Bimetallic nanotubes enhanced MALDI-TOF MS for rapid detection of carbapenemase activity in Klebsiella pneumoniae.

The global spread of carbapenemase-producing Klebsiella pneumoniae represents a critical threat to public health, necessitating the development of rapid, sensitive, and reliable diagnostic methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has gained attention as a high-throughput, rapid diagnostic tool for detecting carbapenemase-mediated hydrolysis. However, the effectiveness of conventional organic matrices is limited by inadequate stability, low detection sensitivity, and substantial background interference. In this study, we developed novel bimetallic nanotubes (N-CNT@Zn-Ga/MOF@Au) as a MALDI matrix by integrating nitrogen-doped carbon nanotubes (N-CNT) with a Zn-Ga-based metal-organic framework (MOF) and gold nanoparticles (AuNPs). This nanocomposite exhibited a high surface area, enhanced light absorption, and reduced background noise, contributing to improved signal intensity, reproducibility, and analytical sensitivity. Leveraging this matrix, we analyzed 204 K. pneumoniae clinical isolates for carbapenemase activity through imipenem hydrolysis assays. The method demonstrated exceptional performance, achieving 97.2% sensitivity and 100% specificity within 15 min, with KPC-2-producing strains detected with 100% accuracy. Extending incubation to 1 h allowed for reliable detection of all carbapenemase types, including weaker enzymes such as OXA-48. These findings demonstrate the potential of N-CNT@Zn-Ga/MOF@Au as an advanced MALDI matrix for rapid and accurate detection of carbapenemase activity, with important applications in resistance monitoring and clinical decision support.