Ratiometric Fluorescence Sensing of Human/Bovine Serum Albumin Using In Situ Formed Fe ³⁺ Complex

The quantification of albumin levels in biological samples is a critical biomarker for diagnosing and monitoring various pathological conditions. This study introduced a vitamin B ₆ cofactor derived Schiff base (E)‐(4‐(((2‐(1H‐benzo[d]imidazol‐2‐yl)phenyl)imino)methyl)‐5‐hydroxy‐6‐methylpyridin‐3‐yl)methyl dihydrogen phosphate (BMLP) via the condensation of pyridoxal 5′‐phosphate with 2‐(1H‐benzo[d]imidazol‐2‐yl)aniline. Schiff base BMLP formed a complex with Fe ³⁺ in 2:1 binding ratio and redshifted its fluorescence emission from 417 to 474 nm, accompanied by slight quenching at 417 nm and fluorescence enhancement at 474 nm. The in situ formed BMLP─Fe ³⁺ complex was employed for the ratiometric fluorescence sensing of albumins, that is, human serum albumin (HSA) and bovine serum albumin (BSA). With the addition of HSA and BSA, the fluorescence emission of BMLP─Fe ³⁺ complex was quenched at 474 nm and enhanced at 417 nm. The selective ratiometric response from the BMLP─Fe ³⁺ complex can be employed to detect HSA and BSA down to 3.8 and 3.6  µM, respectively. The developed fluorescence‐based sensing approach was applied for quantifying HSA and BSA in real biological matrices, including milk and urine, with satisfactory recovery rates.