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We report a facile strategy for developing reagentless amperometric pyruvate biosensors based on enzyme nanoparticles (EnNPs). The EnNPs were prepared using pyruvate oxidase crosslinked with graphene quantum dots. Before EnNP immobilization, screen-printed carbon electrodes (SPCEs) were modified with Prussian blue, a biocompatible coordination polymer. The biosensor system was optimized in terms of the working potential and pH value. At pH 7.0 in 50 mM phosphate-buffered solution, the biosensor showed optimal characteristics under an applied potential of -0.10 V versus an internal pseudo-Ag reference electrode. Using these optimized conditions, the biosensor performance was characterized via the chronoamperometric technique. The EnNP-immobilized SPCE exhibited a dynamic linear range from 10 to 100 μM for pyruvate solution, and a sensitivity of 40.8 μA mM -1 cm -2 was recorded. The observed detection limit of the biosensor was 0.91 μM (S/N = 3) and it showed strong anti-inference capability under the optimized working potential. Furthermore, the practical applicability of the proposed biosensor was studied in fish serum samples.

Reagentless Amperometric Pyruvate Biosensor Based on a Prussian Blue- and Enzyme Nanoparticle-Modified Screen-Printed Carbon Electrode