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 CbiK^P 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 (K_d) 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.

钴（Co ²⁺）是所有生命类型生长发育所需的重要微量营养素。在人类中，钴具有多种病理和生理状况，如变应性皮炎，肺炎，间质纤维化，肺泡炎，心肌病，鼻炎和肺癌，具有细胞毒性和遗传毒性。在这项研究中，我们提出了一种基于遗传编码的荧光共振能量转移（FRET）的钴光学传感器（CobOS），用于确定该金属离子的迁移方案，该方案由摄取/渗出机理组成，并测量实时游离离子的浓度及其在生物体内的分布细胞。CobOS包含来自Desulfo v ibrio v ulgaris的钴感测域CbiK ^P希尔登伯勒与FRET对融合分别增强了N和C端的蓝绿色荧光蛋白（ECFP）和金星。在研究其特异性和选择性时，CobOS提供了钴离子的最大比例读数，从而确立了其作为FRET钴传感器的有效性。该传感器可以抵抗pH的变化，并且不会因添加其他具有生物学意义的金属离子而不受影响。最有效的传感器变体CobOS-86n以0.86×10 ^-6 M的亲和力（K _d）结合钴，涵盖了50 nM至200μM的钴浓度，对于实时测量体内钴水平可能具有实际意义在大肠杆菌（E. coli）中，酵母和哺乳动物细胞使用广域共聚焦荧光显微镜进行非侵入性检测。