Mechanistic aspects of functional layer formation in hybrid one-step designed GOx/Nafion/Pd-NPs nanobiosensors.

Amperometric nanobiosensors are crucial time and cost effective analytical tools for the detection of a wide range of bioanalytes, viz. glucose present in complex environments at very low concentrations. Although the excellent analytical performance of nanobiosensors is undoubted, their exact molecular structure often remains unclear. Here, by combining advanced nanoanalytical approaches with theoretical modeling, we conducted a comprehensive study towards the investigation of the molecular structure of a hybrid GOx/Nafion/Pd-NPs layer deposited by electroplating from the multicomponent electrolyte solution on the surface of screen printed electrodes modified with graphene oxide. Specifically, we revealed that Pd cations were adsorbed on GOx amino acid residues, forming the GOx·nPd enzymatic complex. The highest adsorption energy of Pd cations on GOx was found during their interaction with the side chains of basic amino acids and methionine. In addition, we showed and fully validated the end-structure of the one-step designed GOx/Nafion/Pd-NPs nanobiosensor as a structural model mainly composed of GOx and water molecules incorporated into the metal-polymer scaffold. Our approach will thus serve as a guideline for the study of molecular interactions occurring in complex systems and will contribute to the design of the next generation of hybrid nanobiosensors. The proposed mechanism, driving the self-assembly of the hybrid layer, will allow us to construct modular enzymatic nanoanalytical devices with tailored sequences in the future.