PROJECT ABSTRACT Oral biofilm-related infections remain a persistent and costly clinical problem. Existing treatments are unable to simultaneously kill and physically disrupt biofilms and require manual biofilm removal procedures that are cumbersome with reduced efficacy in difficult to reach areas such as endodontic canal systems. Furthermore, options for sample retrieval for diagnostics during clinical procedures are limited. Efficacious, automated technologies capable of precisely targeting complex anatomical areas are needed to retrieve samples, kill and remove biofilms, and deliver drugs on site. We propose a novel approach combining nanotechnology and robotics to develop the first automated system for targeted disinfection, removal, and sampling of endodontic biofilms. We have designed small-scale robots using catalytic nanoparticles as building blocks that display tether-free controlled motion with multifunctionality. Our approach utilizes iron oxide nanoparticles (IONPs) with dual catalytic-magnetic properties that (i) generate bactericidal and biofilm degrading reactive molecules in situ, and (ii) remove the disrupted biofilm via magnetic-field driven robotic assemblies termed Catalytic Antibiofilm Robots (CARs). Preliminary data demonstrate that CARs locally remove and collect biofilms with high precision and efficacy in comparison to conventional treatment, including confined endodontic spaces. By tuning the magneto-catalytic properties and control of the CARs systems, we will develop robotic device prototypes that fit the oral cavity for simultaneous endodontic biofilm treatment, removal and sample retrieval. We propose to further improve IONP-made robots coupled with a clinical electromagnetic controller to develop two CARs-based oral biodevice platforms. (Aim 1) CAR1s, formed from aggregated IONP, will be used for catalytic bacterial killing, biofilm treatment, and sample retrieval from root canals for diagnostic analysis. We will identify key parameters for CAR1s improvement, assessing magnetic control, bioactivity and visualization/tracking. CAR1s will be evaluated for targeting difficult-to-reach areas, such as C-shaped/curved canals and isthmus, as well as treating and retrieving biofilms. We will characterize and improve CAR1 control first using 3D-printed tooth replicas with diverse canal morphologies to improve movement and controllability, followed by testing our system using ex vivo extracted tooth/typodont and pig jaw models. (Aim 2) CAR2s will be fabricated by 3D micromolding functional polymers with embedded IONPs for biofilm disruption, retrieval, and drug delivery at the apical region. We will optimize magnetic control and tracking, antibiofilm activity and triggered cargo delivery, testing efficacy to remove and retrieve biofilms. We will assess bioactivity using mixed- species biofilms and maneuverability to the apical region of the root canal recapitulated in 3D-printed teeth and ex vivo models, while rigorously eva...