Tailoring Water-Based Graphite Conductive Ink Formulation for Enzyme Stencil-Printing: Experimental Design to Enhance Wearable Biosensor Performance

Herein, we report for the first time an experimental design-based approach to develop water-based graphite conductive ink containing enzymes and redox mediators to obtain fully printed wearable biosensors for lactate and glucose monitoring. The experimental design encompasses both electrochemical parameters, such as electroactive area and electron transfer rate constant, and rheological parameters, including elastic (G′) and viscous (G″) moduli where G″/G′ is expressed as tanδ. Notably, the printed electrodes exhibited an electroactive area AEA of 3.95 ± 0.31 cm2 and a roughness factor, ρ, of 43.8, which is 50 times higher than those of commercially available screen-printed electrodes. Furthermore, lactate oxidase and glucose oxidase are integrated within water-based graphite conductive ink to obtain enzyme-based inks: enzyme-ink (E-INK), to detect lactate, and enzyme mediator-ink (EM-INK), to detect glucose. The resulting biosensors demonstrated high sensitivity and low limit of detection 3.3 μA mM–1 and 0.3 ± 0.1 μM (ferricyanide as electron mediator), and 4.3 μA mM–1 and 3 ± 1 μM, for E-INK and EM-INK, respectively. The biosensors also exhibited excellent selectivity, maintaining their storage stability, with approximately 80–90% of the initial signal retained after 90 days. Overall, this promising system holds potential to be utilized as a flexible and wearable biosensor. Its use of biocompatible water-based inks makes it suitable for applications in sports medicine and remote clinical care.