Dual-readout villus-like scaffold microfluidic platform for quantitative analysis of gut microbiota formation under...

A dual-readout modular microfluidic platform integrating a villus-like scaffold is presented for quantitative analysis of bacterial colonization and functional outputs under perfusion. The gut microbiota plays pivotal roles in host immunity and metabolism, and its assembly is strongly influenced by the order of bacterial arrival and colonization in the intestinal tract. To quantitatively investigate microbiota formation, an analytical system that unifies a three-dimensional (3D) microenvironment with fluidic transport and multimodal readouts is required. Here, we develop a villus-like scaffold-integrated modular microfluidic device enabling dual readout: (i) in situ fluorescence imaging and quantification of bacterial attachment on the scaffold, and (ii) effluent biochemical assays for metabolic and functional outputs. The villus-like scaffold was fabricated by replica molding using an acrylic master and polydimethylsiloxane (PDMS) molds, where ultrasonication-assisted bubble removal and post-gelation dehydration in 99% ethanol improved structural replication. The fabricated villus-like agarose scaffold showed a replication yield of up to 100%, with pillar dimensions of 192.1 ± 2.4 μm in diameter and 522.0 ± 10.3 μm in height. Under perfusion at 32 μL min^-1, the 3D scaffold increased L. plantarum colonization and lactate production compared with the flat hydrogel control, while E. coli exhibited increased scaffold-localized fluorescence despite decreased effluent β-glucuronidase (GUS)  signals over 8 h. This device-oriented system integrates villus-like scaffold architecture, perfusion operation, and dual-readout quantification, providing a scalable foundation for analytical studies of gut microbiota assembly.