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.
                            提供了一种用于检测试剂的能量转移荧光探针,其包括荧光报告分子和猝灭剂分子,这些分子相对于报告分子定位在探针上,以使得当猝灭剂分子处于第一状态时,猝灭剂分子猝灭报告分子的荧光。当试剂将猝灭剂转化为第二状态时,猝灭剂分子具有减少猝灭报告分子的能力。猝灭剂分子从第一状态转化为第二状态的转化示例包括还原、氧化、
水解、
磷酸酶裂解以及酰胺转化为胺。在一种实施例中,猝灭剂分子是酶的底物,该酶将猝灭剂从第一状态转化为第二状态。例如,该酶可以是还原酶、氧化酶、
水解酶、肽酶或
磷酸化酶。该探针用于在样品中荧光检测试剂。根据该方法,将探针与含有试剂的样品接触,其中猝灭剂分子被试剂从能够猝灭报告分子荧光的第一状态转化为减少猝灭报告分子能力的第二状态。通过监测猝灭剂分子被试剂从第一状态转化为第二状态时报告分子的荧光强度来检测试剂。