A ratiometric fluorescent probe based on FRET for selective monitoring of tungsten in living cells

Abstract

Tungsten is considered a critical metal due to its unique properties and economic importance. Recent research has linked high tungsten concentrations to pulmonary diseases and increased cancer risk. Tungsten is on the list of critical metals at high risk maintained by the European Union. We have developed a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for the first time to determine tungsten ion efflux mechanisms and measure its concentration and distribution within living cells in real-time. For the construction of the nanosensor, TupA (tungsten binding protein), a highly selective protein, was sandwiched between ECFP and Venus, a FRET pair. SENTUN (SENsor for TUNgsten) was the name given to the newly developed nanosensor. SENTUN was highly specific and selective for tungsten and stable at physiological pH levels. We also created three mutants: SENTUN-118D, SENTUN-118K, and SENTUN-178W, with SENTUN-118K exhibiting the highest affinity for tungsten and its comprehensive range detection. The novelty of this sensor is that it is genetically encoded and can be used for real-time measurement of tungsten levels in living cells.