Multilayered 3D-printed microfluidic chip integrated with functional unit operations for bacterial viability testing

Biochemical diagnostic assays, such as broth microdilution (BMD), have been miniaturized and implemented in microfluidic formats; however, their broader adoption remains limited by complex device architectures and fabrication protocols. A key challenge is the integration of multiple fluidic unit operations required to execute sequential assay steps, which remains difficult to achieve using conventional soft lithography or standard 3D printing approaches. To address these limitations, we developed a microfluidic system comprising a multilayer 3D-printed microfluidic chip and a tunable fluid actuation module (MagnaLink) for automated on-chip BMD operation. The microfluidic chip integrates valving, mixing, filtration, and plasmonically enhanced colorimetric sensing within a simple fabrication workflow, enabling rapid bacterial viability testing. The MagnaLink module provides reproducible, bidirectional, leak-free on-chip actuation through a reversible magnetic sealing approach (MagnaSeal). The individual components of both the microfluidic chip and MagnaLink module were characterized theoretically, numerically, and experimentally. As a proof of concept, antibiotic susceptibility testing of Pseudomonas aeruginosa was performed against ciprofloxacin and gentamicin, achieving complete agreement with standard BMD assays for minimum inhibitory concentration determination. This platform provides a scalable approach for automating sequential biochemical assays on-chip and has the potential to support the development of rapid, low-cost, and deployable point-of-care diagnostic workflows.