Precise <i>Klebsiella pneumoniae</i> carbapenem resistance prediction by Zn–Cu/MOF-embedded hybrid nanosheet-assisted carbapenemase activity analysis

The global rise of carbapenemase-producing <i>Klebsiella pneumoniae</i> (CPKP) poses a major threat to public health due to its high morbidity and mortality rates, driven largely by the production of carbapenemase enzymes. Rapid and accurate detection of carbapenemase activity is crucial for guiding appropriate antimicrobial therapy and controlling nosocomial transmission. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) enables direct detection of carbapenem hydrolysis but is currently limited by the drawbacks of conventional organic matrices, such as high background interference, toxic solvents, and poor reproducibility. In this study, we report a novel nanocomposite matrix, GNP@Zn-Cu/MOF@Au, synthesized <i>via</i> a green, aqueous-phase method without toxic reagents or extreme conditions. This heterostructured material exhibited a large mesoporous surface area, excellent UV absorption, and thermal responsiveness, which enhanced signal intensity, reproducibility, and analytical sensitivity, significantly improving MALDI-TOF MS performance. When applied to the identification of carbapenemase activity in clinical CPKP isolates, the matrix achieved 100% sensitivity and specificity after 1 hour of incubation and 97.3% sensitivity after just 15 minutes. Notably, KPC-2-producing strains were detected with full sensitivity within 15 minutes, while extended incubation enabled reliable identification of low-activity enzymes such as OXA-48. These findings demonstrate the strong potential of GNP@Zn-Cu/MOF@Au as a reliable and sustainable alternative to conventional matrices for rapid and accurate detection of carbapenemase activity in clinical diagnostics.