Question

The presence of a non-fluorescing compound in a sample that absorbs light at the same wavelength as a target analyte can interfere with a fluorescence measurement. Explain how the non fluorescing compound would interfere?

Answer 1

The presence of a non-fluorescing compound in a sample that absorbs light at the same wavelength as a target analyte can interfere with a fluorescence measurement.

In addition to naturally fluorescing compounds, there are many fluorescent derivatives that can be formed to visualize lower levels of various non-fluorescing analytes. Fluorescence detection enables environmental analysis of trace levels of pesticides such as n-methyl carbamates or glyphosate in drinking water or soil. The high specificity is especially well suited to measurements in complex matrices, such as foods, where co-extractives can often cause interferences.

Fluorescence as a spectroscopic phenomenon is utilized as the output for a range of assay formats used in high-throughput screening (HTS). These include readouts such as fluorescence polarization (FP), analyte quantitation, cell viability, fluorescence resonance energy transfer, and high-content imaging applications. While the wavelength at which light emitted in many fluorescence-based assays can be “tuned”, many small molecules in a small molecule library are optically active and can interfere with assay performance. This interference can be either by absorbing and emitting light in the same window as the assay resulting in false-positive signal (fluorescing) or absorbing light at either the excitation or emission wavelengths resulting in attenuated intensity of signal (quenching).

Non-fluorescing compounds can interfere with fluorescence signal through the “inner filter effect” by absorbing emitted light, particularly at higher concentrations. The non-fluorescing compound inner filter effect can be mitigated by performing a “preread” measuring the absorbance of the compounds at the excitation and emission wavelengths of the fluorophore used in the assay to identify possible interference.

So, a non-fluorescing compound in a sample that absorbs light at the same wavelength as a target analyte must be separated otherwise it might be interfere with its fluorescence response.