Oxygen deficient MoO3 nanoparticles as peroxidase substitutes, their substrate-nanozyme interactions and real time validation in human serum sample

Oxygen deficient molybdenum oxide nanosheet (Od-MO Nshs) was synthesised by hydrothermal method and its peroxidase-like activity was established with 3,3′,5,5′-Tetramethylbenzidine-(TMB) and o-phenylenediamine dihydrochloride-(OPD) as a co-substrate in presence of Hydrogen peroxide (H₂O₂) and catalytic parameters were compared with horseradish peroxidase-(HRP) enzyme. Optimization was carried out for physical and chemical parameters. Synthesised nanoparticles-(NPs) were characterized for size, shape, composition oxidation state etc. NPs crystalline size was calculated using Scherrer’s equation and Williamson-Hall plot and was found to be 18.46 nm and 36.1 nm respectively. Based on the peroxidase-like activity of Od-MO Nshs nanozymes-(NZs), a simple colorimetric method was established for detecting H₂O₂ in human serum samples. Compared to HRP, Od-MO NShs showed linear detection in the fixed time method at 48 times lower H₂O₂ concentration with TMB and 16 times and 4 times lower H₂O₂ concentration accordingly. Od-MO NShs showed 2.7- and 5.3-time lesser Michaelis–Menten constant (K_m) for H₂O₂ compared to HRP with TMB and OPD, respectively indicating higher substrate affinity. The detection and quantification limits for TMB-Od-MO NShs were 0.5 and 1.5 µM, significantly lower than TMB-HRP (9.4 µM, 28.5 mM). For OPD-Od-MO NShs, the limits were 9.4 µM, and 28.5 m, compared to 1.1 µM and 3.4 µM for OPD-Od-MO NShs. in addition, these NPs exhibited photocatalytic performance, achieving effective degradation (>55 %) of selected organic dyes, highlighting their potential environmental remediation application.