Flow cells

Patent metadata

Field	Value
Publication number	US-12534569-B2
Application number	US-202217855325-A
Country	US
Kind code	B2
Filing date	Jun 30, 2022
Priority date	Jul 2, 2021
Publication date	Jan 27, 2026
Grant date	Jan 27, 2026

Abstract

An example of a kit includes a flow cell, a primer fluid, and a cleaving fluid. The flow cell includes at least one surface functionalized with a polymeric hydrogel including azide functional groups or amine functional groups. The primer fluid includes a plurality of alkyne-containing primers, each alkyne-containing primer having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer. The cleaving fluid includes a substance that is reactive with the amino cleavable group.

First claim

What is claimed is:

1 . A kit, comprising:
a flow cell including at least one surface functionalized with a polymeric hydrogel including azide functional groups or amine functional groups; a primer fluid including a plurality of alkyne-containing primers, each alkyne-containing primer having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer; and a cleaving fluid that is reactive with the amino cleavable group,
wherein the amino cleavable group of each of the plurality of alkyne-containing primers is a BOC amide or triphenylmethylamine; and
wherein the cleaving fluid is an acid.

2 . The kit as defined in claim 1 , wherein the surface of the flow cell is non-patterned, and the polymeric hydrogel is positioned within a lane of the non-patterned surface.

3 . The kit as defined in claim 1 , wherein the surface of the flow cell is patterned with depressions separated by interstitial regions, and the polymeric hydrogel is positioned within each depression of the patterned surface.

4 . The kit as defined in claim 1 , wherein the surface of the flow cell is patterned with protrusions separated by interstitial regions, and the polymeric hydrogel is positioned on each protrusion of the patterned surface.

5 . A kit, comprising:
a flow cell including at least one surface functionalized with a polymeric hydrogel including azide functional groups or amine functional groups; a primer fluid including a plurality of alkyne-containing primers, each alkyne-containing primer having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer; a cleaving fluid that is reactive with the amino cleavable group; and a regeneration fluid including i) azide terminated molecules selected from the group consisting of azido-PEG 4 -C2-carboxylic acid, trifluoromethanesulfonyl azide, imidazolium sulfonyl azide hydrochloride, 1-(fluorosulfuryl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate, fluorosulfuryl azide, perfluorobutanesulfonyl azide, azido-PEG 4 -N-Hydroxysuccinimide ester, and imidazole-1-sulfonyl azide hydrochloride, or ii) tetrazine terminated molecules selected from the group consisting of tetrazine-N-Hydroxysuccinimide ester and methyltetrazine-sulfo-N-Hydroxysuccinimide ester.

6 . A kit, comprising:
a flow cell including at least one surface functionalized with a polymeric hydrogel including azide functional groups or amine functional groups; a primer fluid including a plurality of alkyne-containing primers, each alkyne-containing primer having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer; a cleaving fluid that is reactive with the amino cleavable group; and a linker fluid including one of:
a tetrazine molecule to react with the amine functional groups of the flow cell, the tetrazine molecule being selected from the group consisting of tetrazine-N-Hydroxysuccinimide ester, methyltetrazine-sulfo-N-Hydroxysuccinimide ester, and methyltetrazine-PEG n -N-Hydroxysuccinimide ester where n=4 or 5 or 8; or a tetrazine molecule to react with the azide functional groups of the flow cell, the tetrazine molecule being sulfo-6-methyl-tetrazine-dibenzocyclooctyne.

7 . A method, comprising:
grafting a plurality of alkyne-containing primers to respective azide functional groups or tetrazine functional groups of a polymeric hydrogel on a surface of a flow cell, each of the plurality of alkyne-containing primers having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer; performing a nucleic acid analysis involving the grafted plurality of alkyne-containing primers; introducing a cleaving fluid to cleave the grafted plurality of alkyne-containing primers at the amino cleavable group, thereby leaving a plurality of amine functional groups at the surface of the flow cell; and reacting respective azide terminated molecules or tetrazine terminated molecules with at least some of the plurality of amine functional groups to introduce new azide functional groups or new tetrazine functional groups to the surface of a flow cell.

8 . The method as defined in claim 7 , wherein performing the nucleic acid analysis involves:
introducing a sample including a plurality of template nucleic acid strands into the flow cell, whereby at least some of the plurality of template nucleic acid strands respectively hybridize to the primer sequence of at least some of the grafted plurality of alkyne-containing primers; and performing sequencing-by-synthesis.

9 . The method as defined in claim 7 , wherein:
the azide terminated molecules are selected from the group consisting of azido-PEG 4 -C2-carboxylic acid, trifluoromethanesulfonyl azide, imidazolium sulfonyl azide hydrochloride, 1-(fluorosulfuryl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate, fluorosulfuryl azide, perfluorobutanesulfonyl azide, azido-PEG 4 -N-Hydroxysuccinimide ester and imidazole-1-sulfonyl azide hydrochloride; or the tetrazine terminated molecules are selected from the group consisting of tetrazine-N-Hydroxysuccinimide ester, methyltetrazine-sulfo-N-Hydroxysuccinimide ester, sulfo-6-methyl-tetrazine-dibenzocyclooctyne, and methyltetrazine-PEG n -N-Hydroxysuccinimide ester where n=4 or 5 or 8.

10 . The method as defined in claim 7 , wherein:
the flow cell includes the azide functional groups; some of the azide functional groups remain free after grafting; the free azide functional groups are reduced to amine groups during the nucleic acid analysis; and at least some of the azide terminated molecules react with at least some of the plurality of amine functional groups and at least some other of the azide terminated molecules react with the amine groups.

11 . The method as defined in claim 7 , wherein:
the amino cleavable group is a phthalimide group; and the cleaving fluid includes hydrazine or methyl hydrazine.

12 . The method as defined in claim 7 , wherein:
the amino cleavable group is a BOC amide or triphenylmethylamine; and the cleaving fluid includes an acid.

Assignees

• Illumina Inc

Inventors

• Boutell Jonathan Mark
• George Wayne N
• Ma Xiaoyu
• Wu Xiaolin
• Xi Weixian

Classifications

• C08G63/91

  Polymers modified by chemical after-treatment · CPC title

• C08G63/823

  for the preparation of polylactones or polylactides · CPC title

• C08G63/6852 Primary

  derived from hydroxy carboxylic acids · CPC title

• C12Q1/689

  for bacteria · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title