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US 5,017,475 AGranted
Fluorescent detection method based on enzyme activated conversion of a fluorophore precursor substrate
AssigneeMicrobiological Associates, Inc.
InventorsRichard A. Harte, Stephen H. Mastin
Life of this patent
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Abstract
Methods are provided for the fluorescent detection of an analyte of interest wherein an amine-substituted, ortho-fused pyrazine fluorophore, with neither nitrogen of the pyrazine ring being fused or substituted, is produced by enzymatic oxidation of a fluorophore precursor substrate which comprises a nitrogen-substituted, cyclic compound. Also provided are novel fluorophore-labelled compounds.
Claims29
A method for detecting an analyte of interest in a liquid sample which comprises:
- (a) contacting the sample with a first reagent capable of forming a complex with the analyte of interest so as to form a first reagent:analyte complex;
- (b) contacting the first reagent:analyte complex with an oxidase-labelled reagent capable of forming a complex with the first reagent:analyte complex so as to form a first reagent:analyte:oxidase-labelled reagent complex;
- (c) treating the first reagent:analyte:
- oxidase-labelled reagent complex with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted;
- and
- (d) exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation;
- (e) detecting the radiation so emitted, thereby by detecting the analyte of interest.
4 more elements
A method for detecting an analyte of interest present in a liquid sample which comprises:
- (a) contacting the sample with an oxidase-labelled reagent capable of competing with the analyte of interest for binding sites on a complementary material not normally present in the sample, and with the complementary material, the contact being effected under appropriate conditions such that the analyte of interest and the oxidase-labelled reagent competitively bind to the complementary material so as to form analyte of interest:
- complementary material complexes and oxidase-labelled reagent:
- complementary material complexes;
- (b) separating the analyte of interest:
- complementary material complexes and the oxidase-labelled reagent:
- complementary material complexes from unbound analyte of interest and unbound oxidase-labelled reagent so as to obtain mixture of analyte of interest:
- complementary material complexes and oxidase-labelled reagent:
- complementary material complexes:
- (c) treating the mixture with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted;
- (d) exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation;
- and
- (e) detecting the radiation so emitted, thereby by detecting the analyte of interest.
9 more elements
A method for detecting a single-stranded nucleic acid sequence of interest in a liquid sample which comprises:
- (a) contacting the liquid sample with an oxidase-labelled, single-stranded nucleic acid sequence complementary to the nucleic acid sequence of interest under hybridizing conditions so as to form an oxidase-labelled, double-stranded nucleic acid sequence;
- (b) treating the oxidase-labelled, double-stranded nucleic acid sequence with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrzaine ring is fused or substituted;
- (d) exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation;
- and
- (e) detecting the radiation so emitted, thereby by detecting the analyte of interest.
2 more elements
A method for detecting oxidase activity in a liquid sample which comprises:
- (a) treating the liquid sample with a nitrogen-substituted, cyclic compound and an oxidizing agent under conditions such that an oxidase present in the liquid sample enzymatically oxidizes the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrzaine ring is fused or substituted;
- (d) exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation;
- and
- (e) detecting the radiation so emitted, thereby by detecting oxidase activity in the liquid sample.
1 more element
A method for detecting an oxidizing agent in a liquid sample which comprises:
- (a) treating the liquid sample with a nitrogen-substituted, cyclic compound and an oxidase under conditions such that the nitrogen-substituted, cyclic compound is enzymatically oxidized to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrzaine ring is fused or substituted;
- (d) exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation;
- and
- (e) detecting the radiation so emitted, thereby by detecting the oxidizing agent.
1 more element
Dependencies are inferred from the claim text.