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Abstract

An energy transfer fluorescent probe for detecting a reagent is provided which includes a fluorescent reporter molecule and a quencher molecule positioned on the probe relative to the reporter molecule such that the quencher molecule quenches the fluorescence of the reporter molecule when in a first state, the quencher molecule being converted by the reagent to a second state which has a reduced ability to quench the reporter molecule. Examples of conversions of the quencher molecule from a first state to a second state include reductions, oxidations, hydrolyses, phosphate cleavages, and the conversion of amides to amines. In one embodiment, the quencher molecule is a substrate for an enzyme which converts the quencher from a first state to a second state. For example, the enzyme may be an reductase, an oxidase, hydrolytic, a peptidase or a phosphorylase. The probe is used to fluorescently detect a reagent in a sample. According to the method, the probe is contacted with the sample containing the reagent wherein the quencher molecule is converted by the reagent from a first state which is able to quench the fluorescence of the reporter molecule to a second state which has a reduced ability to quench the reporter molecule. The reagent is detected by monitoring the fluorescence intensity of the reporter molecule as the quencher molecule is converted from the first state to the second state by the reagent.

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First Claim