Use of hGluc/tdTomato pair for sensitive BRET sensing of protease with high solution media tolerance

► Highly bright hGluc and tdTomato were genetically linked as a BRET pair. ► hGluc/tdTomato BRET pair showed high tolerance to buffer components and pH. ► The pair possessed good spectral resolution. ► The BRET sensor showed high sensitivity when detecting enterokinase in vitro. ► The BRET sensor sensitively detected enterokinase in serum.