Paper-based graphene bioelectrode enabling third-generation fructose dehydrogenase sensing of inulin

: Here, the first paper-based direct electron-transfer enzymatic biosensor is presented. This is the first biosensor designed to enable simple detection of inulin in biological fluids, serving as an exogenous marker for estimating glomerular filtration rate (GFR). The biosensor relies on a laser-induced graphene (LIG) paper-based sensor integrating Fructose Dehydrogenase (FDH). This enzyme was incorporated into paper for the first time in this work and demonstrated direct electron transfer (DET) capability. Various cellulosic substrates were evaluated as biosensor supports, and a tree-free bamboo-derived paper (FB) was selected for its ability to accommodate both LIG as the transducer film and FDH as the bioreceptor. The FB-LIG, which yielded the highest bioelectrocatalytic response, was employed to determine inulin in human urine and serum in the context of GFR studies. The FB-LIG biosensor was selective, and via matrix-matched calibration was used to determine inulin in samples, obtaining useful dose-response linearity (urine: 1.6-22.7 mg L-1; serum: 4.6-11.4 mg L-1) and the needed sensitivity (LOD: urine = 0.3 mg L-1, serum = 1.0 mg L-1), together with high reproducibility (RSD ≤2.0 %, n = 3). The complete inulin analysis takes less than 10 min and includes a rapid hydrolysis step followed by direct biosensor measurement. The FB-LIG biosensor reliability was demonstrated by measuring inulin in real urine and serum samples at clinically relevant levels, achieving satisfactory recoveries (90-111 %; RSD ≤7.9 %, n = 3). Here, for the first time, a sustainable substrate enabled the development of a paper-based third-generation biosensor, demonstrating the potential of LIG on paper for bioelectrocatalysis.