Fluorescence ‘On‐Off‐On’ Switching of Pepsin Stabilized Copper Nanoclusters by Sequential Addition of Vitamin B6 Cofactor Plp and Histidine

This manuscript introduces an innovative fluorescence approach for detecting histidine (His) using the biomimetic transamination reaction. The blue fluorescence of pepsin‐protected copper nanoclusters (pep‐CuNCs, λ _ex = 350 nm, λ _em = 450 nm) was quenched by vitamin B ₆ cofactor pyridoxal 5 ^′ ‐phosphate (PLP) due to the formation of internal aldimine between the PLP and lysine residue of pepsin‐CuNCs. Subsequently, the addition of His to the in situ formed nano‐assembly PLP_pep‐CuNCs (PL1) restored the fluorescence emission of pep‐CuNCs. The internal aldimine of PL1 is cleaved, and an external aldimine is formed between the PLP and His. The formation of internal aldimine and external aldimine is supported by various studies, including Ultraviolet–visible, fluorescence, fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, and molecular docking. Using PL1, the concentration of His can be detected down to 0.26 µM with a linearity range of 9.9–82 μM. Finally, the PL1 is successfully applied to quantify His in real samples, such as human serum and urine, with the recoveries ranging between 91.66% and 98.62%.