One-step modification of nano-polyaniline/glucose oxidase on double-side printed flexible electrode for continuous glucose monitoring: Characterization, cytotoxicity evaluation and in vivo experiment.

The single-step modification of the nanostructured polyaniline (PANI)/glucose oxidase (GOD) enzyme on double-sided, screen-printed, flexible electrodes doped with Prussian blue (PB), has been achieved and successfully applied in continuous glucose monitoring in vivo, and its biocompatibility has been evaluated systematically. The proposed fabrication procedure is simple, low cost, and suitable for large-scale production. PB doped with carbon ink catalyzes the reduction of hydrogen peroxide (H2O2) in low-voltage conditions, which could help eliminate interferences. And the PANI/GOD nanostructure makes the GOD enzyme more stable for long-term, in vivo monitoring. More importantly, a polyurethane (PU) layer is deposited on the electrode's surface as a diffusion limiting membrane that enhanced the linear range and biocompatibility. In tests in vitro, the proposed biosensor achieved a linear range of 0-12 mM and a good sensitivity of 16.66 μA·mM-1·cm-2(correlation coefficient R2 = 0.9962) with an excellent specificity to glucose. The biosensor exhibits long-term stability, with a maximum lifespan of 14 days when stored in phosphate buffer solution at 4 °C, and achieves a sensitivity of 120%. The biocompatibilities of the electrode materials have also been systematically evaluated in cytotoxicity and cell adhesion tests to ensure the safety of implantation. In experiments in vivo, the biosensor can successfully monitor the glucose level fluctuation of rats after 24 h following implantation. Overall, the biosensor fabricated with the double-side, screen-printing process, satisfies the glucose monitoring range in vivo and eliminates various types of interference, thus establishing a new, large-scale production procedure for flexible in vivo biosensors.