FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Abstract

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 μM and covers the physiological range of 500 nM to 12 μM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.