阅读说明：本技术 试剂盒和流通池 (Kit and flow cell ) 是由 W·N·乔治 B·D·马瑟 A·A·布朗 P·G·拉夫兰克尼 M·C·罗杰·巴西加卢波 A 于 2020-11-25 设计创作，主要内容包括：试剂盒的示例包括流通池和裂解混合物。示例性流通池包括基底；所述基底上的催化性聚合物水凝胶,所述催化性聚合物水凝胶包含解封催化剂；以及附接到所述催化性聚合物水凝胶的扩增引物。所述解封催化剂加速引入所述流通池并掺入附接到所述扩增引物的模板链中的3’OH封端的核苷酸的封端基团的裂解。所述裂解混合物的示例包含用于引发所述封端基团的裂解的组分。(Examples of kits include flow cells and lysis mixtures. An exemplary flow cell includes a substrate; a catalytic polymeric hydrogel on the substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst; and an amplification primer attached to the catalytic polymeric hydrogel. The deblocking catalyst accelerates cleavage of a blocking group of a 3' OH-blocked nucleotide introduced into the flow cell and incorporated into a template strand attached to the amplification primer. Examples of the cleavage mixture include a component for initiating cleavage of the end capping group.)

1. A kit, comprising:

a flow-through cell comprising:

a substrate;

a catalytic polymeric hydrogel on the substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst; and

an amplification primer attached to the catalytic polymeric hydrogel;

wherein the catalyst accelerates cleavage of a blocking group of a 3' OH-terminated nucleotide introduced into the flow cell and incorporated into a template strand attached to the amplification primer; and

a cleavage mixture comprising a component for initiating cleavage of the end-capping group.

2. The kit of claim 1, wherein the deblocking catalyst is selected from the group consisting of acid catalysts, base catalysts, enzymes, peptides, dnases, organic catalysts, and combinations thereof.

3. The kit of claim 1, wherein the deblocking catalyst is a metal attached to a metal-ligand complex of the polymeric hydrogel.

4. The kit of claim 1, wherein the deblocking catalyst is an acid catalyst selected from the group consisting of carboxylic acids, phosphonic acids, sulfonic acids, and combinations thereof.

5. The kit of claim 1, wherein the deblocking catalyst is a base catalyst selected from the group consisting of amines, heterocyclic amines, and combinations thereof.

6. The kit of claim 1, wherein the deblocking catalyst is an enzyme.

7. The kit of claim 1, wherein the deblocking catalyst is a peptide.

8. The kit of claim 1, wherein the deblocking catalyst is a DNase.

9. A method, the method comprising:

introducing the spiking mixture into a flow cell, the flow cell comprising:

a substrate;

a catalytic polymeric hydrogel on the substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst; and

a template strand attached to the catalytic polymeric hydrogel;

thereby incorporating individual nucleotides along the template strand into the corresponding nascent strand, the individual nucleotides comprising:

a dye label attached to the base; and

a 3' OH end-capping group attached to the saccharide;

removing the spiking mixture;

optically imaging incorporation of the individual nucleotides; and

introducing a cleavage mixture comprising a component for initiating cleavage of the 3'OH blocking group into the flow cell, whereby the deblocking catalyst accelerates cleavage of the 3' OH blocking group.

10. The method of claim 9, wherein the catalyst accelerates removal of external protecting groups of the 3'OH end capping groups, and wherein reagents in the cleavage mixture remove internal protecting groups of the 3' OH end capping groups.

11. A flow-through cell, the flow-through cell comprising:

a substrate;

a catalytic polymeric hydrogel on the substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst selected from the group consisting of:

a phosphonic acid;

a heterocyclic amine;

an enzyme;

a peptide;

a DNA enzyme;

the metal of the metal-ligand complex;

an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1, 8-diazabicyclo (5.4.0) undec-7-ene, and combinations thereof; and

a photoacid generator; and

an amplification primer attached to the catalytic polymeric hydrogel.

12. The flow cell of claim 11, wherein the catalyst is integrated into the monomer units of the catalytic polymeric hydrogel.

13. The flow cell of claim 11, wherein the catalyst is grafted to an initial polymer hydrogel.

14. The flow cell of claim 11, wherein the catalytic polymeric hydrogel comprises an initial polymeric hydrogel, and wherein the flow cell further comprises an oligonucleotide attached to the initial polymeric hydrogel, and wherein the deblocking catalyst is attached to a complementary oligonucleotide tether hybridized to the oligonucleotide.

15. A flow-through cell, the flow-through cell comprising:

a substrate;

a polymer hydrogel on the substrate, the polymer hydrogel comprising a first member of a hydrogen bonding pair;

a deblocking catalyst attached to the polymer hydrogel by a second member of the hydrogen bonding pair; and

an amplification primer attached to the catalytic polymeric hydrogel.

16. A method, the method comprising:

applying a catalytic polymeric hydrogel to a surface of a flow cell substrate, the catalytic polymeric hydrogel comprising an unblocking catalyst selected from the group consisting of:

a phosphonic acid;

a heterocyclic amine;

an enzyme;

a peptide;

a DNA enzyme;

the metal of the metal-ligand complex;

an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1, 8-diazabicyclo (5.4.0) undec-7-ene, and combinations thereof; and

a photoacid generator; and

attaching amplification primers to the catalytic polymer hydrogel.

17. The method of claim 16, further comprising forming the catalytic polymeric hydrogel comprising the deblocking catalyst.

18. The method of claim 17, wherein forming the catalytic polymeric hydrogel involves copolymerizing a first monomer comprising a primer grafting functional group with a second monomer comprising the deblocking catalyst.

19. The method of claim 17, wherein forming the catalytic polymeric hydrogel involves:

synthesizing an initial polymer hydrogel; and

grafting the catalyst to the initial polymer hydrogel.

20. The method of claim 17, wherein forming the catalytic polymeric hydrogel involves:

synthesizing an initial polymer hydrogel;

grafting oligonucleotides to the initial polymer hydrogel; and

hybridizing a complementary oligonucleotide tether to the oligonucleotide, wherein the catalyst is attached to the complementary oligonucleotide tether.

21. The method of claim 17, wherein forming the catalytic polymeric hydrogel involves:

synthesizing an initial polymer hydrogel; and

attaching a metal-ligand complex to the initial polymer hydrogel, wherein the metal of the metal-ligand complex is the catalyst.

Background

Various approaches in biological or chemical research involve performing a large number of controlled reactions on a local support surface or within a predefined reaction chamber. The specified reaction can then be observed or detected, and subsequent analysis can help identify or reveal the identity of the chemicals involved in the reaction. In some examples, the controlled reaction produces fluorescence, and thus the optical system can be used for detection. In other examples, the controlled reaction may change charge, conductivity, or some other electrical characteristic, so that the electronic system may be used for detection.

Disclosure of Invention

A first aspect disclosed herein is a kit comprising a flow cell comprising a substrate; a catalytic polymeric hydrogel on a substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst; and an amplification primer attached to the catalytic polymeric hydrogel; wherein the catalyst accelerates cleavage of the blocking group of the 3' OH-terminated nucleotide introduced into the flow cell and incorporated into the template strand attached to the amplification primer; and a cleavage mixture comprising a component for initiating cleavage of the end-capping group.

In one example of the first aspect, the deblocking catalyst is selected from the group consisting of acid catalysts, base catalysts, enzymes, peptides, dnases, organic catalysts, and combinations thereof.

In one example of the first aspect, the deblocking catalyst is a metal attached to a metal-ligand complex of the polymer hydrogel.

In one example of the first aspect, the deblocking catalyst is an acid catalyst selected from the group consisting of carboxylic acids, phosphonic acids, sulfonic acids, and combinations thereof.

In one example of the first aspect, the deblocking catalyst is a base catalyst selected from the group consisting of amines, heterocyclic amines, and combinations thereof.

In one example of the first aspect, the deblocking catalyst is an enzyme.

In one example of the first aspect, the deblocking catalyst is a peptide.

In one example of the first aspect, the deblocking catalyst is a dnase.

It is to be understood that any features of the kits disclosed herein may be combined together in any desired manner and/or configuration to achieve the benefits described in this disclosure, including, for example, enhancing decap kinetics.

In a second aspect disclosed herein, a method includes introducing an admixture mixture into a flow cell, the flow cell comprising: a substrate; a catalytic polymeric hydrogel on a substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst; and a template strand attached to the catalytic polymeric hydrogel; thereby incorporating individual nucleotides along the template strand into the corresponding nascent strand, the individual nucleotides comprising: a dye label attached to the base; and a 3' OH end-capping group attached to the saccharide; removing the incorporation mixture; optically imaging incorporation of the individual nucleotides; and introducing a cleavage mixture comprising a component for initiating cleavage of the 3'OH blocking group into the flow cell, whereby the deblocking catalyst accelerates cleavage of the 3' OH blocking group.

In one example of the second aspect, the catalyst accelerates the removal of the external protecting group of the 3'OH end-capping group, and wherein the reagent in the cleavage mixture removes the internal protecting group of the 3' OH end-capping group.

It should be appreciated that any of the features of the method may be combined in any desired manner. Further, it is understood that any combination of features of the method and/or the kit can be used together and/or in combination with any of the examples disclosed herein to achieve benefits as described herein, including, for example, enhanced decap kinetics.

A third aspect disclosed herein is a flow-through cell comprising: a substrate; a catalytic polymeric hydrogel on a substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst selected from the group consisting of: a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; the metal of the metal-ligand complex; an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1, 8-diazabicyclo (5.4.0) undec-7-ene, and combinations thereof; and a photoacid generator; and an amplification primer attached to the catalytic polymeric hydrogel.

In one example of the third aspect, the catalyst is integrated into the monomer units of the catalytic polymeric hydrogel.

In one example of the third aspect, the catalyst is grafted to the initial polymeric hydrogel.

In one example of the third aspect, the catalytic polymeric hydrogel comprises an initial polymeric hydrogel, and wherein the flow cell further comprises an oligonucleotide attached to the initial polymeric hydrogel, and wherein the deblocking catalyst is attached to a complementary oligonucleotide tether hybridized to the oligonucleotide.

It will be appreciated that any of the features of the flow cell may be combined in any desired manner. Further, it is to be understood that any combination of features of the flow cell and/or features of the kit and/or the method may be used together and/or in combination with any of the examples disclosed herein to achieve benefits as described in the present disclosure, including, for example, the generation of polymeric hydrogels that help to enhance decap kinetics.

A fourth aspect disclosed herein is a flow-through cell comprising: a substrate; a polymer hydrogel on a substrate, the polymer hydrogel comprising a first member of a hydrogen bonding pair; a deblocking catalyst attached to the polymer hydrogel by a second member of a hydrogen bonding pair; and an amplification primer attached to the catalytic polymeric hydrogel.

It will be appreciated that any of the features of the flow cell may be combined in any desired manner. Further, it is to be understood that any combination of features of the flow cell and/or other flow cells and/or features of the kit and/or the method may be used together and/or in combination with any of the examples disclosed herein to achieve benefits as described in the present disclosure, including, for example, enhanced decapsulation kinetics.

A fifth aspect disclosed herein is a method comprising: applying a catalytic polymeric hydrogel to a surface of a flow cell substrate, the catalytic polymeric hydrogel comprising a deblocking catalyst selected from the group consisting of: a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; the metal of the metal-ligand complex; an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1, 8-diazabicyclo (5.4.0) undec-7-ene, and combinations thereof; and a photoacid generator; and attaching amplification primers to the catalytic polymer hydrogel.

One example of the fifth aspect further includes forming a catalytic polymeric hydrogel including a deblocking catalyst.

In one example of the fifth aspect, forming the catalytic polymeric hydrogel involves copolymerizing a first monomer comprising the primer grafting functionality with a second monomer comprising the deblocking catalyst.

In one example of the fifth aspect, forming the catalytic polymeric hydrogel involves: synthesizing an initial polymer hydrogel; and grafting a catalyst to the initial polymer hydrogel.

In one example of the fifth aspect, forming the catalytic polymeric hydrogel involves: synthesizing an initial polymer hydrogel; grafting oligonucleotides to the initial polymer hydrogel; and hybridizing a complementary oligonucleotide tether to the oligonucleotide, wherein the catalyst is attached to the complementary oligonucleotide tether.

In one example of the fifth aspect, forming the catalytic polymeric hydrogel involves: synthesizing an initial polymer hydrogel; and attaching a metal-ligand complex to the initial polymer hydrogel, wherein the metal of the metal-ligand complex is a catalyst.

It should be appreciated 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 method and/or other methods and/or features of the kit and/or the flow cell may be used together and/or in combination with any of the examples disclosed herein to achieve benefits as described herein, including, for example, enhanced decap kinetics.

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, but possibly different, 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. 1 is a schematic flow diagram showing a multi-step process for deblocking an example of a 3' OH-blocked nucleotide;

FIG. 2 is a schematic illustration of an example catalyst attached to a polymer hydrogel by an example of non-covalent bonding;

FIG. 3A is a top view of an example of a flow cell;

FIG. 3B is an enlarged partial cross-sectional view of an example of a flow channel of a flow cell including an example of a polymer hydrogel positioned in the flow channel; and is

Fig. 3C is an enlarged partial cross-sectional view of an example of a flow channel of a flow cell including an example of a polymer hydrogel positioned in a depression formed in the flow channel.

Detailed Description

Some nucleic acid sequencing techniques utilize a 3' OH capping group attached to the oxygen atom of the sugar molecule in the nucleotide. The 3' OH blocking group can be used as a reversible terminator that allows only single base incorporation to occur in each sequencing cycle. More specifically, in some cases, the 3' OH blocking group will be removed to start the next sequencing cycle. The temporary termination of nucleotide incorporation allows time to wash away unincorporated nucleotides, excite a detectable label (e.g., a fluorescent dye) attached to the incorporated nucleotide, and image the excited detectable label, to name a few.

A catalytic polymeric hydrogel is disclosed herein. The catalyst of the catalytic polymeric hydrogel is a reactive molecule or portion thereof that accelerates the cleavage of the 3' OH blocking group from the incorporated nucleotide. The catalyst may be selected such that it is inactive during the incorporation event. The catalyst may be selected for the reactions that occur during deblocking (after incorporation and imaging), and thus may accelerate the cleavage of any 3' OH blocking groups. In certain embodiments, the catalyst may be used for 3' OH blocking groups that are more stable in aqueous solution but tend to exhibit slower deblocking kinetics. In these cases, the catalyst can help reduce the time per sequencing cycle, help reduce the overall sequencing turn around time, and help reduce phasing problems that can occur when deblocking is slow (to name a few benefits).

During the cleavage of the 3' OH blocking group, a series of reactions can occur. Figure 1 depicts an example of this series of reactions for an example of a 3' OH-terminated nucleotide 10. The 3 'OH-terminated nucleotide 10 comprises a heterocyclic base B, a sugar (shown in fig. 1 as deoxyribose), one or more phosphate groups P, and a 3' OH-terminating group 12 attached to the sugar. In this example, the 3' OH end-capping group 12 is an azidomethyl group. In step 1 of FIG. 1, Phosphine (PR) is added₃Wherein R is H or (CH)₂)_nOH (n ═ 1-3)) to react with the azide. This reaction results in nitrogen loss and formation of iminophosphoranes. At step 2, the iminophosphorane is hydrolyzed to form a hemiaminal acetal ether, and at step 3, the hemiaminal acetal ether is hydrolyzed to reveal a 3' OH. For deblocking as shown in FIG. 1, any catalyst that accelerates the hydrolysis step at 2 and/or 3 of FIG. 1 may be incorporated into or onto the polymer hydrogel, or any catalyst that accelerates the reaction in step 1 of FIG. 1 may be incorporated into or onto the polymer hydrogel. When the catalyst is specifically selected to catalyze step 1 of FIG. 1, the catalyst accelerates the external protecting group of the 3' OH blocking group 12Removal of groups (e.g., azide groups as shown in FIG. 1), and reagents in the cleavage mixture can be used to remove internal protecting groups (e.g., -CH as shown in FIG. 1) of the 3' OH blocking group 12₂–)。

Although fig. 1 shows one example of deblocking, it is to be understood that the catalyst used may depend on the 3'OH blocking group and the reaction used to cleave the 3' OH blocking group.

Definition of

It will be understood that the terms used herein will have their ordinary meaning in the relevant art, unless otherwise specified. Several terms used herein and their meanings are listed below.

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

The terms "comprising," "including," "containing," and various forms of these terms are synonymous with one another and are intended to be equally broad.

The terms "top," "bottom," "lower," "upper," and the like 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 orientation, but are used to designate a relative orientation between components. The use of directional terms should not be construed to limit the examples disclosed herein to any particular orientation.

The "acrylamide monomer" is of the structureOr a monomer including an acrylamide group having the structure. Examples of monomers comprising an acrylamide group include azidoacetamidopentylglamide:and N-isopropylacrylamide:other acrylamide monomers may be used, as set forth hereinSome examples of which are described.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double and 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. As an example, the name "C1-C6 alkyl" indicates the presence of one to six 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 can have 2 to 20 carbon atoms.

As used herein, "aralkyl" and "aryl (alkyl)" refer to an aryl group attached as a substituent via a lower alkylene group. The lower alkylene and aryl groups of an aralkyl group may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

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

The "amine" or "amino" functional group is intended to mean the radical-NR_aR_bGroup, wherein R_aAnd R_bEach independently selected from hydrogen (e.g. hydrogen)) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

As used herein, the term "attached" refers to the state of two things being joined, fastened, adhered, connected, or joined to one another, either directly or indirectly. For example, the nucleic acid may be attached to the polymer hydrogel by covalent or non-covalent bonds. Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are physical bonds that do not involve sharing electron pairs, and may include, for example, hydrogen bonds, ionic bonds, van der waals forces, hydrophilic interactions, and hydrophobic interactions.

The "azide" or "azido" functional group is defined as the-N₃。

The terms "block copolymer" and "monomer units formed" in a block "refer to a copolymer formed when two or more monomers are brought together and form a block of repeating units. Each block should have at least one feature and/or at least one block-specific functional group (e.g., azide, for attaching primers, catalysts, etc.) that is not present in the adjacent block.

As used herein, the term "catalytic polymeric hydrogel" refers to a copolymer having a catalyst integrated into one of the monomer units, or to an initial polymeric hydrogel having a catalyst attached thereto. As used herein, the term "initial polymeric hydrogel" refers to a polymerized hydrogel prior to any reaction/interaction that introduces a catalyst.

As used herein, "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When a carbocyclyl group is a ring system, two or more rings may be joined together in a fused, bridged, or spiro joined fashion. 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] octanyl, adamantyl and spiro [4.4] nonanyl.

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

As used herein, "cycloalkyl" refers to a monocyclic or polycyclic hydrocarbon ring system that is fully saturated (no double or triple bonds). When composed of two or more rings, the rings may be joined together in a fused fashion. Cycloalkyl groups may contain 3 to 10 atoms in the ring. In some embodiments, cycloalkyl groups may contain 3 to 8 atoms in the ring. Cycloalkyl groups may be unsubstituted or substituted. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, "cycloalkenyl" or "cycloalkene" refers to a carbocyclic ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene.

As used herein, "cycloalkynyl" or "cycloalkyne" refers to a carbocyclic ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. One example is cyclooctyne. Another example is bicyclononylene.

As used herein, the term "deposition" refers to any suitable application technique, which may be manual or automated, and in some cases, results in modification of surface properties. Generally, deposition can be carried out using vapor deposition techniques, coating techniques, grafting techniques, and the like. Some specific examples include Chemical Vapor Deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, bubble or dip coating, knife coating, whipping dispensing, flow-through coating, aerosol printing, screen printing, micro-contact printing, ink jet printing, and the like.

As used herein, the term "depressions" refers to discrete concave features in a substrate or patterned resin having surface openings at least partially surrounded by interstitial regions of the substrate or patterned resin. The depressions can have any of a variety of shapes at the openings in their surfaces, including, for example, circular, oval, square, polygonal, star-shaped (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, conical, angular, etc. For example, the recess may be a well or two interconnected wells. The depressions may also have more complex architectures such as ridges, step features, and the like.

When used to refer 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 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 (e.g., 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 enables detection of a reaction occurring in the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of the array, optically labeled molecules, and the like.

As used herein, a "flow channel" or "channel" may be a region defined between two bonded or otherwise attached components that can selectively receive a liquid sample. In some examples, a flow channel may be defined between the patterned or unpatterned substrate and the cover, and thus may be in fluid communication with one or more recesses defined in the patterned resin. The flow channel may also be defined between two patterned or unpatterned substrate surfaces that are bonded together.

As used herein, "heterocyclic amine" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing an amine nitrogen as one or the only heteroatom in the ring backbone.

As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing 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 ring system is aromatic. Heteroaryl groups can have 5 to 18 ring members (i.e., the number of atoms making up the ring backbone).

As used herein, "heterocyclyl" refers to a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. The heterocyclic groups may be joined together in a fused, bridged or spiro-connected fashion. 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, heteroatoms may be present in non-aromatic or aromatic rings. A 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-NHNH₂A group.

As used herein, the term "hydrazone" or "hydrazino" refers toGroup, wherein R_aAnd R_bEach independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

As used herein, "hydroxy" refers to an-OH group.

As used herein, the term "interstitial regions" refers to regions of discrete recesses, such as a substrate, patterned resin, or other support. For example, a gap region may separate one recess of an array from another recess of the array. The two recesses, which are separated from each other, may be discrete, i.e. not in physical contact with each other. In many examples, the interstitial regions are continuous, while the depressions are discrete, as is the case, for example, with a plurality of depressions defined in an otherwise continuous surface. In other examples, the gap regions and features are discrete, for example, as is the case with multiple trenches separated by respective gap regions. The separation provided by the gap region may be partial or complete. The gap region may have a surface material that is different from a surface material of the recess defined in the surface. For example, the recess may have a polymer and a primer set therein, and the gap region may have neither a polymer nor a primer set thereon.

As used hereinBy "nitrile oxide" is meant "R_aC≡N⁺O^-"group, wherein R_aAs defined herein. Examples of the preparation of the nitrile oxide include treatment with chloramide-T or treatment with a basic group at imide chloride [ RC (Cl) ═ NOH]Or generated in situ from an aldoxime by reaction between hydroxylamine and an aldehyde.

As used herein, "nitrone" refers toGroup, wherein R₁、R₂And R₃May be R as defined herein_aGroup and R_bAny of the groups.

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 the 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), as well as 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. The nucleic acid analog can have any of an altered phosphate backbone, a sugar, or a nucleobase. Examples of nucleic acid analogs include, for example, universal base or phosphate-sugar backbone analogs, such as Peptide Nucleic Acids (PNAs).

"patterning resin" refers to any polymer that can have recesses defined therein. Specific examples of resins and techniques for patterning the resins are described further below.

As used herein, the term "phosphonic acid" refers to R-PO₃H₂。

As used herein, a "primer" is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA or single-stranded RNA). Some of the primers, referred to herein as amplification primers, serve as a starting point for template amplification and cluster generation. Other primers, referred to herein as sequencing primers, serve as a point of initiation of DNA or RNA synthesis. The 5' end of the primer may be modified to allow a coupling reaction with a functional group of the polymer. The primer length can be any number of bases in length and can include a variety of non-natural nucleotides. In one example, the sequencing primer is a short strand, ranging from 10 to 60 bases or 20 to 40 bases.

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

The term "substrate" refers to a structure to which various components of a flow cell (e.g., catalytic polymer hydrogels, primers, etc.) can be added. The substrate may be a wafer, panel, rectangular sheet, die, or any other suitable configuration. The substrate is generally rigid and insoluble in aqueous liquids. The substrate may be inert to the chemistry used to modify the depressions or present in the depressions. For example, the substrate may be inert to the chemistry used to form the catalytic polymer hydrogel, attach primers, and the like. The substrate may be a single layer structure or a multi-layer structure (e.g., including a support and a patterned resin on the support). Examples of suitable substrates are described further below.

As used herein, the term "sulfonic acid" refers to-S (═ O)₂-OH。

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.

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

Catalytic polymeric hydrogels

The polymeric backbone in any example of the catalytic polymeric hydrogel can be a semi-rigid polymeric material that is permeable to liquids and gases. In some examples, the catalytic polymeric hydrogel includes an initial polymeric hydrogel having a catalyst attached thereto. In these examples, a post-polymerization treatment is used to add the catalyst to the initial polymer hydrogel. In other examples, the catalytic polymeric hydrogel may be a copolymer that includes a catalyst in one of its monomeric components.

Examples of initial polymeric hydrogels include acrylamide copolymers such as poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) PAZAM. PAZAM and some other forms of acrylamide copolymers are represented by the following structure (I):

wherein:

R^Aselected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxyl, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol;

R^Bis H or optionally substituted alkyl;

R^C、R^Dand R^EEach independently selected from the group consisting of H and optionally substituted alkyl;

-(CH₂)_peach of-may be optionally substituted;

p is an integer in the range of 1 to 50;

n is an integer ranging from 1 to 50,000; and is

m is an integer ranging from 1 to 100,000.

One of ordinary skill in the art will recognize that the arrangement of repeating "n" and "m" features in structure (I) is representative, and that the monomeric subunits may be present in the polymer structure in any order (e.g., random, block, patterned, or combinations thereof).

The molecular weight of PAZAM and other forms of acrylamide copolymers may range from about 5kDa to about 1500kDa or about 10kDa to about 1000kDa, or in one specific example may be about 312 kDa.

In some examples, PAZAM and other forms of acrylamide copolymers are linear polymers. In some other examples, PAZAM and other forms of acrylamide copolymers are lightly crosslinked polymers.

In other examples, the initial polymeric hydrogel may be a variation of structure (I). In one example, the acrylamide units may be N, N-dimethylacrylamideAnd (6) replacing. In this example, the acrylamide units in structure (I) may be substituted with one or more groups selected from the group consisting ofIn the alternative, wherein R^D、R^EAnd R^FEach is H or C1-C6 alkyl, and R^GAnd R^HEach being a C1-C6 alkyl group (rather than H in the case of acrylamide). In this example, q may be an integer in the range of 1 to 100,000. In another example, N-dimethylacrylamide may be used in addition to the acrylamide units. In this example, structure (I) may include, in addition to recurring "n" and "m" featuresWherein R is^D、R^EAnd R^FEach is H or C1-C6 alkyl, and R^GAnd R^HEach is a C1-C6 alkyl group. In this example, q may be an integer in the range of 1 to 100,000.

As another example of an initial polymeric hydrogel, the recurring "n" feature in structure (I) may be replaced by a monomer comprising a heterocyclazido group having structure (II):

wherein R is¹Is H or C1-C6 alkyl; r₂Is H or C1-C6 alkyl; l is a linker comprising a straight chain, whichHaving 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and 10 optional substituents on carbon and any nitrogen atoms in the chain; e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atom in the chain; a is an N-substituted amide having H attached to N or C1-C4 alkyl; and Z is a nitrogen-containing heterocycle. Examples of Z include 5 to 10 ring members present as a single cyclic structure or a fused structure. Some specific examples of Z include pyrrolidinyl, pyridinyl, or pyrimidinyl.

As yet another example, the initial polymeric hydrogel may comprise repeating units of each of structures (III) and (IV):

wherein R is^1a、R^2a、R^1bAnd R^2bEach of which is independently selected from hydrogen, optionally substituted alkyl, or optionally substituted phenyl; wherein R is^3aAnd R^3bEach of which is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl or optionally substituted C7-C14 aralkyl; and L is¹And L²Each of which is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

It will be appreciated that other molecules may be used to form the initial polymer hydrogel, so long as they are functionalized to graft oligonucleotide primers thereto. Other examples of suitable polymer layers include those having a colloidal structure, such as agarose; or a polymer network, such as gelatin; or crosslinked polymer structures such as polyacrylamide polymers and copolymers, Silane Free Acrylamide (SFA) or azidated versions of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing vinyl groups, or from monomers that form a [2+2] cycloaddition reaction. Other examples of suitable initial polymeric hydrogels include mixed copolymers of acrylamide and acrylate. A variety of polymer architectures comprising acrylic monomers (e.g., acrylamide, acrylate, etc.) can be used in examples disclosed herein, such as branched polymers, including star polymers, star-shaped or star-block polymers, dendrimers, and the like. For example, monomers (e.g., acrylamide, catalyst-containing acrylamide, etc.) can be incorporated randomly or in blocks into the branches (arms) of the star-shaped polymer.

The initial polymeric hydrogel may be formed using any suitable copolymerization process. A catalyst may then be attached to the initial polymer hydrogel to form a catalytic polymer hydrogel. A variety of different post-polymerization techniques can be used to attach the catalyst to the initial polymer hydrogel.

In one example, a catalyst may be grafted to the initial polymer hydrogel. In this exemplary method, forming the catalytic polymeric hydrogel involves synthesizing an initial polymeric hydrogel (which does not contain a catalyst); and grafting a catalyst to the initial polymer hydrogel. In one example, grafting involves a click chemistry reaction, such as copper catalyzed click chemistry, or the use of strain-promoted catalyst-free click chemistry, such as with bicyclo [6.1.0] non-4-yne (BCN). In one instance, a click chemistry reaction results in covalent attachment of a catalyst to the initial polymer hydrogel. It will be appreciated that the reaction which takes place will depend on the chemistry of the initial polymer hydrogel and the catalyst.

Any reactive molecule, functional group, etc. that increases the rate of chemical reaction that cleaves the 3' OH blocking group from the incorporated nucleotide can be used as a catalyst. It is to be understood that any acid catalyst, base catalyst, organic catalyst, enzyme catalyst, peptide catalyst, or dnase catalyst may be grafted to the initial polymer hydrogel. Some of the listed catalysts may belong to two of the listed catalyst classes. In some specific examples, the deblocking catalyst is selected from the group consisting of: a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; the metal of the metal-ligand complex; an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1, 8-diazabicyclo (5.4.0) undec-7-ene, and combinations thereof; and a photoacid generator.

In some examples, the catalyst is an acid catalyst selected from the group consisting of carboxylic acids, phosphonic acids, sulfonic acids, and combinations thereof. The acid catalyst may catalyze the hydrolysis reaction that occurs during deblocking.

In other examples, the catalyst is a base catalyst selected from the group consisting of amines, heterocyclic amines, and combinations thereof. Lewis bases (such as thioethers, triazoles, imidazoles, etc.) may be particularly useful for catalyzing the phosphazide reaction (step 1 in fig. 1). The base catalyst may catalyze the hydrolysis reaction that occurs during deblocking.

The organic catalyst consists of carbon, hydrogen, sulfur and/or other non-metallic elements present in the organic compound and increases the rate of the deprotection chemical reaction. Exemplary organic catalysts may be selected from the group consisting of urea, thiourea, imidazole, guanidine, 1, 8-diazabicyclo (5.4.0) undec-7-ene, and combinations thereof.

The enzyme catalyst may be a hydrolase enzyme. Examples of suitable hydrolases include phosphatases, esterases (e.g., acetylcholinesterase, lipase, etc.), sequence-specific proteases (e.g., TEV protease or thrombin), and glycosidases (which do not degrade ribose, such as cellulose or amylase). Another suitable hydrolase is carbonic anhydrase. Another enzyme catalyst is a serine protease, which cleaves peptide bonds in proteins and can catalyze the hydrolysis of amides and esters.

Other catalysts include dnases, which are also known as deoxyribozymes. Exemplary dnases include those that target ester group hydrolysis, such as DNA mimics of enzymes, such as esterases. In these examples, the deprotection chemical reaction is catalyzed by dnase.

While several examples have been provided, it is believed that any useful enzyme or engineered enzyme may be used, provided that the enzyme is capable of catalyzing the deprotection of the 3' OH blocking group.

Examples of peptide catalysts include supramolecular β -barrel esterases with hydrophobic exterior and histidine-rich pore interior, self-assembling peptide catalysts, and other catalytic peptide assemblies. In these examples, the deprotection chemistry is catalyzed by peptides.

As another example, the catalyst may be incorporated into the surface of the initial polymer hydrogel via non-covalent attachment. Non-covalent attachment may include, for example, hybridization with oligonucleotides grafted to the surface of the initial polymer hydrogel, hydrogen-bonded arrays, biotin-streptavidin or other similar bonds, metal-ligand complexation, and the like.

Figure 2 schematically shows one example of non-covalent attachment of a catalyst. Specifically, fig. 2 shows the oligonucleotide 14 attached to the initial polymer hydrogel 16, and the catalyst 18 attached to a complementary oligonucleotide tether 20 that is hybridized to the oligonucleotide 14. Oligonucleotides 14 attached to the initial polymer hydrogel 16 may be grafted as described herein with reference to fig. 3A-3C for the amplification primers (e.g., by click chemistry at the terminal azides). Oligonucleotide 14 can have 5 to 20 nucleotides (as shown by N in fig. 2), and this sequence can be the same as or different from the sequence of the amplification primer (discussed below). The catalyst 18 is attached to a tether 20 having a sequence complementary to the oligonucleotide 14. The catalyst 18 that may be attached according to the example shown in fig. 2 includes any of an acid catalyst, a base catalyst, an organic catalyst (e.g., imidazole, guanidine, etc.), a copper catalyst, an enzyme catalyst, a peptide catalyst, or a dnase catalyst.

Suitable copper catalysts may include copper (I) catalysts such as copper (I) acetate, copper (I) bromide, copper (I) chloride, copper (I) iodide, bis (1, 3-bis (2, 6-diisopropylphenyl) imidazol-2-ylidene) copper (I) tetrafluoroborate, copper (I) bis [ (tetrabutylammonium iodide) ], [ bis (trimethylsilyl) acetylene ] (hexafluoroacetylacetonato) copper (I), tris (triphenylphosphine) copper (I) bromide, copper (I) chloride (1, 5-cyclooctadiene) dimer, copper (I) 3-methylsalicylate, and the like.

One example of a method for forming the catalytic polymer hydrogel 16' shown in FIG. 2 involves synthesizing an initial polymer hydrogel 16 (which does not contain the catalyst 18); grafting oligonucleotides 14 to the initial polymer hydrogel 16; and hybridizing a complementary oligonucleotide tether 20 to the oligonucleotide 14, wherein the catalyst 18 is attached to the complementary oligonucleotide tether 20.

Another example of non-covalent attachment of the catalyst 18 to the initial polymer hydrogel 16 involves a hydrogen bonding array. In this example, the catalyst 18 hydrogen bonds with the polymer hydrogel 16. Any hydrogen bonding pair may be used, wherein one component of the pair is covalently attached to the initial polymer hydrogel and the other component of the pair is attached to the catalyst 18. Examples of hydrogen bonding pairs include O … H, NH … N and CH … N (to name a few).

Yet another example of non-covalent attachment of the catalyst 18 to the initial polymer hydrogel 16 involves metal-ligand complexation. In one example, the catalyst 18 is a metal attached to a metal-ligand complex of the initial polymer hydrogel 16. The catalyst 18 may be any suitable catalytic metal such as copper, palladium, ruthenium, and the like. The catalytic metal complexes with a ligand that is attached to the initial polymer hydrogel 16. Examples of metal-ligand complexes include complexes of copper (II) with ligands such as bis (2-pyridylmethyl) -amine or pyridine functionalized cyclodextrins.

One example of this exemplary method for forming the catalytic polymeric hydrogel 16' involves synthesizing the initial polymeric hydrogel 16; and attaching a metal-ligand complex to the initial polymer hydrogel 16, wherein the metal of the metal-ligand complex is the catalyst 18.

Other catalysts 18 that may be attached to the initial polymer hydrogel 16 include photoacid generators. When a photoacid generator is used as the catalyst 18, catalytic activity may be initiated or enhanced upon exposure to an external stimulus, such as a photon of a wavelength matched to the particular photoacid generator.

The photoacid generator catalyst can be attached to the polymer hydrogel 16 'by a guest-host chemistry in which the catalyst 18 is a guest and the host is capable of i) attaching to the polymeric hydrogel 16' and ii) holding the guest until subjected to an external stimulus. Exemplary host compounds include cucurbituril and cyclodextrins. One example of a guest-type photoacid generator catalyst is a blue/visible light-based photoacid generator. Photoinitiated catalysts (e.g., photoacid generators) may also be used as guests in the guest-host example.

The guest-host molecules may be attached to the initial polymeric hydrogel 16 using any of the post-polymerization techniques disclosed herein (e.g., grafting, non-covalent attachment).

While several examples have been provided, it should be understood that the catalyst 18 attached to the initial polymer hydrogel 16 may depend in part on the chemistry of the initial polymer hydrogel 16 and the chemistry of the end-capping group 12 contained on the nucleotide 10 to be introduced into the catalytic polymer hydrogel 16'.

In other examples disclosed herein, the initial polymer hydrogel 16 is not used. In contrast, the copolymerization product is the catalytic polymer hydrogel 16'. In these examples, the catalyst 18 is integrated into the monomer units used during copolymerization.

In these examples, the catalyst 18 may be part of any of the acrylamide monomeric units described herein for the initial polymer hydrogel 16. For example, R in structure (I)^AThere may be carboxyl groups, which is one example of an acid catalyst. As another example, R in structure (II)₂May be a C1-C6 alkyl group, and catalyst 18 may be incorporated as the end group of a C1-C6 alkyl chain. The catalyst-containing monomer units can be copolymerized (randomly or in blocks) with any of the acrylamide monomer units described herein, so long as one of these units contains a primer grafting functionality. One example of a catalyst-containing acrylamide monomer unit that can be used is shown in structure (V):

wherein R is⁴Is H or CH₃And X is a functional group used as a catalyst. In some examples, the catalyst (X) can be any of the acid catalysts, base catalysts, or organic catalysts disclosed herein. In some examples, a linking group (such as an alkyl group, a short poly (ethylene glycol) chain, etc.) may be located between the NH and the catalyst (X).

Other examples of the catalyst-containing monomer unit that can be used include (meth) acrylic monomers (e.g., acrylic acid, methacrylic acid) having the catalyst (X) attached thereto.

In one exemplary method, forming the catalytic polymeric hydrogel 16' involves copolymerizing a first monomer (e.g., azide) comprising a primer grafting functional group with a second monomer comprising a catalyst 18. In some examples, a third acrylamide monomer unit may also be used in the copolymerization process. One example of a copolymer formed by this method is shown in structure (VI):

wherein R is^A、R^B、R^C、R^D、R^EAnd p is as described for structure (I), X is as described for structure (V), and n and r are independently in the range of 5 to 30 mol%, and m is the remaining mol%.

In one example, the surface concentration of the catalyst 18 can range from about 0.01% to about 50% of the repeating units of the polymer hydrogel 16'. These concentrations may exceed the actual catalyst solution concentration, which helps achieve the high efficiencies of the examples disclosed herein.

In other examples, the nucleophile-responsive functional group may function as an anchor for catalyst 18 or an anchor for an in situ generated catalyst. In some examples, the nucleophile-responsive functional group is a cyclic sulfonate ester (such as a sultone ring), 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). An example of a sultone ring-opening reaction is:

in one example, the ring-opening reaction may be performed during deblocking such that catalyst 18, such as an acid or base, is generated in situ. The nucleophile-responsive functional group may be considered a catalyst precursor attached to the catalytic polymeric hydrogel 16'.

In another example, "Nu" is the catalyst 18 and the ring-opening reaction forms a covalent bond with the previous sultone. This reaction may be formed prior to sequencing such that catalyst 18 is present when deblocking is performed.

Other examples of nucleophile-responsive functional groups have the following structure:

wherein (a) Y is SO₂And Y' is CH₂(ii) a Or (b) Y and Y' are both C (O). In other aspects, the nucleophile-responsive functional group is:suitable nucleophiles include primary alkylamines and alkyl alcohols.

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

in a specific example, the monomer is:

flow cell

The polymer hydrogel 16 and the incorporated catalyst 18 may be used in a flow cell 22, an example of which is shown in fig. 3A. The flow cell 22 includes a substrate 24 and a catalytic polymeric hydrogel 16' on the substrate 24.

The substrate 24 may be a single layer/material. Examples of suitable single layer substrates include: epoxysiloxanes, glass and modified or functionalized glasses, plastics (including acrylics, polystyrenes and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as that available from Chemours)) Cycloolefin/cycloolefin polymers (COP) (such as those from Zeon)) Polyimide, etc.), nylon (polyamide), ceramic/ceramic oxide, silicon dioxide, fused or silicon dioxide-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p + silicon), silicon nitride (Si)₃N₄) Silicon oxide (SiO)₂) Tantalum pentoxide (Ta)₂O₅) Or other tantalum oxide (TaO)_x) Hafnium oxide (HfO)₂) Carbon, metal, inorganic glass, and the like. The substrate 24 may also be a multi-layer structure. Some examples of multilayer structures include glass or silicon with a coating of tantalum oxide or another ceramic oxide at the surface. Other examples of multilayer structures include an underlying support (e.g., glass or silicon) having a patterned resin thereon. Other examples of multilayer substrates may include silicon-on-insulator (SOI) substrates.

In one example, the substrate 24 may have a diameter in the range of about 2mm to about 300mm, or a rectangular sheet or panel having a maximum dimension of up to about 10 feet (about 3 meters). In one example, the substrate 24 is a wafer having a diameter in the range of about 200mm to about 300 mm. In another example, the substrate 24 is a die having a width in a range from about 0.1mm to about 10 mm. While exemplary dimensions have been provided, it should be understood that a substrate 24 having any suitable dimensions may be used. As another example, a panel that is a rectangular support having a larger surface area than a 300mm circular wafer may be used.

In the example shown in fig. 3A, the flow cell 22 includes a flow channel 26. Although several flow channels 26 are shown, it should be understood that any number of channels 26 (e.g., a single channel 26, four channels 26, etc.) may be included in the flow cell 22. Each flow channel 26 is an area defined between two attached components (e.g., the base 24 and the cover or two bases 24) into which a fluid (e.g., those described herein) can be introduced and removed. Each flow channel 26 may be isolated from other flow channels 26 such that fluid introduced into any particular flow channel 26 does not flow into any adjacent flow channel 26. Some examples of fluids introduced into the flow channel 26 may introduce reaction components (e.g., polymerases, sequencing primers, nucleotides, etc.), wash solutions, deblocking agents, and the like.

The flow channels 26 may be defined in the substrate 24 using any suitable technique depending in part on the material of the substrate 24. In one example, the flow channels 26 are etched into the glass substrate 24. In another example, the flow channels 26 may be patterned into the resin of the multilayer substrate 24 using photolithography, nanoimprint lithography, or the like. In yet another example, a separate material (not shown) may be applied to the substrate 24 such that the separate material defines the walls of the flow channel 26 and the substrate 24 defines the bottom of the flow channel 26.

In one example, the flow channel 26 has a rectangular configuration. The length and width of the flow channel 26 may be less than the length and width of the substrate 24, respectively, such that the portion of the substrate surface surrounding the flow channel 26 may be used for attachment to a cover (not shown) or another substrate 24. In some cases, the width of each flow channel 26 may be at least about 1mm, at least about 2.5mm, at least about 5mm, at least about 7mm, at least about 10mm, or greater. In some cases, the length of each channel 20 may be at least about 10mm, at least about 25mm, at least about 50mm, at least about 100mm, or greater. The width and/or length of each flow channel 26 may be greater than, less than, or intermediate to the values specified above. In another example, the flow channels 26 are square (e.g., 10mm by 10 mm).

When microcontact, aerosol or inkjet printing is used to deposit the individual materials defining the flow channel walls, the depth of each flow channel 26 may be as small as a monolayer thick. For other examples, the depth of each flow channel 26 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or greater. In one example, the depth may be in a range of about 10 μm to about 100 μm. In another example, the depth is about 5 μm or less. It should be understood that the depth of each flow channel 26 may be greater than, less than, or intermediate to the values specified above.

Different examples of architectures within the flow channels 26 of the flow cell 22 are shown in fig. 3B and 3C.

In the example shown in fig. 3B, the flow cell 22 includes a single layer substrate 24A and a flow channel 26 defined in the single layer substrate 24A. In this example, the catalytic polymeric hydrogel 16' is located within the flow channel 26.

To introduce the catalytic polymeric hydrogel 16 'into the flow channels 26, a mixture of the catalytic polymeric hydrogel 16' may be created and then applied to the substrate 24 (having the flow channels 26 defined therein). In one example, the catalytic polymeric hydrogel 16' may be present in a mixture (e.g., with water or with ethanol and water). The mixture may then be applied to the substrate surface (including in the flow channels 26) using spin coating or dipping or dip coating or a material flow under positive or negative pressure or another suitable technique. These types of techniques blanket deposit the catalytic polymer hydrogel 16' on the substrate 24 (e.g., in the flow channels 26 and on the interstitial regions 28). Other selective deposition techniques (e.g., involving masks, controlled printing techniques, etc.) may be used to specifically deposit the catalytic polymeric hydrogel 16' in the flow channels 26 rather than on the interstitial regions 28.

In some examples, the substrate surface (including the portion exposed in the flow channel 26) may be activated, and then the mixture (including the catalytic polymeric hydrogel 16') may be applied thereto. In one example, the silane or silane derivative (e.g., norbornenesilane) can be deposited on the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface can be exposed to plasma ashing to generate surfactants (e.g., -OH groups) that can adhere to the catalytic polymeric hydrogel 16'.

Depending on the chemistry of the catalytic polymeric hydrogel 16', the applied mixture may be exposed to a curing process. In one example, curing may be performed at a temperature in the range of room temperature (e.g., about 25 ℃) to about 95 ℃ for a time in the range of about 1 millisecond to about several days.

Polishing may then be performed to remove the catalytic polymeric hydrogel 16 'from the interstitial regions 28 at the periphery of the flow channel 26 while leaving the catalytic polymeric hydrogel 16' on the surface at least substantially intact in the flow channel 26.

In some examples, the as-deposited catalytic polymer hydrogel 16' already has the catalyst 18 attached thereto, so no additional treatment is performed to introduce the catalyst 18 into the flow channel 26. An example of this is when the catalyst 18 is part of a monomeric unit of the polymer hydrogel backbone. Another example of this is when the catalyst 18 has been attached after polymerization. Accordingly, one exemplary method disclosed herein includes forming a catalytic polymeric hydrogel 16' comprising a catalyst 18; and applying the catalytic polymeric hydrogel 16' to the surface of the substrate 24.

In other examples, the as-deposited polymer hydrogel is the initial polymer hydrogel 16 without the catalyst 18 attached thereto. Because the initial polymer hydrogel 16 does not contain the catalyst 18, an additional treatment is performed to introduce the catalyst 18 into the initial polymer hydrogel 16 to form the catalytic polymer hydrogel 16' in the flow channels 26. In these examples, the initial polymer hydrogel 16 is deposited into the flow channel 26 and polished, and then the catalyst 18 may be introduced into the initial polymer hydrogel 16 using any of the post-polymerization attachment techniques described herein. Because the initial polymer hydrogel 16 is present in the flow channel 26 rather than on the interstitial regions 28, the catalyst 18 will preferentially attach to the initial polymer hydrogel 16 in the flow channel 26.

The flow cell 22 also includes amplification primers 30 attached to the catalytic polymer hydrogel 16'. The following discussion of primer attachment refers to the catalytic polymer hydrogel 16'. It will be appreciated that when the initial polymer hydrogel 16 is introduced into the flow channel 26, the catalyst 18 may be introduced before or after the amplification primers 30. Thus, the discussion of primer attachment also applies to the initial polymer hydrogel 16.

A grafting process may be performed to graft the amplification primers 30 to the catalytic polymer hydrogel 16' in the flow channel 26. In one example, the primer 30 may be immobilized to the catalytic polymer hydrogel 16 'by a single point covalent attachment at or near the 5' end of the amplification primer 30. This attachment leaves i) the adaptor-specific portion of the primer 30 free to anneal to its cognate, ready-to-sequence, nucleic acid fragment, and ii) the 3' hydroxyl group free for primer extension. Any suitable covalent attachment may be used for this purpose. Examples of termination primers that can be used include alkyne termination primers (e.g., which can be attached to the azide surface portion of the catalytic polymer hydrogel 16'), phosphorothioate termination primers (e.g., which can be attached to the bromine surface portion of the catalytic polymer hydrogel 16'), or azide termination primers (e.g., which can be attached to the alkyne surface portion of the catalytic polymer hydrogel 16 ').

Specific examples of suitable primers 30 include the P5 and P7 primers 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 other instrument platforms.

In one example, grafting may involve flow-through deposition (e.g., using a temporarily bonded or permanently bonded lid), bubble coating, spray coating, whipped dispensing, or by another suitable method of attaching the primers 30 to the catalytic polymer hydrogel 16' in the flow channels 26. Each of these exemplary techniques may utilize a primer solution or mixture that may include the primer 30, water, a buffer, and a catalyst. With either of the grafting methods, the primers 30 react with the reactive groups of the catalytic polymeric hydrogel 16' in the flow channel 26 and have no affinity for the surrounding substrate 24. Thus, the primers 30 selectively graft to the catalytic polymer hydrogel 16' in the flow channels 26.

In the example shown in fig. 3C, flow cell 22 includes a multilayer substrate 24B including a support 32 and a patterned material 34 on support 32. Patterned material 34 defines recesses 36 separated by gap regions 28. A recess 36 is located within each of the flow channels 26.

In the example shown in fig. 3C, patterned material 34 is located on support 32. It should be understood that any material that can be selectively deposited, or deposited and patterned to form recesses 36 and gap regions 28, may be used to pattern material 34.

As one example of patterned material 34, inorganic oxides may be selectively applied to support 32 by vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta)₂O₅) Alumina (e.g., Al)₂O₃) Silicon oxide (e.g. SiO)₂) Hafnium oxide (e.g., HfO)₂) And the like.

As another example of the patterning material 34, a resin may be applied to the support 32 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, bubble coating, spin coating, spray coating, whipped dispensing, ultrasonic spray coating, knife coating, aerosol printing, screen printing, microcontact printing, and the like. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, imprinting techniques, molding techniques, microetching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resin (POSS) -based resins, non-POSS epoxy resins, poly (ethylene glycol) resins, polyether resins (e.g., ring-opening epoxy resins), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., from Bellex)) And combinations thereof.

As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a polyhedral oligomeric silsesquioxane (Si O) that is silicon dioxide (SiO)₂) With organosilicon (R)₂SiO) hybrid intermediates (e.g. RSiO)_1.5) The chemical composition of (1). An example of a POSS may be the POSS described by Kehagias et al in Microelectronic Engineering 86(2009), pp 776-778, which is incorporated by reference in its entirety. In one example, the composition is of the formula [ RSiO_3/2]_nWherein the R groups may be the same or different. Exemplary R groups of POSS include epoxy, azide/azide, and,Thiols, poly (ethylene glycol), norbornenes, tetrazines, acrylates and/or methacrylates, or additionally include, for example, alkyl, aryl, alkoxy, and/or haloalkyl groups. The resin compositions disclosed herein may comprise one or more different cage or core structures as monomeric units. The polyhedral structure may be T₈Structures, such as:and is represented by the following formula:the monomer unit generally has eight functional arms R₁To R₈。

The monomer unit may have a cage structure with 10 silicon atoms and 10R groups, referred to as T₁₀Such as:or may have a cage structure with 12 silicon atoms and 12R groups, referred to as T₁₂Such as:POSS-based materials alternatively can include T₆、T₁₄Or T₁₆A cage structure. The average cage content can be adjusted during synthesis and/or controlled by purification methods, and the cage size distribution of the monomer units can be used in the examples disclosed herein.

In some POSS examples disclosed herein, R₁To R₈Or R₁₀Or R₁₂At least one of which comprises an epoxy group. R₁To R₈Or R₁₀Or R₁₂May or may not be the same, and in some examples, R₁To R₈Or R₁₀Or R₁₂At least one of which contains an epoxy group, and R₁To R₈Or R₁₀Or R₁₂At least one other of which is a non-epoxy functional group. The non-epoxy functional groups may be (a) orthogonally reactive with epoxy groups (i.e., not in the presence of epoxy groups)The same conditions) that serves as a handle for coupling the resin to an amplification primer, polymer, or polymerization agent; or (b) groups that modulate mechanical or functional properties of the resin (e.g., modulate surface energy). In some examples, the non-epoxy functional group is selected from the group consisting of azide/azide, thiol, poly (ethylene glycol), norbornene, tetrazine, amino, hydroxyl, alkynyl, ketone, aldehyde, ester, alkyl, aryl, alkoxy, and haloalkyl.

As shown in fig. 3C, patterned material 34 includes recesses 36 defined therein and gap regions 28 separating adjacent recesses 36. Many different layouts of the recesses 36 are contemplated, including regular, repeating, and non-regular patterns. In one example, the depressions 36 are arranged in a hexagonal grid for tight packing and improved density. Other layouts may include, for example, rectilinear (rectangular) layouts, triangular layouts, and the like. In some examples, the layout or pattern may be an x-y format of the recesses 36 in rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of recesses 36 and/or interstitial regions 28. In other examples, the layout or pattern may be a random arrangement of recesses 36 and/or interstitial regions 28. The pattern may include dots, pads, wells, pillars, bars, swirls, lines, triangles, rectangles, circles, arcs, checkerboards, squares, diagonals, arrows, squares, and/or cross-hatching.

The layout or pattern of the recesses 36 may be characterized relative to the density of the recesses 36 (the number of recesses 36) in a defined area. For example, the depressions 36 may be about 200 ten thousand/mm²The density of (a) exists. The density can be adjusted to different densities, including, for example, about 100/mm²About 1,000 pieces/mm²About 10 ten thousand/mm²About 100 ten thousand/mm²About 200 ten thousand/mm²About 500 ten thousand/mm²About 1000 ten thousand/mm²About 5000 ten thousand/mm²Or a higher or lower density. It should also be understood that the density of recesses 36 in patterned material 34 may be between one of the lower and one of the upper values selected from the ranges described above. For example, a high density array may be characterized as having recesses 36 spaced less than about 100nm apart, and a medium density array may be characterizedCharacterized as having recesses 36 spaced about 400nm to about 1 μm apart, and the low density array may be characterized as having recesses 36 spaced greater than about 1 μm apart. While exemplary densities have been provided, it should be understood that any suitable density may be used. The density of the recesses 36 may depend in part on the depth of the recesses 36. In some cases, it may be desirable for the spacing between the recesses 36 to be even greater than the examples listed herein.

The layout or pattern of the recesses 36 may also or alternatively be characterized in terms of an average pitch or spacing from the center of a recess 36 to the center of an adjacent recess 36 (center-to-center spacing) or from the edge of one recess 36 to the edge of an adjacent recess 36 (edge-to-edge 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, about 50nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or greater or less. The average pitch of the particular pattern of depressions 36 may be between one of the lower and upper values selected from the ranges described above. In one example, the depressions 36 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 be used.

The dimensions of each recess 36 may be characterized by its volume, open area, depth, and/or diameter.

Each recess 36 may have any volume capable of confining a fluid. The minimum or maximum volume may be selected, for example, to accommodate the expected throughput (e.g., multiplexing), resolution, nucleotide 10, or analyte reactivity for downstream use of the flow cell 22. For example, the volume can be at least about 1X 10^-3μm³At least about 1X 10^-2μm³At least about 0.1 μm³At least about 1 μm³At least about 10 μm³At least about 100 μm³Or larger. Alternatively or additionally, the volume may be up to about 1 x 10⁴μm³Up to about 1X 10³μm³Up to about 100 μm³At most about 10μm³Up to about 1 μm³Up to about 0.1 μm³Or smaller.

The area occupied by each recessed opening may be selected based on criteria similar to those set forth above for the volume. For example, the area of each recessed opening can be at least about 1 × 10^-3μm²At least about 1X 10^-2μm²At least about 0.1 μm²At least about 1 μm²At least about 10 μm²At least about 100 μm²Or larger. Alternatively or additionally, the area may be up to about 1 × 10³μm²Up to about 100 μm²Up to about 10 μm²Up to about 1 μm²Up to about 0.1 μm²Up to about 1X 10^-2μm²Or smaller. The area occupied by each recessed opening may be greater than, less than, or intermediate to the values specified above.

The depth of each recess 36 may be large enough to accommodate some catalytic polymer hydrogel 16'. In one example, the depth can be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or greater. Alternatively or additionally, the depth may be up to about 1 × 10³μ m, up to about 100 μm, up to about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each recess 36 may be greater than, less than, or intermediate to the values specified above.

In some cases, the diameter or length and width of each depression 36 may be at least about 50nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or greater. Alternatively or in addition, the diameter or length and width may be up to about 1 × 10³μ m, up to about 100 μm, up to about 10 μm, up to about 1 μm, up to about 0.5 μm, up to about 0.1 μm, or less (e.g., about 50 nm). In some examples, the diameter or length and width is about 0.4 μm. The diameter or length and width of each recess 36 may be greater than, less than, or intermediate to the values specified above.

In the example shown in FIG. 3C, a catalytic polymeric hydrogel 16' is disposed within each recess 36. The catalytic polymeric hydrogel 16 'may be applied as described with reference to FIG. 3B such that the catalytic polymeric hydrogel 16' is present in the depressions 36 and not on the surrounding interstitial regions 28.

In the example shown in FIG. 3C, primers 30 can be grafted to the catalytic polymer hydrogel 16' within each recess 36. The primer 30 may be applied as described with reference to FIG. 3B, so that the primer will graft to the catalytic polymer hydrogel 16' and not to the surrounding interstitial regions 28.

Although not shown in fig. 3A, 3B, or 3C, it should be understood that the flow cell 22 may also include a cover attached to the base 24. In one example, the cover may be bonded to at least a portion of the base 24, for example, at some of the gap regions 28. The bond formed between the cover and the substrate 24 may be a chemical bond or a mechanical bond (e.g., using fasteners, etc.).

The cover may be any material that is transparent to the excitation light directed to the substrate 24. For example, the cover can 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.

The lid may be bonded to the substrate 24 using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, glass frit bonding, or other methods known in the art. In one example, a spacer layer may be used to bond the cover to the substrate 24. The spacer layer may be any material that seals at least some of the substrate 24 and the cover together. In some examples, the spacer layer may be a radiation absorbing material that helps bond the substrate 24 and the cover.

In other examples, flow cell 22 may also include additional patterned or unpatterned substrates 24 attached to substrate 24.

Nucleotide, its preparation and use

The nucleotide used with the example of flow cell 22 is 3' OH-terminated nucleotide 10 (see fig. 1). As described herein, a 3 'OH-terminated nucleotide 10 includes a nucleotide and a 3' OH-terminating group 12 attached to the sugar of the nucleotide. The nucleotide can be any of the examples described herein.

The 3' OH-blocking group 12 may be attached to the oxygen atom of the sugar molecule in the nucleotide. The 3' OH blocking group 12 may be a reversible terminator that only allows single base incorporation to occur in each sequencing cycle. Reversible terminators prevent the incorporation of additional bases into the nascent strand that is complementary to the template polynucleotide strand. This enables detection and identification of a single incorporated base. The 3' OH blocking group 12 can then be removed, enabling additional sequencing cycles to occur at each template polynucleotide strand. The cleavage reaction used to remove the 3' OH blocking group 12 is catalyzed by catalyst 18.

Examples of different 3'OH end-capping groups 12 include 3' -ONH₂Reversible terminator, 3' -O-allyl reversible terminator (-CH ═ CHCH)₂) And 3' -O-azidomethyl reversible terminator (-CH)₂N₃). Other suitable reversible terminators include ortho-nitrobenzyl ether, alkyl ortho-nitrobenzyl carbonates, ester moieties, other allyl moieties, acetals (e.g., t-butoxy-ethoxy), MOM (-CH)₂OCH₃) Partial, 2, 4-dinitrobenzene sulfinyl, tetrahydrofuran ether, 3' phosphoric acid, ether, -F, -H₂、-OCH₃、-N₃、-HCOCH₃And 2-nitrophenylcarbonate. For an allyl reversible terminator, the ligand on the surface of the polymer hydrogel 16' may bind a palladium (Pd (0)) catalyst, a ruthenium catalyst. For esters and acetals, any of the acid and/or base catalysts described herein may be used.

The 3' OH-terminated nucleotide 10 is a fully functional nucleotide that may also comprise a detectable label attached to the base B of the nucleotide. The detectable label can be any optically detectable moiety, including a luminescent moiety, a chemiluminescent moiety, a fluorescent moiety, a fluorogenic substrate moiety, a chromophoric moiety, and/or a chromophoric substrate moiety. Some examples of suitable optically detectable moieties include fluorescein labels, rhodamine labels, cyanine labels (e.g., Cy3, Cy5, etc.) and Alexa family fluorescent dyes and other fluorescent and fluorogenic substrate dyes).

Any suitable linking molecule may be used to attach the detectable label to base B of the nucleotide. The linker molecule is cleavable and a series of similar reactions can occur for the removal of the deblocking group 12. In one example, the linker molecule is of the formula ((CH) — (CH)₂)₂O)_n-wherein n is an integer between 2 and 50.

In some applications, it may be desirable to utilize a different type of detectable label for each nucleotide 10 that includes a different base, such as A, T, G, C (as well as U or I). For example, fluorescent or fluorogenic substrate labels may be selected such that each label absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from the other label set. Such distinguishable analogs provide the ability to simultaneously monitor the presence of different labels in the same reaction mixture. In some examples, one of the four nucleotides of the sequence may not comprise a label, while the other three nucleotides comprise different labels.

Sequencing method

Examples of flow cell 22 may be used for global sequencing technologies, such as Sequencing By Synthesis (SBS). In bulk sequencing, the amplification primers 30 may be used to form a template polynucleotide strand (not shown) to be sequenced on the flow cell 22. At the beginning of template polynucleotide strand formation, a library template can be prepared from any nucleic acid sample (e.g., a DNA sample or an RNA sample). Nucleic acid samples can be fragmented into single-stranded, similarly sized (e.g., <1000bp) DNA or RNA fragments. Adapters may be added to the ends of these fragments during preparation. By reducing cycling amplification, different motifs can be introduced in the adaptor, such as sequencing binding sites, indices, and regions complementary to the primers 30 in the recess 36. The final library template comprises DNA or RNA fragments and adapters at both ends. In some examples, fragments from a single nucleic acid sample have the same adaptors added thereto.

Multiple library templates may be introduced into the flow cell 22. A plurality of library templates are hybridized to one of two types of primers 30, for example, immobilized in the flow channel 26 or in a recess 36 in the flow channel 26.

Cluster generation may then proceed. In one example of cluster generation, library templates are replicated from hybrid primers by 3' extension using high fidelity DNA polymerase. The initial library template is denatured, leaving behind copies immobilized in the flow channel 26 or recess 36. Isothermal bridge amplification or some other form of amplification may be used to amplify the immobilized copies. For example, the replicated template loops back to hybridize to the adjacent complementary primer 30, and the polymerase replicates the replicated template to form double-stranded bridges, which are denatured to form two single strands. The two strands loop back and hybridize to adjacent complementary primers 30 and are extended again to form two new double-stranded loops. This process was repeated for each template copy through isothermal denaturation and amplification cycles to generate dense clonal clusters. Each cluster of the double-stranded bridge is denatured. In one example, the antisense strand is removed by specific base cleavage, leaving a forward template polynucleotide strand. Clustering results in the formation of several template polynucleotide strands in the flow channel 26 or in each depression 36. This example of clustering is bridge amplification and is one example of amplifications that can be performed. It should be understood that other amplification techniques may be used, such as the exclusive amplification (Examp) workflow (Illumina Inc.).

A sequencing primer that hybridizes to a complementary sequence on a template polynucleotide strand may be introduced. The sequencing primer prepares the template polynucleotide strand for sequencing.

To initiate sequencing, the spiking mixture may be added to flow cell 22. In one example, the incorporation mixture comprises a liquid carrier, a polymerase, and a 3' OH-terminated nucleotide 10. It will be appreciated that in some instances, the incorporation mixture is selected such that it does not activate the catalyst 18, as it is not desirable to initiate cleavage of the end-capping group 12 prior to incorporation and imaging. For the guest-host example, the incorporation mixture can include a guest catalyst (e.g., a metal) in an unactivated state. The guest catalyst may be bound to the initial polymer hydrogel 16' (e.g., via a ligand), and may then be stimulated during deblocking by orthogonal means (e.g., exposure to a particular wavelength) to convert the guest catalyst to an activated form.

When the spiking mixture is introduced into the flow cell 22, the fluid enters the flow channel 26 and/or the recess 36 (where the template polynucleotide chain is present).

The 3' OH-terminated nucleotides 10 are added to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner 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. More specifically, one of the nucleotides is incorporated into the nascent strand of the extended sequencing primer and complementary to the template polynucleotide strand by the corresponding polymerase. In other words, in at least some of the template polynucleotide strands that pass through the flow cell 22, the respective polymerase extends the hybridized sequencing primer by one of the nucleotides incorporated into the mixture.

In this exemplary method, after the nucleotide base is incorporated into the nascent strand, the incorporation mixture, including any unincorporated 3' OH-terminated nucleotides 10, may be removed from the flow cell 22. This can be achieved using a wash solution (e.g., buffer).

As referred to herein, the 3 'OH-terminated nucleotide 10 includes a reversible termination feature (e.g., a 3' OH-blocking group 12) that terminates further primer extension once the nucleotide is added to the sequencing primer. Without further incorporation, the recently incorporated nucleotide 10 can be detected by an imaging event. During an imaging event, an illumination system (not shown) may provide excitation light to the flow channel 26 and/or the recess 36.

The lysis mixture may then be introduced into the flow cell 22. In the examples disclosed herein, the cleavage mixture is capable of i) removing the 3' OH blocking group 12 from the incorporated nucleotide, and ii) cleaving any detectable label from the incorporated nucleotide. The catalyst present on the polymer hydrogel 16 'can accelerate the reaction that occurs during the removal of the 3' OH blocking group. Removal of the 3' OH blocking group 12 enables subsequent sequencing cycles to be performed, and speeding up this reaction with the catalyst 18 may make the overall sequencing process more efficient.

Some examples of catalysts accelerate intermediate steps during the deblocking reaction. Thus, the catalyst performs its function when incorporated into the cleavage mixture and initiates the deblocking reaction. When a photoacid generator is used as the catalyst 18, additional light exposure may be used to initiate catalytic activity.

Examples of 3' OH blocking groups and suitable deblocking agents/components in the cleavage mixture may include: an ester moiety removable by basic hydrolysis; nal, trimethylchlorosilane and Na can be used₂S₂O₃Or allyl moieties removed with acetone/water solution of hg (ii); azidomethyl groups cleavable with phosphines such as tris (2-carboxyethyl) phosphine (TCEP) or tris (hydroxypropyl) phosphine (THP); acetals cleavable with acidic conditions, such as tert-butoxy-ethoxy; available LiBF₄And CH₃CN/H₂MOM (-CH) with O cleavage₂OCH₃) A moiety; 2, 4-dinitrobenzene sulfinyl that is cleavable with nucleophiles such as thiophenol and thiosulfate; tetrahydrofuranyl ethers cleavable with Ag (I) or Hg (II); and a 3' phosphate cleavable by a phosphatase (e.g., a polynucleotide kinase).

Washing may occur between various 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. In some examples, paired-end sequencing may be used, in which the forward strand is sequenced and removed, and then the reverse strand is constructed and sequenced.

Although SBS has been described in detail, it is to be understood that the flow cell 22 described herein may be used in conjunction with other sequencing protocols for genotyping, or in other chemical and/or biological applications. In some cases, the primers of the flow cell may be selected to enable paired-end sequencing to be performed simultaneously, with both the forward and reverse strands present on the catalytic polymer hydrogel 16', allowing base detection of each read at the same time. Sequencing paired ends sequentially and simultaneously facilitates detection of genomic rearrangements and repetitive sequence elements as well as gene fusions and new transcripts. In another example, the flow cell 10 disclosed herein can be used to generate libraries on a flow cell.

Reagent kit

Any of the examples of flow cells 22 described herein may be part of a kit. Thus, any of the examples of the polymer hydrogel 16' disclosed herein can be part of a kit. Some examples of kits include a flow cell 22 comprising a substrate 24; a catalytic polymeric hydrogel 16 'on a substrate 24, the catalytic polymeric hydrogel 16' comprising a catalyst 18; and an amplification primer 30 attached to the catalytic polymeric hydrogel 16'; wherein the catalyst 18 is used to accelerate cleavage of the blocking group 12 of the 3' OH-terminated nucleotide 10 introduced into the flow cell 22 and incorporated into the template strand attached to the amplification primer 30; and a cleavage mixture comprising a component for initiating cleavage of the end-capping group 12.

Additional description

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided 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 is also to be understood that the terms explicitly employed herein, which may also appear in any disclosure incorporated by reference, are to be accorded the most consistent meanings with the specific concepts disclosed herein.

Reference throughout this specification to "one example," "another example," "an example," etc., 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. Furthermore, it should be understood that the elements described for any example may be combined in any suitable manner in the various examples, unless the context clearly dictates otherwise.

It should be understood that ranges provided herein include the recited range as well as any value or subrange within the recited range, as if such value or subrange were explicitly recited. For example, a range of about 2mm to about 300mm should be interpreted to include not only the explicitly recited limits of about 2mm to about 300mm, but also to include individual values (such as about 40mm, about 250.5mm, etc.) and sub-ranges (such as about 25mm to about 175mm, etc.). Further, when values are described using "about" and/or "substantially," they are intended to encompass minor variations (up to +/-10%) from the stated values.

While several examples have been described in detail, it should be understood that modifications to the disclosed examples may be made. Accordingly, the above description should be considered non-limiting.