Biosensor and method for immobilizing a physiologically active substance

Abstract

An object of the present invention is to provide a biosensor and a method for immobilizing a physiologically active substance, by which preconcentration effects can be obtained at a pH that is equivalent to or higher than the isoelectric point of the physiologically active substance and the physiologically active substance can be covalently bound to the surface. The present invention provides a biosensor comprising a solid substrate to which a polymer having a primary or secondary amino group is bound, by which a physiologically active substance can be chemically immobilized following preconcentration of the substance at a pH that is equivalent to or higher than the isoelectric point of the substance.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sensorgram obtained by setting sample 1 (comparative example) and sample 2 (the present invention) prepared in Example 1 in a surface plasmon measurement device and then applying a pepsin solution (pH 7.4) for 5 minutes.

FIG. 2 shows the sensorgram obtained by setting sample 1 (comparative example) and sample 2 (the present invention) prepared in Example 1 in a surface plasmon resonance device and then applying a BSA solution (pH7.4) for 5 minutes.

FIG. 3 shows the sensorgram obtained by setting sample 1 (comparative example) and sample 2 (the present invention) prepared in Example 1 in a surface plasmon resonance device and then applying a CA solution (pH7.4) for 5 minutes.

FIG. 4 shows the results obtained through examination of: the amount of pepsin immobilized on sample 3 prepared by washing without allowing an EDC (0.4 M)/NHS (0.1 M) aqueous solution (carboxylic acid activator) to come into contact with sample 2 upon which pepsin (pH 7.4) had been preconcentrated for 5 minutes; and the amount of pepsin immobilized on sample 4 prepared by washing after causing an EDC (0.4M)/NHS (0.1 M) aqueous solution to come into contact with sample 2 and remain in contact therewith for 5 minutes.

FIG. 5 shows the results obtained through examination of the preconcentration degrees of BSA (pH 7.4, 0.1 mg/ml) on sensor chips prepared using various polyamines.

Claims

1. A biosensor comprising a solid substrate to which a polymer having a primary or secondary amino group is bound, by which a physiologically active substance can be chemically immobilized following preconcentration of the substance at a pH that is equivalent to or higher than the isoelectric point of the substance.

2. The biosensor according to claim 1, wherein the polymer having a primary or secondary amino group is a polymer obtained by causing a polymer having a carboxyl group to react with a polyamine.

3. The biosensor according to claim 2, wherein the polymer having the carboxyl group is carboxymethyldextran.

4. The biosensor according to claim 1, wherein the solid substrate to which the polymer having the primary or secondary amino group is bound is a solid substrate to which a water-soluble polymer is bound, a solid substrate to which a hydrophobic polymer is bound, or a solid substrate on which a self-assembled monomolecular film is formed.

5. The biosensor according to claim 1, wherein a layer of the polymer having the primary or secondary amino group is formed on a metal.

6. The biosensor according to claim 5, wherein the metal is gold, silver, copper, platinum, or aluminium.

7. The biosensor according to claim 1, which is used for nonelectrochemical detection.

8. The biosensor according to claim 1, which is used for surface plasmon resonance analysis.

9. A method for immobilizing a physiologically active substance, which comprises preconcentrating a physiologically active substance having a carboxyl group at a pH that is equivalent to or higher than the isoelectric point of the substance, on a solid substrate to which a polymer having a primary or secondary amino group is bound, causing a carboxylic acid activator to come into contact with the substrate, so as to bind the polymer having the amino group to the physiologically active substance.

10. The method for immobilizing a physiologically active substance according to claim 9, wherein the polymer having the primary or secondary amino group is a polymer obtained by causing a polymer having a carboxyl group to react with a polyamine.

11. The method for immobilizing a physiologically active substance according to claim 10, wherein the polymer having the carboxyl group is carboxymethyldextran.

12. The method for immobilizing a physiologically active substance according to claim 9, wherein the solid substrate to which the polymer having the primary or secondary amino group is bound is a solid substrate to which a water-soluble polymer is bound, a solid substrate to which a hydrophobic polymer is bound, or a solid substrate on which a self-assembled monomolecular film is formed.

13. The method for immobilizing a physiologically active substance according to claim 9, wherein a layer of the polymer having the primary or secondary amino group is formed on a metal.

14. The method for immobilizing a physiologically active substance according to claim 13, wherein the metal is gold, silver, copper, platinum, or aluminium.

15. A method for detecting or measuring a substance that interacts with a physiologically active substance, which comprises a step of causing a test substance to come into contact with the biosensor according to claim 1, on the surface of which a physiologically active substance is covalently bound.

16. The method according to claim 15, wherein a substance that interacts with a physiologically active substance is detected or measured by a nonelectrochemical method.

17. The method according to claim 15, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.