A genetically encoded probe for monitoring and detection of iron in real-time†

Abstract

Iron, the most abundant transition metal in the body, regulates cellular function but can be harmful in excess, leading to reactive oxygen species production and cellular damage. Intracellular Fe²⁺ exerts a significant impact on cellular function, potentially contributing to various critical diseases. To address this, detection methods need high selectivity, sensitivity, and real-time monitoring capabilities, essential for comprehending disease progression. This necessitates advancements beyond conventional detection approaches. Frataxin, a crucial mitochondrial protein, is indispensable for sustaining life, contributing not only to iron metabolism but also to the formation of iron–sulfur clusters critical for cellular function. Its deficiency is implicated in neurodegenerative diseases. We have developed a nanosensor, based on fluorescence resonance energy transfer (FRET), designed to probe iron efflux mechanisms and facilitate dynamic monitoring of iron concentration and its spatial distribution within living cells. To construct this nanosensor, we strategically positioned CyaY, a bacterial frataxin ortholog, between ECFP and Venus, forming a FRET pair. This innovative nanosensor, designated as FeOS (iron optical sensor), demonstrates exceptional selectivity for iron and maintains stability under physiological pH conditions. Additionally, we engineered three mutant variants: I17C, AD10-I17C, and D76H, with A10D-I17C displaying the highest affinity for iron and a broad detection range. The distinguishing feature of this sensor is that it is genetically encoded, facilitating real-time detection of iron levels within living cells.