EP-4735160-A2 - FLOW CELLS WITH DENDRON ARCHITECTURE

Abstract

An example of a flow cell includes a substrate including a surface and a dendron architecture. The dendron architecture includes a functionalized focal point of attachment that is attached to the substrate surface and a plurality of peripheral functional groups that are orthogonal to the functionalized focal point of attachment. The flow cell further includes a primer set attached to the dendron architecture via the plurality of peripheral functional groups.

Inventors

• GEORGE, Wayne N.
• MARIA, Iuliana Petruta
• SMITH, RANDALL
• BROWN, ANDREW A.

Assignees

• Illumina, Inc.

Dates

Publication Date

20260506

Application Date

20240621

Claims (20)

1. What is claimed is: 1. A flow cell, comprising: a substrate including a surface; a dendron architecture including: a functionalized focal point of attachment that is attached to the substrate surface; and a plurality of peripheral functional groups that are orthogonal to the functionalized focal point of attachment; and a primer set attached to the dendron architecture via the plurality of peripheral functional groups.
2. 2. The flow cell as defined in claim 1, wherein the substrate surface includes a silane that attaches the functionalized focal point of attachment to the substrate surface.
3. 3. The flow cell as defined in claim 1, wherein the substrate surface is free of silane.
4. 4. The flow cell as defined in any one of claim 1 through claim 3, wherein the substrate surface includes a regenerating moiety, and wherein the functionalized focal point of attachment is attached to the substrate surface via bonding with the regenerating moiety.
5. 5. The flow cell as defined in any one of claim 1 through claim 4, wherein: the functionalized focal point of attachment includes a functional group that is selected from the group consisting of an azide, an aryl azide, an amine, a norbornene, a tetrazole, a tetrazine, a sulfonyl fluoride, a thiol, an epoxy, a phosphine having at least two phenyl groups that are capable of undergoing a Staudinger reaction, an acrylate, an alkyne, a cyclooctyne based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction, a cyclooctene based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction or an inverse electron demand Diels Alder a terminal alkene, an activated ester, and an aryl fluorosulfate; and each of the plurality of peripheral functional groups includes a functional group that is selected from the group consisting of an azide, an aryl azide, an amine, a norbornene, a tetrazole, a tetrazine, a sulfonyl fluoride, a thiol, an epoxy, a phosphine having at least two phenyl groups that are capable of undergoing a Staudinger reaction, an acrylate, an alkyne, a cyclooctyne based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction, a cyclooctene based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction or an inverse electron demand Diels Alder reaction, a terminal alkene, an activated ester, and an aryl fluorosulfate.
6. 6. The flow cell as defined in any one of claim 1 through claim 4, wherein: the functionalized focal point of attachment is a tetrazine; the plurality of peripheral functional groups is a plurality of azides; and the substrate surface includes a norbornene functional group that attaches the functionalized focal point of attachment to the substrate surface.
7. 7. The flow cell as defined in any one of claim 1 through claim 6, wherein the number of peripheral functional groups included in the dendron architecture ranges from 2 to 20.
8. 8. A method, comprising: introducing a dendron architecture to a surface of a substrate, wherein the dendron architecture includes: a functionalized focal point of attachment that attaches to the substrate surface; and a plurality of peripheral functional groups that are orthogonal to the functionalized focal point of attachment.
9. 9. The method as defined in wherein a primer set is pre-grafted to the plurality of peripheral functional groups.
10. 10. The method as defined in claim 8, further comprising grafting a primer set to the plurality of peripheral functional groups.
11. 11. The method as defined in claim 8, wherein prior to introducing the dendron architecture to the substrate surface, the method further comprises grafting a regenerating moiety to the substrate surface, and wherein the functionalized focal point of attachment attaches to the regenerating moiety.
12. 12. The method as defined in any one of claim 8 through claim 11, further comprising silanizing the substrate surface prior to introducing the dendron architecture thereto.
13. 13. The method as defined in any one of claim 8 through claim 12, wherein: the functionalized focal point of attachment includes a functional group that is selected from the group consisting of an azide, an aryl azide, an amine, a norbornene, a tetrazole, a tetrazine, a sulfonyl fluoride, a thiol, an epoxy, a phosphine having at least two phenyl groups that are capable of undergoing a Staudinger reaction, an acrylate, an alkyne, a cyclooctyne based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction, a cyclooctene based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction or an inverse electron demand Diels Alder reaction, a terminal alkene, an activated ester, and an aryl fluorosulfate; and each of the plurality of peripheral functional groups includes a functional group that is selected from the group consisting of an azide, an aryl azide, an amine, a norbornene, a tetrazole, a tetrazine, a sulfonyl fluoride, a thiol, an epoxy, a phosphine having at least two phenyl groups that are capable of undergoing a Staudinger reaction, an acrylate, an alkyne, a cyclooctyne based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction, a cyclooctene based molecule that is capable of a strain-promoted alkyne-azide cycloaddition reaction or an inverse electron demand Diels Alder reaction, a terminal alkene, an activated ester, and an aryl fluorosulfate.
14. 14. The method as defined in any one of claim 8 through claim 12, wherein: the functionalized focal point of attachment is a tetrazine; the plurality of peripheral functional groups is a plurality of azides; and the substrate surface includes a norbornene functional group that attaches the functionalized focal point of attachment to the substrate surface.
15. 15. The method as defined in any one of claim 8 through claim 14, wherein the number of peripheral functional groups included in the dendron architecture ranges from 2 to 20.
16. 16. A method, comprising: depositing a polymeric hydrogel directly over a surface of a substrate; and introducing a dendron architecture to a surface of the polymeric hydrogel, wherein the dendron architecture includes: a functionalized focal point of attachment that attaches to the polymeric hydrogel surface, a plurality of peripheral functional groups that are orthogonal to the functionalized focal point of attachment, and a primer set grafted to the plurality of peripheral functional groups prior to the introduction of the dendron architecture to the polymeric hydrogel surface.
17. 17. The method as defined in claim 16, wherein prior to introducing the dendron architecture to the polymeric hydrogel surface, the method further comprises grafting a regenerating moiety to the polymeric hydrogel surface, and wherein the functionalized focal point of attachment attaches to the regenerating moiety.
18. 18. The method as defined in of claim 16 or claim 17, further comprising silanizing the substrate surface prior to depositing the polymeric hydrogel thereon.
19. 19. The method as defined in any one of claim 16 through claim 18, wherein: the functionalized focal point of attachment includes a functional group that is selected from the group consisting of an azide, an aryl azide, an amine, a norbornene, a tetrazole, a tetrazine, a sulfonyl fluoride, a thiol, an epoxy, a phosphine having at least two phenyl groups that are capable of undergoing a Staudinger reaction, an acrylate, an alkyne, a cyclooctyne based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction, a cyclooctene based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction or an inverse electron demand Diels Alder reaction, a terminal alkene, an activated ester, and an aryl fluorosulfate; and each of the plurality of peripheral functional groups includes a functional group that is selected from the group consisting of an azide, an aryl azide, an amine, a norbornene, a tetrazole, a tetrazine, a sulfonyl fluoride, a thiol, an epoxy, a phosphine having at least two phenyl groups that are capable of undergoing a Staudinger reaction, an acrylate, an alkyne, a cyclooctyne based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction, a cyclooctene based molecule that is capable of undergoing a strain-promoted alkyne-azide cycloaddition reaction or an inverse electron demand Diels Alder reaction, a terminal alkene, an activated ester, and an aryl fluorosulfate.
20. 20. The method as defined in any one of claim 16 through claim 18, wherein: the functionalized focal point of attachment is an N-hydroxysuccinimide; the plurality of peripheral functional groups is a plurality of azides; and the polymeric hydrogel surface includes an amine functional group that attaches the functionalized focal point of attachment to the polymeric hydrogel surface.

Description

FLOW CELLS WITH ARCHITECTURE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Serial Number 63/511,458, filed June 30, 2023, the content of which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING [0002] The Sequence Listing submitted herewith is hereby incorporated by reference in its entirety. The name of the file is ILI264BPCT_IP-2640- PCT_Sequence_Listing.xml, the size of the file is 14,914 bytes, and the date of creation of the file is June 21, 2024. BACKGROUND [0003] Some available platforms for sequencing nucleic acids and other biomolecules utilize a sequencing-by-synthesis approach. With this approach, a nascent strand is synthesized, and the addition of each monomer (e.g., nucleotide) to the growing strand is detected optically and/or electronically. As a template strand directs synthesis of the nascent strand, one can infer the sequence of the template DNA from the series of nucleotide monomers that were added to the growing strand during the synthesis process. In some examples of sequencing-by-synthesis, sequential paired-end sequencing may be used, where forward strands are sequenced and removed, and then reverse strands are constructed and sequenced. SUMMARY [0004] Examples of the flow cells and methods disclosed herein utilize a substrate including a surface, where the substrate surface has a dendron architecture attached thereto. The dendron architecture includes a plurality of peripheral groups, where each peripheral group is capable of forming a chemical bond with an oligonucleotide primer. Because each peripheral functional group is chemically available for oligonucleotide primer attachment, a concentration of the primers utilized in flow cell (relative to flow cell surface area) may be controlled to enhance signal strength during sequencing operations. [0005] In some examples, the flow cells and methods further utilize a regenerating moiety that enables the flow cell to be used multiple times. As such, these flow cells further enable sequencing operations to be performed with resource efficiency. BRIEF DESCRIPTION OF THE DRAWINGS [0006] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. [0007] Fig.1A is a top view of an example of a flow cell; [0008] Fig.1B is an enlarged, perspective, and partially cutaway view of an example of an architecture within a flow channel of the flow cell; [0009] Fig.1C is an enlarged, perspective, and partially cutaway view of another example of an architecture within a flow channel of the flow cell; [0010] Fig.1D is an enlarged, perspective, and partially cutaway view of still another example of an architecture within a flow channel of the flow cell; [0011] Fig.2A through Fig.2D are schematic views that together illustrate an example of a method disclosed herein, where Fig.2A depicts the formation of a depression in a resin layer of a substrate, Fig.2B depicts the application of a polymeric hydrogel over the structure of Fig.2A, Fig.2C depicts polishing of the polymeric hydrogel from interstitial regions of the structure of Fig.2B, and Fig.2D depicts the introduction of a dendron architecture including pre-grafted primers to a surface of the polymeric hydrogel within the depression; [0012] Fig.3A through Fig.3F are views that together illustrate an example of another method disclosed herein, where Fig.3A depicts the formation of a depression in a resin layer, Fig.3B depicts the application of a sacrificial layer over the structure of Fig.3A, Fig.3C depicts the removal of the sacrificial layer from within the depression, Fig.3D depicts the application of a polymeric hydrogel over the structure of Fig.3C, Fig.3E depicts removing remaining sacrificial layer material from interstitial regions of the structure of Fig.3D, and Fig.3F depicts introducing a dendron architecture including pre- grafted primers to a surface of the polymeric hydrogel. [0013] Fig.4 is schematic flow diagram that illustrates two examples of yet another method disclosed herein, where “A” depicts the formation of a depression in a resin layer, “B” depicts introducing a dendron architecture to a surface of the resin layer within the depression, and “C” depicts primers that are grafted to peripheral groups of the dendron architecture; [0014] Fig.5 is a graphical representation of the results of fluorescence intensity measurements of a flow cell including a synthesized dendron architecture disclosed herein versus a flow cell including a comparative dendron architecture, with incubation and dendritic conditions of each flow cell lane being sho