Rapid, efficient and green method of Pd nanoparticles (PdNPs) synthesis on TEMPO-oxidized cellulose nanofibril (TCNF) is demonstrated here. The nanohybrid (PdNPs/TCNF) exhibited peroxidase and oxidase-like activities evident by the oxidation of three chromogenic substrates. Enzyme kinetic studies using 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation uncovered the excellent kinetic parameters (low K and high V) and good specific activities of 215 U/g and 107 U/g for peroxidase and oxidase-like activities, respectively. A colorimetric assay for ascorbic acid (AA) detection is proposed based on its ability to reduce oxidized TMB to its colorless form. However, presence of nanozyme caused re-oxidation of TMB to its blue colored form within few minutes resulting in time limitation and inaccurate detection. Thanks to the film forming nature of TCNF; this limitation was overcome by employing PdNPs/TCNF film strips that can be easily removed before AA addition. The assay allowed AA detection in the linear range of 0.25-10 μM with a detection limit of 0.039 μM. The results of AA detection in commercial beverages and vitamin C tablets are matching with the specified values. Further the nanozyme exhibited high tolerance to pH (2-10) and temperature (up to 80 °C) and good recyclability for five cycles.
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