Dark Quencher - Mode of Function

Mode of Function

Dark quenchers are dyes with no native fluorescence. Until the last few years, quenchers have typically been a second fluorescent dye, for example, fluorescein as the reporter and rhodamine as the quencher (FAM/TAM probes). However, quencher fluorescence can increase background noise due to overlap between the quencher and reporter fluorescence spectra. This limitation often necessitates the use of complex data analysis and optical filters. Dark quenchers offer a solution to this problem because they do not occupy an emission bandwidth. Furthermore, dark quenchers enable multiplexing (when two or more reporter-quencher probes are used together).

Fluorescent dyes absorb light, which places the dye in an excited state; the dye returns to the ground state from the excited state by emitting light (fluorescence). In a reporter – quencher system the dye nonradiatively (without light) transfers energy to the quencher. This returns the dye to the ground state and generates the quencher excited state. The quencher then returns to the ground state through emissive decay (fluorescence) or nonradiatively (dark quenching). In nonradiative or dark decay, energy is given off via molecular vibrations (heat). With the typical μM or less concentration of probe, the heat from radiationless decay is too small to affect the temperature of the solution.