Designing of nanosensor and its applications for spatiotemporal visualization and real time quantification of biotin in biological samples

Abstract

Biotin is a 244 Da water-soluble vitamin involved in vital metabolic processes by acting as an essential cofactor for several enzymes. When biotin levels are low, critical enzymes such as pyruvate carboxylase, 3-methylcrotonyl-CoA, and propionyl-CoA carboxylase become dysfunctional leading to fatal human disease. Traditional methods for measuring biotin in biological fluids yield valuable results for particular applications, but the majority of them are intrusive, expensive, and lack sensitivity and specificity. A highly effective genetically encoded FRET-based nanosensor OSenB (Optical Sensor for Biotin) is developed using FRET pair enhanced cyan fluorescent protein (ECFP) and Venus attached to the N- and C-termini of the avidin module, allowing real-time monitoring and quantification of biotin at a trace level. OSenB exhibits discernible FRET ratio shifts, measures biotin with excellent specificity and selectivity, provides a rapid detection response, is resilient to pH fluctuations, and produces a very precise FRET signal with a detection range of 12 pM to 165 nM biotin quantification. Developed mutant sensor OSenB-44 with 0.717 nM apparent affinity (Kd), was found to be the most successful nanosensor. Confocal images show that OSenB-44 was successfully expressed in vivo and dynamically measured biotin in Escherichia coli, yeast, and human embryonic kidney (HEK)− 293T in real-time.