Electrochemical β-Lactam Probes for Rapid Detection of β-Lactamases: From Solution-Phase Activation toward Surface-Integrated Biosensing

Rapid detection of antibiotic-resistant bacteria is a crucial tool in the global fight against antimicrobial resistance, helping to limit the spread of resistance and guide treatment decisions. Here, we report the design, synthesis, and electrochemical evaluation of β-lactam-based redox-activatable probes for detecting β-lactamase activity. The probes incorporate a β-lactam core linked to redox reporters through cleavable linkages, enabling signal generation upon enzymatic hydrolysis. High-performance liquid chromatography and differential pulse voltammetry analyses were used to assess time-dependent activation and concentration-dependent responses against commercial β-lactamase blends and metallo-β-lactamases. Selected probes, bearing cephalosporin recognition motifs and maltol redox reporters, were further evaluated against clinical isolates, demonstrating selective activation in carbapenemase-producing strains. To extend the platform toward solid-state biosensing, an azide-functionalized analog was clicked on alkyne-modified glassy carbon electrodes. Stepwise surface functionalization and immobilization were validated electrochemically using model redox reporters, confirming their activity. The immobilized probe retained responsiveness, demonstrating the feasibility of integrating this sensing strategy into solid-state diagnostic devices. By integrating stable cephalosporin scaffolds with redox-reporter signaling, this work introduces a novel probe system that unites chemical probe design with surface-based electrochemical sensing, providing a strong foundation for the development of portable, point-of-care diagnostics for β-lactamase-mediated antibiotic resistance.