A Dual-Function 3D-Printed Cyclonic Collector/Reactor-Integrated Colorimetric Biosensor for Automated Detection of Airborne Bacteria

Rapid detection of airborne bacteria is crucial for preventing disease transmission and safeguarding public health. However, conventional methods for bioaerosol collection and detection often rely on complex and expensive equipment with low integration and automation, limiting their use for real-time applications. Herein, a dual-function 3D-printed cyclonic collector/reactor-integrated colorimetric biosensor was innovatively developed for the collection, separation, and detection of airborne bacteria. This platform incorporated a custom-designed wet cyclone, which served as a bioaerosol collector and an immunoassay reactor by utilizing the cyclonic vortex for simultaneous bioaerosol collection and active mixing with immunoreagents. Airborne bacteria were first collected into the phosphate-buffered saline at a flow rate of 12 L/min, followed by pumping the immunomagnetic nanoparticles (MNPs) and palladium/platinum (Pd/Pt) nanozymes. Then, the flow rate was changed to 5 L/min within the wet cyclone to facilitate the mixing for forming MNP–bacteria–nanozyme sandwich complexes. After magnetic separation, a colorless mixture of 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) was pumped to resuspend these complexes, which were catalyzed by the Pd/Pt nanozymes on the complexes to produce the blue TMBox. The resulting absorbance was finally measured by a self-developed portable microreader, which showed high consistency with the traditional microplate reader. The entire processes, from bioaerosol collection to detection, were automatically controlled via a microcontroller, minimizing manual operation and cross-contamination risks. This platform was demonstrated to complete the airborne bacteria detection in 2.0 h with a detection limit of 459 CFU/m3. This highly integrated platform offers a promising tool for rapid and automated detection of bioaerosols.