One-pot assembling pyrroloquinoline quinone glucose dehydrogenase with polydopamine to overcome the reproducibility issues of layer-by-layer electrode development.

The reproducibility of enzyme-based biosensors remains a critical challenge, particularly in clinical and wearable applications. Here, we present a novel one-pot polydopamine (PDA)-assisted immobilization strategy for pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) on graphite electrodes to address the limitations of conventional layer-by-layer (LbL) methods. The (PQQ-GDH/PDA)_OPA/G platform demonstrated a uniform and nanostructured enzyme-polymer matrix, confirmed by SEM and spectroscopic characterization, resulting in enhanced surface coverage and enzyme stabilization. Electrochemical analyses revealed an onset potential of +0.19 ± 0.01 V and a maximum current of 0.87 ± 0.08 μA in the presence of glucose. Amperometric calibration yielded a linear range of 0.4-1.2 mM, a sensitivity of 0.47 μA mM^-1, and a low detection limit of 26 ± 2 μM. Michaelis-Menten kinetic analysis provided an I _max of 1.13 ± 0.07 μA and a K ^app _M of 3.11 ± 0.59 mM. Reproducibility was excellent, with relative standard deviations below 8% for all key parameters. The biosensor retained full functionality under physiological conditions (pH 7.2, 37 °C) and exhibited high selectivity against common interferents, including dopamine, uric acid, and ascorbic acid, with signal variations below 5%. Remarkably, the sensor maintained stable responses in artificial serum for over 67 days, confirming its long-term operational stability. These findings highlight the one-pot PDA-based approach as a scalable, reproducible, and biocompatible platform for next-generation glucose biosensors suitable for real-world biomedical monitoring.