Click-Functionalized Biochar as a Spacer-EngineeredInterface for Electrochemical Biosensing

Abstract Biochar, a carbon-rich material produced through biomass pyrolysis, is gaining significant attention in electrochemical sensor development. This material offers dual advantages: it enhances electrode performance while providing functional groups for binding with recognition elements such as antibodies, enzymes, and aptamers. Despite growing interest, practical applications encounter several challenges. The biochar heterogeneous surface could lead to random immobilization of interaction sites and fouling, both of which limit sensor performance, particularly in complex matrices. In addition, surface modification strategies specifically designed to introduce molecular spacers on biochar for electrochemical biosensing remain comparatively underexplored. This work introduces a multistep chemical modification strategy designed to add reactive functional groups to the biochar surface and, in particular, is focused on the development of a molecular spacer on spent coffee ground-derived biochar (pristine biochar) by introducing a covalently bound molecular moiety through azide–alkyne cycloaddition (clicked biochar). Each reaction intermediate was characterized through in-depth chemical–physical characterization, confirming successful functionalization at each step. Crucially, the molecular spacer extends recognition elements further into solution while limiting large protein adsorption. To validate this approach, the performance of an enzymatic biosensor using alkaline phosphatase (AP) and a model substrate was evaluated. The functionalized biochar exhibits improved antifouling properties and better analytical performance compared to that of its unmodified counterpart. A kinetic study conducted using chronoamperometry (CA) shows an approximately 2.2-fold increase in the apparent maximum catalytic current for clicked biochar compared with pristine biochar, while the apparent Michaelis–Menten constants remained comparable, confirming improved enzyme accessibility and enhanced catalytic efficiency of the covalently immobilized enzyme. Furthermore, electrochemical impedance spectroscopy (EIS) confirmed the antifouling ability of this material, resulting in a higher reduction in nonspecific adsorption compared to pristine biochar, as highlighted by a change in charge transfer resistance (Rct).