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EP-4245864-B1 - CHIP, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF

EP4245864B1EP 4245864 B1EP4245864 B1EP 4245864B1EP-4245864-B1

Inventors

  • WANG, QI
  • SUN, LEI
  • FENG, Diewen
  • GAO, JINHONG
  • LIN, Zhifeng
  • LIU, LEI
  • CHEN, FANG

Dates

Publication Date
20260506
Application Date
20211111

Claims (14)

  1. A chip, comprising: a substrate having a surface grafted with an amino group, the amino group being a primary amino group or a secondary amino group; a first compound grafted to the surface via the amino group, the first compound comprising the following structure: wherein R 1 and R 2 are each independently selected from -OH and C1-C5 alkoxy, and n is 1 or 2, characterised in that the chip further comprises: a polymer linked to the first compound, wherein the polymer comprises a plurality of structural units, at least one of the structural units comprising an amino group; and a nucleic acid sequence linked to the polymer, the polymer and the nucleic acid sequence are linked via a linker group, wherein the linker group comprises a first terminus capable of bonding to the nucleic acid sequence and a second terminus capable of linking to the amino group of the polymer, the second terminus comprises at least one of -NHS, an epoxy group and an isocyanate group, and the nucleic acid sequence has a -DBCO or -N 3 modification, and the first terminus comprises -DBCO or -N ; wherein when the nucleic acid sequence has a -DBCO modification, the first terminus comprises -N 3 , and when the nucleic acid sequence has a -N 3 modification, the first terminus comprises -DBCO.
  2. The chip according to claim 1, wherein i) R 1 and R 2 are each independently selected from -OH, -OCH 3 and -OCH 2 CH 3 ; or ii) the first compound comprises one of the following structures:
  3. The chip according to any one of claims 1 or 2, wherein the amino group is derived from aminosilane; wherein optionally the aminosilane is selected from at least one of (3-aminopropyl)triethoxysilane, 3-aminopropyl trimethoxysilane, 3-aminopropyl methyldiethoxysilane and aminoethylaminopropyl triethoxysilane.
  4. The chip according to any one of claims 1-3, wherein the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimer, polyacrylamide and polyethyleneimine compounds.
  5. A method for preparing a chip, comprising: obtaining a substrate having a surface grafted with an amino group, the amino group being a primary amino group or a secondary amino group; linking a first compound to the surface via the amino group, the first compound comprising the following structure: wherein R 1 and R 2 are each independently selected from -OH and C1-C5 alkoxy, preferably -OH, -OCH 3 and -OCH 2 CH 3 ; and n is 1 or 2; characterised in that the method further comprises the following steps: linking a polymer to the first compound, wherein the polymer comprises a plurality of structural units, at least one of the structural units comprising an amino group; and linking a nucleic acid sequence to the polymer via a linker group , wherein the linker group comprises a first terminus capable of linking to the nucleic acid sequence and a second terminus, the second terminus comprises at least one of an -NHS group, an epoxy group and an isocyanate group, the nucleic acid sequence has a -DBCO or -N 3 modification, and the first terminus comprises -DBCO or -N 3 ; wherein when the nucleic acid sequence has a -DBCO modification, the first terminus comprises -N 3 , and when the nucleic acid sequence has a -N 3 modification, the first terminus comprises -DBCO.
  6. The method according to claim 5, wherein R 1 and R 2 are each independently selected from -OH, - OCH 3 and -OCH 2 CH 3 ; wherein optionally the first compound comprises one of the following structures:
  7. The method according to any one of claims 5 or 6, wherein the amino group is derived from aminosilane; wherein optionally the aminosilane is selected from at least one of (3-aminopropyl)triethoxysilane, 3-aminopropyl trimethoxysilane, 3-aminopropyl methyldiethoxysilane and aminoethylaminopropyl triethoxysilane.
  8. The method according to claim 7, wherein a reaction is performed at room temperature for 1-8 h to allow the aminosilane to be linked to the surface, in order to obtain the substrate with the surface grafting amino group.
  9. The method according to any one of claims 5-8, wherein a reaction is performed at room temperature for 3-5 h to allow the amino group to be linked to the first compound.
  10. The method according to any one of claims 5-7, wherein the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimer, polyacrylamide and polyethyleneimine compounds, wherein optionally a reaction is performed at 30-40°C for 1-5 h to allow the polymer to be linked to the first compound.
  11. The method according to any one of claims 5-10, wherein a reaction is performed at room temperature for 0.5-5 h to allow the second terminus of the linker group to be linked to the amino group of the polymer.
  12. The method according to claim 11, wherein a reaction is performed at 30-65 °C for 1-48 h to allow the first terminus of the linker group to be linked to the nucleic acid sequence.
  13. The method according to any one of claims 5-12, further comprising: treating the surface with a blocking reagent selected from at least one of NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3 and acetic anhydride.
  14. Use of the chip according to any one of claims 1-4 or the chip prepared by the method according to any one of claims 5-3 in nucleic acid detection, wherein optionally, the nucleic acid detection is sequencing.

Description

TECHNICAL FIELD The present application relates to the technical field of nucleic acid detection, and in particular, to a chip, a preparation method therefor and application thereof. BACKGROUND In recent years, with the rapid development in such fields as big health and precision medicine, the upgrading and progress of nucleic acid detection technologies are receiving more and more attention. Nucleic acid detection chips and related technologies, including capturing of target nucleic acids, specific molecular biological reactions, nucleic acid sequence determination, etc., performed on the surface of a chip, are key factors that determine the performance and application range of nucleic acid detection technologies. In the process of realizing development and upgrading of nucleic acid sequence determination (sequencing) technologies based on chip detection, the first-generation Sanger sequencing, the second-generation sequencing, and the third-generation or fourth-generation single-molecule sequencing are developed. Various sequencing technologies or sequencing platforms that realize sequencing based on chip detection have their own requirements on the properties of the surface of the chip, the amount of linkage/immobilization or distribution of compounds or sequences thereon, etc. Therefore, for each sequencing technology or sequencing platform, it is generally necessary to develop and design a specific chip that meets specified requirements or can be adapted to the corresponding sequencing platform or sequencing technology. US 2002/022228 A1 and US 6 602 692 B1 disclose a chip comprising a squaric acid derivative immobilised to the chip by amino groups grafted on the surface of the chip. Biomolecules like antibodies or nucleic acids are immobilised to the chip directly via a covalent bond with the squaric acid derivative. US 7 241 883 B2 discloses immobilisation techniques using squaric or croconic acid for use in DNA assays.. SUMMARY The invention is set out in the appended claims directed to a chip (claim 1), a method of its manufacture (claim 5) and use thereof (claim 14). The chip has a uniformly modified surface with high biochemical activity, which is beneficial for controlling the amount and/or density of the subsequently loaded oligonucleotide sequence (primer or probe) and/or nucleic acid molecule under test that are indirectly linked to the first compound. The chip is particularly suitable for the application that poses high requirements on surface properties and needs stable and controllable surface performance. The chip with the surface properties described above is suitable for a sequencing platform that realizes sequencing based on chip detection and by using the sequencing by synthesis (SBS) principle, such as a single-molecule sequencing platform or a high-throughput sequencing platform, specifically, for example, a platform that allows a nucleic acid molecule under test to be linked to the surface of the chip for single-molecule detection directly without amplification, or for example, a platform that allows a nucleic acid molecule under test to be linked to the surface of the chip and then detected after being amplified into clusters (amplifying signals) on the surface. The chip is suitable for a mainstream platform for realizing sequencing based on the SBS principle currently on the market, such as sequencing platforms of ILLUMINA, BGI and the like. In addition, by using the method according to the above embodiments, the chips with stable and consistent surface properties can be easily and controllably prepared in batches, and have strong industrial practicability. BRIEF DESCRIPTION OF THE DRAWINGS The aforementioned and/or additional aspects and advantages of the embodiments of the present application will become apparent and easily understood from the description of the embodiments in reference to the following drawings, among which: FIG. 1 is a schematic top view of a chip according to an embodiment of the present application;FIG. 2 is an image of a certain field of view on the surface of a chip according to an embodiment of the present application;FIG. 3 is an image of a certain field of view on the surface of a chip according to an embodiment of the present application;FIG. 4 is an image of a certain field of view on the surface of a chip according to an embodiment of the present application;FIG. 5 is an image of a certain field of view on the surface of a chip according to an embodiment of the present application; andFIG. 6 is an image of a certain field of view on the surface of a chip according to an embodiment of the present application. DETAILED DESCRIPTION The chip of the present application, the preparation method therefor and the application thereof are described in further details below with reference to specific examples. The embodiments described below with reference to the accompanying drawings are illustrative and are merely intended to explain the present application,