Cascade Detection of Vitamin B6 Cofactors Pyridoxal 5′-Phosphate and Pyridoxine Using Trypsin-Stabilized Fluorescent Nickel Nanoclusters

Abstract Vitamins are essential micronutrients, and their deficiency can cause serious health issues, making rapid and sensitive analytical detection crucial. This study presents a synthesis method to obtain fluorescent trypsin (Tryp)-stabilized nickel nanoclusters (NiNCs) for the cascade detection of VB6 PLP and PN via the displacement approach. The fluorescent Tryp−NiNCs were synthesized using a simple one-pot chemical reduction method, with Tryp protein acting both as a stabilizing and reducing agent. The synthesized Tryp−NiNCs exhibit cyan-blue fluorescence emission at 490 nm (λex = 380 nm). For the sensing application, pyridoxal-5′-phosphate (PLP) was functionalized over the surface of Tryp−NiNCs in situ via the formation of a weakly fluorescent internal aldimine complex, PLP_Tryp−NiNCs. Further, when the PLP_Tryp−NiNCs complex was interacted with 2-aminophenylboronic acid (2APBA), the internal aldimine complex converted into an external aldimine complex 2APBA_PLP that restored the fluorescence emission of Tryp−NiNCs at 490 nm. The fluorescent nanoassembly of 2APBA_PLP@Tryp−NiNCs incorporates 2APBA_PLP and Tryp−NiNCs. Subsequently, when PN was introduced into the 2APBA_PLP@Tryp−NiNCs nanoassembly, the 2APBA_PLP complex hydrolyzed and displaced PLP, which quenched the fluorescence emission at 490 nm due to the reversible formation of PLP_Tryp−NiNCs. The detection limit for PN was estimated to be 0.37 μM with a linearity range of 2.50−26.75 μM. The developed method was applied to quantify PN in human urine (98.64−103.06%) and serum (96.42−101.36%) using the spiking method.