Biochip substratum and method for production thereof

Patent metadata

Field	Value
Publication number	US-9857369-B2
Application number	US-201414338205-A
Country	US
Kind code	B2
Filing date	Jul 22, 2014
Priority date	Jun 30, 2008
Publication date	Jan 2, 2018
Grant date	Jan 2, 2018

Abstract

A substrate for biochips which has a high probe loading amounts and a uniform immobilization density, and which further has a high detection sensitivity and a high reproducibility by preventing a non-specific adsorption of proteins, when used as a substrate for biochips for immobilizing probes composed of biologically relevant substances such as proteins and nucleic acids, is disclosed. Amino groups can be bound to the surface of the substrate uniformly, at a high density and stably by covalently immobilizing an amino group-containing polymer on the surface of the substrate. The probe immobilization rate is high and immobilizing density was uniform by immobilizing a probe composed of a biologically relevant substance such as a protein or nucleic acid by utilizing the amino groups. Further, detection sensitivity and reproducibility are high by inhibiting non-specific adsorption of proteins.

First claim

The invention claimed is:

1. A substrate for biochips, comprising:
(i) a substrate body having a surface composed of carbon;
(ii) an amino group-containing polymer comprising primary amino groups, the amino group-containing polymer being formed by polymerizing monomers comprising amino group-containing vinyl monomers, and being covalently bound directly to the surface of the substrate body; and
(iii) a hydrophilic polymer covalently bound directly to the surface of the substrate body,
wherein the amino group-containing polymer is bound to the carbon substrate in a radical reaction between the carbon surface and an amino group-containing vinyl monomer-derived unit in the amino group-containing polymer, and
wherein a homogeneous mixture of the amino group-containing polymer and the hydrophilic polymer is immobilized on the surface of the substrate body.

2. The substrate according to claim 1 , wherein each of 50% or more of monomeric units which constitute said amino group-containing polymer are derived from monomer(s) having at least one amino group.

3. The substrate according to claim 2 , wherein each of 90% or more of the monomeric units which constitute said amino group-containing polymer are derived from the monomer(s) having at least one amino group.

4. The substrate according to claim 1 , wherein said amino group-containing polymer is immobilized in an amount such that a density of a number of the primary amino groups in the amino group-containing polymer is 4.22×10 16 amino groups/cm 2 or more.

5. The substrate according to claim 4 , wherein the amount of said amino group-containing polymer immobilized is 40 μg/cm 2 or less.

6. The substrate according to claim 1 , wherein said amino group-containing polymer has a weight-average molecular weight such that said amino group-containing polymer has 17 or more primary amino groups per one molecule.

7. The substrate according to claim 6 , wherein the weight-average molecular weight of said amino group-containing polymer is 60,000 or less.

8. The substrate according to claim 1 , wherein said hydrophilic polymer is at least one selected from the group consisting of polyethylene glycol, polyacrylamide and agarose.

9. The substrate according to claim 1 , wherein an amount of said amino group-containing polymer immobilized is such that a density of a number of the primary amino groups in the amino group-containing polymer is 4.22×10 16 amino groups/cm 2 or more and said hydrophilic polymer is 40 μg/cm 2 or less.

10. The biochip according to claim 1 , wherein amino group-containing vinyl monomers constitute 50% or more of the constituent units in the amino group-containing polymer.

11. The biochip according to claim 1 , wherein amino group-containing vinyl monomers constitute 90% or more of the constituent units in the amino group-containing polymer.

12. A biochip comprising the substrate of claim 1 ; and a biologically relevant substance covalently bound directly or via a linker to an amino group of the substrate.

13. The biochip according to claim 12 , wherein each of 50% or more of monomeric units which constitute said amino group-containing polymer are derived from monomer(s) having at least one amino group.

14. The biochip according to claim 13 , wherein each of 90% or more of the monomeric units which constitute said amino group-containing polymer are derived from the monomer(s) having at least one amino group.

15. The biochip according to claim 12 , wherein said amino group-containing polymer is immobilized in an amount such that a density of a number of the primary amino groups in the amino group-containing polymer is 4.22×10 16 amino groups/cm 2 or more.

16. The biochip according to claim 15 , wherein the amount of said amino group-containing polymer immobilized is 40 μg/cm 2 or less.

17. The biochip according to claim 12 , wherein said amino group-containing polymer has a weight-average molecular weight such that said amino group-containing polymer has 17 or more primary amino groups per one molecule.

18. The biochip according to claim 17 , wherein the weight-average molecular weight of said amino group-containing polymer is 60,000 or less.

19. The biochip according to claim 12 , wherein said hydrophilic polymer is at least one selected from the group consisting of polyethylene glycol, polyacrylamide and agarose.

20. The biochip according to claim 12 , wherein an amount of said amino group-containing polymer immobilized is such that a density of a number of the primary amino groups in the amino group-containing polymer is 4.22×10 16 amino groups/cm 2 or more and the total amount of said amino group-containing polymer and said hydrophilic polymer is 40 μg/cm 2 or less.

21. The biochip according to claim 12 , wherein the biologically relevant substance is selected from the group consisting of proteins, nucleic acids, peptide derivatives, sugar chains and derivatives thereof, natural products and small molecule compounds.

22. A method of producing the substrate for biochips of claim 1 , said method comprising:
coating the surface of the substrate body with said amino group-containing polymer and said hydrophilic polymer; and subsequently irradiating the surface of the substrate body with light under reduced pressure or under an inert gas atmosphere.

23. A method of producing the substrate for biochips of claim 1 , the method comprising:
a plasma irradiation or a light irradiation on the surface of said substrate body; and subsequently bringing said substrate body into contact with said amino group-containing polymer and said hydrophilic polymer.

24. The method according to claim 23 , further comprising a light irradiation on the surface of the substrate body under reduced pressure or under an inert gas atmosphere after bringing said substrate body into contact with said amino group-containing polymer.

Assignees

• Hipep Laboratories
• Nippon Light Metal Co

Inventors

• Nokihara Kiyoshi
• Hirata Akiyoshi
• Oka Yasuo
• Takebayashi Yasushi

Classifications

• B01J2219/00605

  the compounds being directly bound or immobilised to solid supports · CPC title

• Y10T428/273

  of coating · CPC title

• B01J2219/00596

  Solid-phase processes · CPC title

• G01N33/54353

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• G01N33/545 Primary

  Synthetic resin · CPC title