Polymeric mediator‐integrated bienzymatic electrochemical biosensor for <scp>ATP</scp> detection

Abstract Adenosine triphosphate (ATP) is the primary energy currency and a critical biomarker for assessing cellular viability and metabolic reprogramming in malignant tumors. While electrochemical biosensors offer a cost‐effective alternative to traditional bioluminescence assays, they often suffer from low signal amplification. In this study, we developed a bienzymatic cascade electrochemical biosensor for ATP by integrating a polymeric mediator architecture. Amine‐reactive phenazine ethosulfate was covalently conjugated to poly(ethyleneimine) and anchored onto an indium tin oxide electrode, followed by the co‐immobilization of hexokinase and glucose‐6‐phosphate dehydrogenase with a protective chitosan overlayer. This integrated configuration improved spatial proximity between the catalytic enzymes and the redox mediator, effectively overcoming the kinetic barriers of NADH oxidation and minimizing diffusion‐related signal loss. Systematic optimization of the system (including enzyme ratios, Mg 2+ cofactor concentration, and incubation conditions) achieved a high signal‐to‐background ratio. The sensor demonstrated high sensitivity in phosphate‐buffered saline with a detection limit of 3 μM. Furthermore, the platform's analytical applicability in a complex biological matrix was demonstrated using MCF‐7 human breast cancer cell lysates. Despite the complexity of the biological matrix, the sensor achieved a detection limit of 14 μM, which sufficiently covers the physiologically relevant range of ATP in diluted samples. The sensor also exhibited high selectivity toward ATP over structurally related nucleotides and nucleosides. These results indicate that the polymeric mediator‐integrated bienzymatic architecture provides a stable and sensitive platform for monitoring cellular metabolism.