Luminescence-Based Strategies for Detecting β-Lactamase Activity: A Review of the Last Decade

Rapid detection of β-lactamase activity is becoming increasingly important as β-lactam resistance spreads at an alarming rate and conventional diagnostics often require several hours to deliver actionable results. Over the past decade, methods based on luminescence, bioluminescence, chemiluminescence, and fluorescence have become powerful tools for the functional assessment of resistance resulting from β-lactamase activity. These approaches provide highly sensitive, activity-based readouts, often within minutes, and frequently rely on simple optical instrumentation. In this review, we summarize recent developments in luminescent probe design between 2015 and 2025, with emphasis on reaction mechanisms, analytical performance, and the ability of these systems to discriminate between different β-lactamases, including narrow-spectrum enzymes, AmpC, ESBL, and carbapenemases. We also discuss their applications in bacterial cultures, clinical isolates, complex biological matrices and, in some cases, in vivo models. While luminescent assays are not yet positioned to replace standard susceptibility testing, they offer a practical and increasingly robust complement to culture-based and molecular methods. The emerging trends highlighted here, such as self-immobilizing fluorogenic probes, chemiluminescent relay systems, nanomaterial-based sensors and AI-assisted mobile platforms, suggest that luminescent β-lactamase detection could play a meaningful role in future rapid diagnostics and resistance surveillance.