of the patterned substrate 12 through the spacers 28.

The cover 26 and the patterned flow cell substrate 12 together define a flow channel 30, the flow channel 30 being in selective fluid communication with the recesses 14, 14'. The flow channels 30 may be used, for example, to selectively introduce reactive components or reactants to the surface chemistries 20, 22 to initiate a specified reaction in/at the recesses 14, 14'.

Prior to performing a sequencing operation, flow cell 10 may be exposed to a predetermined stimulus of polymer coating 20 in order to transition polymer coating 20 from a current state to a more hydrophilic state (e.g., from a hydrophobic state to a hydrophilic state), from a neutral state to a charged state, and/or from a collapsed state to an extended state. The predetermined stimulus used will depend on the polymeric coating 20 and the stimulus-responsive functional groups it comprises. Exposing the polymeric coating 20 to a predetermined stimulus can involve heating the polymeric coating 20; exposing the polymer coating 20 to a solution of a predetermined pH; exposing the polymer coating 20 to a solution comprising a sugar; exposing the polymer coating 20 to a nucleophile; or exposing the polymer coating 20 to a salt solution. Exposing the polymer coating 20 to any solution while the cap 26 is attached can be accomplished by a flow through process. For example, a basic or acidic solution, a sugar solution (e.g., glucose), or a salt solution may be introduced into the flow cell channel 30 through the input port, allowed to incubate for a time sufficient for the desired property change to occur, and then removed from the channel 30 through the output port. In examples, the incubation time may be from a few seconds to several minutes. When the stimulus-responsive functional group is thermally responsive, the entire flow cell 10 can be heated, or the heated solution can be exposed to the polymeric coating 20 using a flow-through process.

The predetermined stimulus will make the polymer coating 20 more compatible with the conditions of the subsequent sequencing operation.

Examples of the solution of the predetermined pH may include an alkaline solution such as 0.1M NaOH, TRIS-HCL buffer, or carbonate buffer, or an acidic solution such as citrate buffer (pH6) or 2- (N-morpholino) ethanesulfonic acid (MES) buffer. Examples of sugar solutions include glucose solutions having a concentration in the range from about 1mM to about 100 mM. Examples of salt solutions include saline-sodium citrate buffer and Phosphate Buffered Saline (PBS) buffer.

Referring now to fig. 1G and 1H, another example of a method includes bonding a lid 26 to the patterned flow cell support 12 before the primer 22 is grafted.

As shown in fig. 1G, the polymer coating 20 has been applied (e.g., deposited and polished) as described in fig. 1D. At least some of the polished gap regions 16 may define a bonding region 25, and a cover 26 may be bonded to the bonding region 25. The cover 26 may be any material and may have any configuration described herein. The cover 26 may be bonded to the bonding region 25 via any of the techniques described herein.

In the example shown in fig. 1G, the cover 26 includes a top portion 27 integrally formed with a sidewall 29. The sidewalls 29 are bonded to the bonding regions 25 of the patterned substrate 12 through the spacers 28. After the cap 26 is bonded, a flow channel 30 is formed between the cap 26 and the patterned substrate 12. Flow channel 30 may be used to selectively introduce a variety of fluids into flow cell 10' (fig. 1H).

In this example, the primer 22 is then grafted to the polymer coating 20 in the recess 14, as shown in fig. 1H. Any of the primers 22 described herein may be used. In this example, grafting may be accomplished by a flow-through process. During flow-through, the primer solutions or mixtures described herein can be introduced into the flow channel 30 through respective input ports (not shown), can be maintained in the flow channel 30 for a time (i.e., an incubation period) sufficient to attach the primers 22 to the polymeric coating 20 in the one or more recesses 14, and can then be removed from respective output ports (not shown). After attachment of the primer 22, additional fluid may be directed through the flow channel 30 to wash the now functionalized recess and the flow channel 30.

Prior to performing a sequencing operation, the flow cell 10' may be exposed to a predetermined stimulus of the polymeric coating 20 in order to transition the polymeric coating 20 from a current (e.g., more hydrophobic) state to a more hydrophilic state than the current state, from a neutral state to a charged state, and/or from a collapsed state to an extended state. The predetermined stimulus used will depend on the polymeric coating 20 and the stimulus-responsive functional groups it comprises. Exposing the polymeric coating 20 to a predetermined stimulus can involve heating the polymeric coating 20; exposing the polymer coating 20 to a solution of a predetermined pH; exposing the polymer coating 20 to a solution comprising a sugar; exposing the polymer coating 20 to a nucleophile; or exposing the polymer coating 20 to a salt solution. Exposing the polymer coating 20 to any solution when the cover 26 is attached may be accomplished by a flow-through process as previously described herein. When the stimulus-responsive functional group is thermally responsive, the entire flow cell 10' can be heated, or the heated solution can be exposed to the polymeric coating 20 using a flow-through process.

The predetermined stimulus will make the polymer coating 20 more compatible with the conditions of the subsequent sequencing operation.

In other examples, exposing the polymeric coating 20 to a predetermined stimulus can occur prior to grafting of the primers 22. In examples where the predetermined stimulus exposure occurs before primer 22 grafting in fig. 1E, techniques other than flow-through processes, such as dip coating or dip coating, may be used. For example, the silanized, coated, and polished patterned support shown in fig. 1D can be immersed in an alkaline or acidic solution, a sugar solution (e.g., glucose), or a salt solution for a time sufficient for the desired property change to occur. For another example, the silanized, coated, and polished patterned support shown in fig. 1D can be heated to a desired temperature to induce a state transition. In the example where the predetermined stimulus exposure occurs before primer 22 grafting in fig. 1H, a flow-through process can be used for such exposure. For another example, a silanized, coated, and polished patterned support with a cover 26 attached thereto as shown in fig. 1G may be heated to a desired temperature to induce a state transition. The heating may be carried out in the presence of water or a buffer.

As mentioned above, surface chemistries 20, 22 may also be added to the non-patterned support, and this example will be described with reference to fig. 2A-2D. In the case of an unpatterned support 12 ', the continuous surface would comprise the same surface chemistries 20, 22 found in the wells 14' of fig. 1E, 1F, and 1H. Any of the supports disclosed herein may be used as the non-patterned substrate 12 'unless the non-patterned substrate 12' does not include the recesses 14 or the gap regions 16. In this exemplary method, a cover 26 (shown in fig. 2B) is initially bonded to the non-patterned substrate 12' to form a flow channel 30. The cover 26 may be any material and in any configuration described herein. The cover 26 may also be bonded to the unpatterned substrate 12' via any of the techniques described herein.

In the example shown in fig. 2B, the cover 26 includes a top portion 27 integrally formed with a sidewall 29. The sidewalls 29 are bonded to the bonding regions 25 of the non-patterned substrate 12' through the spacers 28. The bonded area 25 may be at the perimeter of the non-patterned substrate 12' or at any region where it is desirable to form a boundary of the flow channel 30. In other examples, the spacer layer 28 may form a sidewall and may be attached to the at least substantially planar cover 26.

The cover 26 (including the sidewalls 29) and the non-patterned substrate 12' together define a flow channel 30. The flow channels 30 may be used, for example, to selectively introduce fluids to form the surface chemistries 20, 22 and to selectively introduce reactive components or reactants to the surface chemistries 20, 22 to induce a state transition of the polymer coating 20 and/or to induce other specified reactions within the flow channels 30.

Prior to forming the polymer coating 20 (shown in fig. 2C), the method may involve exposing the non-patterned substrate 12 'to a cleaning process (via a flow-through process) and/or another process (e.g., silanization) that prepares the exposed surface of the non-patterned substrate 12' for subsequent deposition of the stimulus-responsive polymer.

Silylation of the unpatterned substrate 12' is shown in fig. 2B. In this example, silanization attaches the silane or silane derivative 18 to the exposed portions of the non-patterned wafer surface 12' present in the flow channels 30.

Silanization can be accomplished using any silane or silane derivative 18. The choice of silane or silane derivative 18 may depend in part on the stimulus-responsive polymer used to form the polymeric coating 20 (shown in fig. 2C), as it may be desirable to form a covalent bond between the silane or silane derivative 18 and the polymeric coating 20. The method for attaching the silane or silane derivative 18 to the substrate 12' may be a flow-through process.

As shown in fig. 2C, in this example, a polymer coating 20 is then formed on the silane or silane derivative 18, or other chemical that has been deposited, to prepare the exposed surface of the non-patterned substrate 12' within the flow channel 30.

Any of the stimulus responsive polymers described herein can be used, and combinations of stimulus responsive polymers can be used together. In an example, the formation of the polymer coating may be accomplished by a flow-through process. During flow-through, the stimuli-responsive polymer may be introduced into the flow channel 30 through the respective input port, and may or may not be cured. The polymer coating 20 will form on the exposed surface of the non-patterned substrate 12' and no polishing occurs.

As shown in fig. 2D, the primer 22 is grafted to the polymer coating 20 in the flow channel 30. In this example, grafting may be accomplished by a flow-through process. During flow-through, a primer solution or mixture may be introduced into the flow channel 30 through the respective input port, and may be maintained in the flow channel for a time sufficient to attach the primer 22 to the attachment group of the polymeric coating 20 (i.e., an incubation period). The remaining primer solution or mixture may then be removed from the corresponding output port. After primer attachment, additional fluid may be directed through the flow channel to wash the now functionalized flow channel 30. The resulting flow cell 10 "in this example is shown in fig. 2D.

Prior to performing a sequencing operation, the flow cell 10 "may be exposed to a predetermined stimulus of the polymeric coating 20 in order to transition the polymeric coating 20 from a current state to a more hydrophilic state (e.g., from a hydrophobic state to a hydrophilic state, from a hydrophilic state to a more hydrophilic state), from a neutral state to a charged state, and/or from a collapsed state to an extended state. The predetermined stimulus used will depend on the polymeric coating 20 and the stimulus-responsive functional groups it comprises. Exposing the polymeric coating 20 to a predetermined stimulus can involve heating the polymeric coating 20; exposing the polymer coating 20 to a solution of a predetermined pH; exposing the polymer coating 20 to a solution comprising a sugar; exposing the polymer coating 20 to a nucleophile; or exposing the polymer coating 20 to a salt solution. Because the cover 26 is attached, exposing the polymer coating 20 to any solution can be accomplished by a flow-through process as previously described herein. When the stimulus-responsive functional group is thermally responsive, the entire flow cell 10 "can be heated, or the heated solution can be exposed to the polymeric coating 20 using a flow-through process.

The predetermined stimulus will make the polymer coating 20 more compatible with the conditions of the subsequent sequencing operation. Sequencing operations are the process of determining the nucleotide sequence in a sample of DNA or RNA. In an example, the sequencing operation is sequencing-by-synthesis, which involves imaging a fluorescently labeled reversible terminator as nucleotides are added to the template strand, and then cleaving the fluorescently labeled reversible terminator to allow incorporation of the next base.

In other examples using the flow cell 10 ", exposing the polymer coating 20 to a predetermined stimulus may occur prior to grafting of the primers 22. Again, because the cap 26 is attached prior to application of the polymer coating 20, the flow-through process can be used for a predetermined stimulus exposure. For another example, the silanized and coated non-patterned support as shown in fig. 2C may be heated to a desired temperature to induce a state transition. Heating may be accomplished in an aqueous environment (e.g., water or buffer).

Although not shown, it is understood that the patterned support 12 or the non-patterned support 12' may include inlet and outlet ports that will fluidly engage other ports (not shown) for directing fluids into and out of respective flow channels (e.g., from a reagent cartridge or other fluid storage system) (e.g., to a waste removal system).

Further, although not shown, it is understood that the functionalized support (having surface chemistries 20, 22 thereon) may be bonded to another functionalized substrate having surface chemistries 20, 22 thereon, without being bonded to the cover 26. The two functionalized surfaces may face each other and may have a flow channel defined therebetween. A spacer layer and suitable bonding methods can be used to bond the two functionalized substrates together.

The flow cells 10, 10', 10 "disclosed herein may be used in a variety of sequencing methods or techniques, including techniques commonly referred to as sequencing-by-synthesis (SBS), cycle array sequencing, sequencing-by-ligation, pyrosequencing, and the like. With either of these techniques and in instances where a patterned support 12 is used, amplification will be limited to the functionalized recesses due to the presence of the polymer coating 20 and attached primers 22 in the functionalized recesses (i.e., 14' with surface chemistries 20, 22 thereon) rather than on the interstitial regions 16. Sequencing generally involves hybridizing a nucleic acid template to a flow cell, amplifying the nucleic acid template, and detecting a signal when a nucleotide or oligonucleotide is associated with the amplified nucleic acid template.

As an example, sequencing-by-synthesis (SBS) reactions can be performed in a system such as HISEQ from Illumina (San Diego, Calif.)^TM、HISEQX^TM、MISEQ^TM、MISEQDX^TM、MINISEQ^TM、NOVASEQ^TM、NEXTSEQDX^TMOr NEXTSEQ^TMRun on the sequencer system.

SBS sequencing operations typically include introducing nucleic acid library templates to the flow cell support 12, whereby the nucleic acid library templates hybridize to primers 22 attached to the polymer coating 20; generating nucleic acid template strands from the hybridized nucleic acid library templates; introducing a sequencing primer complementary to an adaptor of the nucleic acid template strand; introducing fluorescently labeled nucleotides and a polymerase to the flow cell support 12, whereby one fluorescently labeled nucleotide is incorporated to extend the sequencing primer along the nucleic acid template strand; and detecting a fluorescent signal from the incorporated one fluorescently labeled nucleotide.

In SBS, more than one nucleic acid library template may be introduced into the flow cell 10, 10', 10 ". A plurality of nucleic acid library templates are hybridized, for example, with one of two types of primers 22 immobilized on the flow cell 10, 10', 10 ". Cluster generation may then be performed. In one example of cluster generation, nucleic acid library templates are copied by 3' extension from hybridized primers 22 using high fidelity DNA polymerase. The original nucleic acid library template is denatured, allowing the copy to be immobilized in the place where primer 22 is located. Isothermal bridge amplification can be used to amplify the immobilized copies. For example, the copied template is looped over and hybridized to adjacent complementary primers 22, and the polymerase copies the copied template to form a double-stranded bridge, which is denatured to form two single strands. The two strands loop over and hybridize to adjacent complementary primers 22 and are extended again to form two new double-stranded loops. This process was repeated on each template copy through a cycle of isothermal denaturation and amplification to produce dense clonal clusters. Each cluster of the double-stranded bridge is denatured. In the example, the reverse strand is removed by specific base cleavage, leaving the forward template strand.

The 3' end of the template and any primers 22 may be blocked to prevent unwanted priming. Sequencing primers may be introduced into the flow cell 10, 10', 10 ". Because the sequencing primer is complementary to the adaptor of the nucleic acid template strand, it will hybridize to the adaptor (e.g., a read 1 sequencing primer of the template).

Extension of a nucleic acid primer (e.g., a sequencing primer) along a nucleic acid template (e.g., a forward template polynucleotide strand) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process may be polymerization (e.g., catalyzed by a polymerase) or ligation (e.g., catalyzed by a ligase). In certain polymerase-based SBS procedures, fluorescently labeled nucleotides are added to the template in a template-dependent manner (thereby extending the sequencing primer), such that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, or the like can be transported into/through the flow channel 30 or the like that houses the array of primers 22 having template strands attached thereto. Sequencing primer extension results in incorporation of the labeled nucleotide, and this incorporation can be detected by an imaging event. During an imaging event, an illumination system (not shown) may provide excitation light to the flow cell 10, 10', 10 ".

In some examples, after a nucleotide has been added, the nucleotide may also include reversible termination properties that further terminate sequencing primer extension. For example, a nucleotide analog having a reversible terminator moiety can be added to the sequencing primer along the template strand such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for examples using reversible termination, the deblock reagent may be delivered to the flow channel 30 or the like (before or after detection occurs).

The washing may occur between multiple fluid delivery steps. The SBS cycle can then be repeated n times to extend the sequencing primer n nucleotides, thereby detecting sequences of length n.

Although SBS has been described in detail, it is to be understood that the flow cells 10, 10', 10 "described herein may be used in genotyping with other sequencing protocols such as flow cell-based library preparation, or in other chemical and/or biological applications.

To further illustrate the present disclosure, exemplary embodiments and prophetic examples are presented herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure.

Non-limiting working and prophetic examples

Example 1 Synthesis of sugar-responsive switchable heteropolymer (boronic acid).

A mixture of brepa (N- (5-bromoacetamidopentyl) acrylamide), sodium azide and Dimethylformamide (DMF) was placed in a DrySyn bath and the solution was heated at 20 ℃ under nitrogen with stirring for 3h to form AzAPA (N- (5-azidoacetamidopentyl) acrylamide). Acrylamide and 3- (acrylamido) phenylboronic acid were dissolved in deionized water. The prepared AzAPA solution was then added to an acrylamide/3- (acrylamido) phenylboronic acid solution and mixed thoroughly before being filtered through a 0.2 μm filter. The filtered solution was then transferred to a 500mL round bottom flask equipped with a stir bar and nitrogen was bubbled through the mixture for 30 min. While degassing the acrylamide/AzAPA premix, the desired amount of potassium persulfate in deionized water was prepared and then transferred to the monomer mixture. The mixture was then treated with the co-initiator TEMED (tetramethylethylenediamine). The solution was stirred at 35 ℃ under nitrogen for 1.5 h. At the end of the polymerization, the nitrogen line was removed to expose the reaction flask to air. The crude mixture was then slowly added to 2-propanol. The crude polymer was then isolated by filtration.

Prophetic example 2. synthesis of pH-responsive switchable heteropolymers (anions).

The mixture of acrylate derivatives and sultones is reacted to form sulfonate derivatized acrylate monomers. The monomer was converted to a heteropolymer as described in example 1.

Prophetic example 3. Synthesis of nucleophile-responsive switchable heteropolymer (sultone).

The sultone-derived acrylate monomer was converted to a heteropolymer as described in example 1.

Prophetic example 4. Synthesis of nucleophile-responsive switchable heteropolymers (cyclic anhydrides).

The succinic anhydride-derived acrylate monomer was converted to a heteropolymer as described in example 1.

Example 5 Synthesis of salt-responsive switchable heteropolymers (zwitterions).

The heteropolymers shown were prepared from suitable monomers as described in example 1. The heteropolymers may have improved dry storage robustness.

Example 6 synthesis of pH-responsive switchable heteropolymers (anions).

The heteropolymers shown were prepared from suitable monomers as described in example 1. The heteropolymers may have improved dry storage robustness.

Example 7 sequencing operations using switchable heteropolymers.

The four heteropolymers were coated separately on the channel surfaces of four single-channel, unpatterned flow cells using a flow-through process.

Comparison: poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide), also known as PAZAM

Test 1: zwitterionic switchable heteropolymer of example 5

And (3) testing 2: example 6 Anionically switchable heteropolymer

And (3) testing: switchable heteropolymer of sugar example 1

18 μ M to 26 μ M primers were grafted onto each polymer layer in a separate flow cell.

Prior to sequencing, the polymer of example 1 was exposed to a solution of glucose, which transformed the polymer of example 1 from its neutral and relatively hydrophobic state to its negatively charged and more hydrophilic state. In this example, the polymer of example 5 and the polymer of example 6 were not switched.

More than 300 sequencing cycles were performed in each channel using the PhiX library. Read 1 corresponds to cycles 1-151 and read 2 corresponds to cycle 152-302. The sequencing data collected included error rate (percentage) (shown in fig. 3A) and quality score (percentage greater than Q30) (shown in fig. 3B). Q30 corresponds to the probability of 1 of 1000 incorrect base calls. This means that the base call accuracy (i.e., the probability of correct base calls) is 99.9%. A lower base call accuracy of 99% (Q20) would have an incorrect base call probability of 1/100, which means that sequencing reads would likely contain one error every 100 base pairs. When the sequencing quality reached Q30, almost all reads would be perfect, with zero errors and zero ambiguity. As shown in fig. 3A and 3B, each of the example polymers performed similarly to the comparative/control example. These results indicate that the polymers of example 1, example 4 and example 5 are capable of supporting sequencing-by-synthesis techniques with properties that roughly match those of the control examples. It is believed that similar results can be obtained with all types of sequencing libraries.

Additional description

It should be recognized that all combinations of concepts described herein and in the appended claims (assuming such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terms explicitly used herein that may also appear in any disclosure incorporated by reference should be accorded the most consistent meaning with the specific concepts disclosed herein.

While several examples have been described in detail, it should be understood that the disclosed examples can be modified. Accordingly, the foregoing description should be considered as non-limiting.