Requirements of a Fluorescent Indicator

There are a number of important criteria in the design of a fluorescent indicator, 

Sensitivity. The measured fluorescence parameter of an indicator should be sensitive to changes of analyte in the desired concentration range, as summarized in Table 10.1 for a number of analytes. The indicator should have high extinction coefficient for efficient excitation and high quantum yield for a good signal-to-noise ratio. 

Selectivity. The measured fluorescence parameter should respond selectively to the analyte of interest. 

Stability. The indicator should be chemically stable and insensitive to pho-tobleaching. 

Calibration method. The measured fluorescence parameter should be independent of indicator concentration, geometry of sample, and sensitivity of detection system. Thus, an intensity-based method requires wavelength-ratiometric probes. Lifetime and anisotropy methods do not require wavelength-ratiometric probes, but the lifetime or anisotropy must be sensitive to analyte. 

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