Ultrasensitive Aptamer-Based Metal–Organic Framework-on-Metal–Organic Framework Platform for Clinical Detection of KPC-2 Klebsiella pneumoniae and Multidrug-Resistant Acinetobacter baumannii

The escalating crisis of hospital-acquired multidrug-resistant (MDR) infections, particularly Klebsiella pneumoniae carbapenemase 2-expressing K. pneumoniae (KPC-2 KP) and MDR-Acinetobacter baumannii (AB), demands rapid diagnostic solutions. We developed a dual nanozyme-powered colorimetric aptasensor leveraging a cascade amplification mechanism, a metal–organic framework (MOF)-on-MOF nanostructure with peroxidase-like activity. Cu-MOF@PMOF(Fe) integrates catechol oxidase-like activity, with the Cu-MOF core oxidizing catechol to generate H2O2, and the PMOF(Fe) shell utilizes H2O2 to oxidize the TMB substrate, producing dual-wavelength signals at 370 and 652 nm for ultrasensitive detection. Functionalized with pathogen-specific aptamers, the system achieves selective bacterial capture within 40 min, quantifying 10–108 CFU/mL with detection limits of 7 CFU/mL for KPC-2 KP and 6 CFU/mL for MDR-AB. Clinical validation using cerebrospinal fluid, peritoneal fluid, serum, and bile samples demonstrated robust performance in complex matrices (91.2–112.2% recovery rates). Therefore, this platform provides a rapid (<1 h), sensitive, and clinically adaptable solution for combating MDR bacterial infections, bridging advanced materials with diagnostic microbiology.