Composition containing fluorescence-generating substrate

Details Composition containing fluorescence-generating substrate Composition containing fluorescence-generating substrate

1999-03-09

2001-01-30

Riley, Jezia (Department: 1656)

Chemistry of hydrocarbon compounds

Compound or reaction product mixture

Aromatic

C585S025000, C585S026000, C435S005000, C435S006120, C536S023100, C536S025300, C536S025320

Reexamination Certificate

active

06180844

ABSTRACT:

BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a composition containing a fluorescence-generating substrate or fluorescent dye precursor. More specifically, it relates to a composition and methods useful in detecting biological analytes.
BACKGROUND OF THE INVENTION
Heretofore, radioactive isotopes, coloring substrates, as well as fluorescent dyes and their precursors (or fluorescence generating substrates) have been widely used to detect biological analytes. In particular, fluorescence-generating substrates are now widely used and studied for enzyme-linked immunosorbent assays, in which an alkali phosphatase or peroxidase is used as the labeling enzyme.
For example, 2′, 7′-clichlorofluorescein is a fluorescence-generating substrate (or fluorescence-generating compound) capable of being used in reaction systems comprising peroxidase and hydrogen peroxide (A. S. Ferrer, et al. Anal. Biochem., 187, 129 (1990)). In that reaction system, in general, coloring substrates such as diaminobenzidine derivatives are often used because of their color stability, although their sensitivity is lower than that of fluorescence-generating substrates (for example, see P. D. Josephy, et al., J. Biol. Chem., 258, 5561 (1983); E. Sheldon, et al., Clin. Chem., 33, 1368 (1987)).
3-(p-hydroxyphenyl) propionic acid (K. Zaitsu, et al., in Anal. Biochem., 109, 109-113 (1980)), N-alkylcarbonyl-p-hydroxyanilides (Japanese Patent Publication (JP-B) Hei-7-108238) and N-aroyl-p-aminoanilides (S. Fujita, et al., in Chemistry-y Letters, 1075-1076 (1997)) have relatively simple structures, and are interesting fluorescence-generating substrates capable of reacting in a system that comprises peroxidase and hydrogen peroxide.
Use of these fluorescence-generating substrates only partially achieves the desired object. However, where hydrogen peroxide is reacted with peroxidase on a solid support in the presence of any of such substrates, and it is desired that the fluorescent dye formed by the reaction is kept on the support, a high-resolution assay is not always possible. For example, the fluorescent region will broaden because of the relatively high solubility of the dye in aqueous media.
SUMMARY OF THE INVENTION
Tile object of the present invention is to provide a novel means of using fluorescence-generating substrates, which is free from the problems of the prior art, noted above. In particular, this invention solves the shortcomings of fluorescence-generating substrates that are used on solid supports. Specifically, the object of the invention is to provide improved fluorescence-generating substrates which generate fluorescent dyes having reduced mobility on solid supports, and also to provide a method of using these substrates.
After studying the behavior of various compounds in reaction systems comprising peroxidase and hydrogen peroxide, we have found that various p-hydroxyphenyl derivatives, which have lower water solubility than that of 3-(p-hydroxyphenyl)propionic acid, noted above, can form substantially water-insoluble fluorescent dyes in the reaction system, and that the possibility of those dyes being formed on solid biological samples is extremely low.
Accordingly, to attain the object noted above, the invention provides a composition for forming fluorescent dyes in oxidative environments, which is characterized by containing a fluorescence-generating substrate (or fluorescent dye precursor) of a formula (1):
where R
1
and R
2
each independently represent a hydrogen atom, 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 lower alkyl groups, or an aryl group;
L represents —NHCO— or —CONH—;
Y represents an -alkylene-COOH group, or a group of structure (II):
 where R
3
, R
4
and R
5
each independently represent a group as defined above for R
1
and R
2
, or a carboxyl group; or two of R
3
, R
4
and R
5
are groups that form, together with the adjacent carbon atoms to which they bond, a fused 5-membered or 6-membered hydrocarbon ring;
alkylene is defined as a linear or branched C
1-6
alkylene group; and
a represents an integer of 0 or 1.
Another embodiment of the invention also provides for a method of detecting an analyte, which comprises:
preparing a biological sample that may contain an analyte to be detected,
applying a peroxidase to the sample containing hydrogen peroxide,
reacting the peroxidase enzyme with hydrogen peroxide in the presence of the fluorescence-generating substrate of formula (I) mentioned above,
and measuring the intensity of the fluorescence of the fluorescent dye thus formed, thereby detecting the presence or absence of the analyte, or measuring the concentration of the analyte.


REFERENCES:
Shiga et al. “A novel method for determining peroxidaes activities using p-acetamidophenol analogs” Analytical Sciences, vol. 11, pp. 195-201, Apr. 1995.
Alvaro Sanchez Ferrer et al, “Fluorescence Detection of Enzymatically Formed Hydrogen Peroxide in Aqueous Solution and in Reversed Micelles”, Analytical, Biochemistry 187, 129-132 (1990).
Kiyoshi Zaitsu et al, “New Fluorogenic Substrates for Horseradish Peroxidase: Rapid and Sensitive Assays for Hydrogen Peroxide and the Peroxidase”, Analytical Biochemistry 109, 109-113 (1990).
P. David Josephy et al, “Co-Oxidation of Benzidine by Prostaglandin Synthase and Comparison with the Action of Horseradish Peroxidase,” The Journal of Biological Chemistry, vol. 258, No. 9, Issue of May 10, pp. 5561-5569 (1983).
Satoshi Fujita et al, “A Novel Fluorogenic Substrate for Horseradish Peroxidase: Efficient Detection of Membrane-Bound Nucleic Acids and Simultaneous Detection of DNAs”, The Chemical Society of Japan, Chemistry Letters pp. 1075-1076 (1997).
Edward Sheldon et al, “Nonisotopic M13 Probes for Detecting the Beta-Globin Gene: Application To Diagnosis of Sickle Cell Anemia”, Clin. Chem. 33/8, 1368-1371 (1987).
Derwent Abstract, “Method for Determining Peroxidase and Substance Having Catalyst Equal to That of the Same” Accession No. 95-039393, 1995.
Derwent Abstract, “Quantitative Measurement of Peroxidase and Equiv. Catalysing Substance—Using P-Hydroxy Acetanilide Based Fluorescence Reagent in Presence of Hydrogen Peroxide, Peroxidase and Catalysing Substance” Accession No. 95-117868/16, 1995.

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