Ratiometric imaging of flux dynamics of cobalt with an optical sensor

Abstract

Cobalt (Co2+) is a vital micronutrient needed for growth and development by all life types. In humans, cobalt is cytotoxic and genotoxic associated with various pathological and physiological conditions such as allergic dermatitis, pneumonia, interstitial fibrosis, alveolitis, myocardiopathy, rhinitis and lung cancer. In this study, we present a genetically encoded fluorescence resonance energy transfer (FRET)-based Cobalt Optical Sensor (CobOS) to determine this metal ion’s trafficking scheme consisting of uptake/efflux mechanisms and measuring the real time free-ion concentration and distribution within living cells. CobOS comprise of a cobalt-sensing domain CbiKP from Desulfovibrio vulgaris Hildenborough fused with the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus at N- and C-terminus respectively. When studied for its specificity and selectivity, CobOS provides maximum ratiometric readout for cobalt ions, thereby establishing its effectiveness as a FRET cobalt sensor. The sensor is resistant to pH changes and remains unaffected by adding other metal ions that are biologically significant. CobOS-86n, the most efficient sensor variant, binds cobalt with an affinity (Kd) of 0.86 × 10−6 M covering cobalt concentrations of 50 nM to 200 μM and can be of practical interest for real time measurement of in vivo cobalt levels in Escherichia coli (E. coli), yeast and mammalian cells non-invasively using widefield confocal fluorescent microscopy.