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Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis. However, so far, the measurement of bicarbonate concentration changes in living cells has only been possible by measuring intracellular pH changes. In this article, we report the development of a genetically encoded pH-independent fluorescence-based single-use sensory cellular test system for monitoring intracellular bicarbonate concentration changes in living cells. We describe the usefulness of the developed biosensor in characterizing the