FRET-based probe for ratiometric detection and imaging of folic acid in real-time

Inadequate folic acid intake is linked to diseases such as megaloblastic anemia, neural tube defects, and hyperhomocysteinemia, increasing the risk of vascular disease and thrombosis. Folic acid, a cofactor in various enzymes, can be produced by plants and bacteria, but not by humans and other animals. L-5-methyl-tetrahydrofolate (L-5-methyl-THF) is the primary dietary folate form, transported in circulation for cellular metabolism. Traditional methods of determining folic acid levels are unreliable and time-consuming. SenFol (Sensor for folic acid) is a fluorescence resonance energy transfer (FRET)-based nanosensor that we have developed by inserting folic acid-binding protein (FolT) as the folate detecting domain between the pair of enhanced cyan fluorescent protein (ECFP) and Venus. The developed sensor is highly specific, produces a quick signal, which is pH stable, and delivers precise, ratiometric readings in cell-based experiments. The projected affinity score of folic acid with FolT was -7.4 kcal/mol. The apparent affinity (K_d) of SenFol for folic acid is 28.49 × 10^-9 M, with a detection range of 5 × 10^-9 M to 5 × 10^-7 M, and a maximum FRET ratio change of 0.45. WT SenFol, a highly efficient folic acid nanosensor, can dynamically detect intracellular folic acid content in E. coli, yeast, and HEK-293 T cells, confirming its potential.