Composition containing fluorescence-generating substrate

• US 6,180,844 B1
• Filed: 03/09/1999
• Issued: 01/30/2001
• Est. Priority Date: 03/09/1998
• Status: Expired due to Fees

• Current Assignee: Aisin Seiki Co., Ltd. (Aisin Corp.)
• Original Assignee: Aisin Seiki Co., Ltd. (Aisin Corp.)
• Inventors: Toru, Takeshi, Fujita, Satoshi, Kondoh, Yasumitsu, Kagiyama, Naoto, Reddy, Paidi Yella
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/264,678
• Time in Patent Office: 693 Days
• Field of Search: 536/25.3, 536/23.1, 536/25.32, 435/5, 435/6, 585/24, 585/25, 585/26
• US Class Current: 585/24
• CPC Class Codes: C12Q 1/28 involving peroxidase

Abstract

A fluorescent dye-forming composition is provided that contains a fluorescence-generating substrate of formula (I):

where R¹ and R² are each independently a hydrogen atom, or an electron-donating group selected from a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, and an aryl group;

L is selected from —NHCO— or —CONH—;

Y is selected from -alkylene-COOH or formula (II):

 where R³, R⁴ and R⁵ each independently represent a hydrogen atom, carboxyl group, or an electron-donating group selected from a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, and an aryl group. Alternatively, two of R³, R⁴ and R⁵ form, together with the adjacent carbon atoms to which they bond, a fused 5-membered or 6-membered hydrocarbon ring. Alkylene represents a linear or branched C_1-6 alkylene group; and a represents an integer of 0 or 1. The substrate forms a fluorescent dye having good binding to biological samples.

Claims

1. A composition for forming fluorescent dyes in oxidative environments, which comprises a fluorescence-generating substrate or fluorescent dye precursor of formula (I):

wherein R¹ and R² are each independently selected from the group consisting of a hydrogen atom, or an electron-donating substituent;

wherein the electron-donating substituent is selected from the group consisting of a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, or an aryl group;

and, wherein L is selected from the group consisting of —

NHCO— and

, —

CONH—

;

and wherein Y is selected from the group consisting of -alkylene-COOH and formula (II);

 

wherein R³, R⁴ and R⁵ are each independently selected from the group consisting of a hydrogen atom, a carboxyl group, a 5- or 6-membered hydrocarbon ring fused to the aromatic ring formed by any two of R³, R⁴ and R⁵, together with the adjacent carbon atoms to which they bond, and an electron-donating substituent;

wherein the electron-donating substituent is selected from the group consisting of a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, and an aryl group;

and, wherein, -alkylene- is selected from the group of linear and branched C_1-6 alkylene groups;

and, wherein a is 1.

2. The composition of claim 1, wherein L, is —

NHCO—

;

Y is formula (II);

wherein R³, R⁴ and R⁵ are each independently selected from the group consisting of a hydrogen atom, a carboxyl group, a 5- or 6-membered hydrocarbon ring fused to the aromatic ring formed by any two of R³, R⁴ and R⁵, together with the adjacent carbon atoms to which they bond, and an electron-donating substituent;

wherein the electron-donating substituent is selected from the group consisting of a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, and an aryl group;

and a is 0.

3. The composition of claim 1, wherein L is —

NHCO—

;

Y is -alkylene-COOH, wherein, -alkylene- is selected from the group of linear and branched C_1-6 alkylene groups;

and a is 1.

4. The composition of claim 1, wherein the oxidative-environment is a reaction system comprising peroxidase and hydrogen peroxide.

5. The composition of claim 2, wherein the oxidative-environment is a reaction system comprising peroxidase and hydrogen peroxide.

6. The composition of claim 3, wherein the oxidative-environment is a reaction system comprising peroxidase and hydrogen peroxide.

7. The composition of claim 4, wherein the oxidative environment is a reaction system comprising peroxidase bonded to a solid support, and hydrogen peroxide.

8. The composition of claim 5, wherein the oxidative environment is a reaction system comprising peroxidase bonded to a solid support, and hydrogen peroxide.

9. The composition of claim 6, wherein the oxidative environment is a reaction system comprising peroxidase bonded to a solid support, and hydrogen peroxide.

10. The composition of claim 7, wherein the solid support is a biological sample, and peroxidase is bonded to said biological sample via immune complex formation.

11. The composition of claim 8, wherein the solid support is a biological sample, and peroxidase is bonded to said biological sample via immune complex formation.

12. The composition of claim 9, wherein the solid support is a biological sample, and peroxidase is bonded to said biological sample via immune complex formation.

13. The composition of claim 10, wherein the biological sample is a chromosome, cell or body tissue sample.

14. The composition of claim 11, wherein the biological sample is a chromosome, cell or body tissue sample.

15. The composition of claim 12, wherein the biological sample is a chromosome, cell or body tissue sample.

16. The composition of claim 7, wherein the solid support is a porous carrier or polymer membrane having a nucleic acid fixed thereon, and peroxidase is bonded to said support via immune complex formation with the nucleic acid, or via hybridization with a nucleic acid probe and said nucleic acid.

17. The composition of claim 8, wherein the solid support is a porous carrier or polymer membrane having a nucleic acid fixed thereon, and peroxidase is bonded to said support via immune complex formation with the nucleic acid, or via hybridization with a nucleic acid probe and said nucleic acid.

18. The composition of claim 9, wherein the solid support is a porous carrier or polymer membrane having a nucleic acid fixed thereon, and peroxidase is bonded to said support via immune complex formation with the nucleic acid, or via hybridization with a nucleic acid probe and said nucleic acid.

19. A method for detecting and measuring the concentration of an analyte, which comprises the steps of preparing a biological sample that may contain the analyte to be detected, applying a peroxidase to said biological sample in the presence of hydrogen peroxide, reacting said peroxidase with hydrogen peroxide in the presence of a fluorescence-generating substrate of claim 1, and measuring the intensity of the fluorescence of the fluorescent dye thus formed.

20. The method of claim 19, wherein the peroxidase is bonded to a solid biological sample through formation of an immune complex.

21. The method of claim 19, wherein the analyte to be detected in the sample is a nucleic acid, and the peroxidase is bonded to the sample via immune complex formation with the nucleic acid or via hybridization with a nucleic acid probe and the nucleic acid in said sample.

22. A composition for forming fluorescent dyes in oxidative environments, which comprises a fluorescence-generating substrate or fluorescent dye precursor of formula (I):

wherein R¹ and R² are each independently selected from the group consisting of a hydrogen atom, or an electron-donating substituent;

wherein the electron-donating substituent is selected from the group consisting of a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, or an aryl group;

and, wherein L is selected from the group consisting of —

NHCO— and

,—

CONH—

,

and wherein Y is formula (II);

 

wherein R³, R⁴ and R⁵ are each independently selected from the group consisting of a hydrogen atom, a carboxyl group, a 5- or 6-membered hydrocarbon ring fused to the aromatic ring formed by any two of R³, R⁴ and R⁵, together with the adjacent carbon atoms to which they bond, and an electron-donating substituent;

wherein the electron-donating substituent is selected from the group consisting of a lower alkyl group, a lower alkoxy group, a hydroxyl group, an alkylcarbonyloxy group, an amino group di-substituted with a lower alkyl group, and an aryl group

and, wherein a is selected from 0 or 1, and when a is 0, no more than one of R³, R⁴ and R⁵ is a hydrogen atom.