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Vitamin B12 (cobalamin) is a cobalt-containing compound that acts as an essential co-factor for various enzymes involved in the metabolic processes of the living cells. The constructed FRET Sensor for Vitamin Anemia Linked (SenVitAL) displayed marginal FRET efficiency. Here, we report the development of a molecular SenVitAL containing enhanced cyan fluorescent protein (ECFP) and venus as FRET pair to improve the FRET efficiency for optical imaging and screening of already developed sensor by our group. The sensor is the improved version of previously reported SenVitAL and consists of ECFP/venus as FRET pair instead of the originally used pair CFP/YFP. To increase the physiological range of vitamin B12 measurement, affinity mutants were created. Compared to the wild type, SenVitAL-5 with W44Q mutation has higher affinity and displayed large dynamic detection range (0.10–480 µM) in response to vitamin B12 binding. For cell-based monitoring and dynamic measurement of vitamin B12 flux rates, SenVitAL-5 was successfully expressed in cytosol of yeast and mammalian cells. Changes in the emission intensities of the two fluorophores were detected using confocal microscopy in both cell types in response to vitamin B12. With the addition of 50 µM extracellular vitamin B12 to the cells, the emission intensity of venus increased and that of ECFP decreased over the time. Furthermore, the results show that the variant SenVitAL-5 measures the vitamin B12 in a concentration-dependent manner, showing the resulting increase in the FRET ratio and thus confirming its utility as an ideal fluorescent indicator for the detection of vitamin B12 in eukaryotic systems in real time. [ABSTRACT FROM AUTHOR]