Simple 3D-printed, cleanroom-free microfluidic assay for evaluating chemotaxis in motile microorganisms

Precise spatiotemporal control of chemical gradients is crucial for studying microbial chemotaxis. However, traditional microfluidic platforms using poly(dimethylsiloxane) and soft lithography present a high barrier to entry due to their reliance on cleanrooms, specialized molds, and complex external pumping systems. To address these challenges, we introduce a rapid, cleanroom-free, 3D-printed microfluidic assay that simplifies the quantification of microbial chemotaxis and motility. Manufactured in under 4 h using high-resolution stereolithography 3D printing, the device maintains a standard 25 × 75 mm2 footprint for effortless microscope integration. Its monolithic, three-chamber design incorporates a central truncated-cone agar well that mechanically secures a hydrogel barrier, generating well-defined, diffusion-based transient chemical gradients without the need for external equipment. Physical characterization via visual flow tests with high-contrast dye confirmed robust leak prevention and unidirectional transient gradient formation within a quantified operational time window. As a biological proof-of-concept, the platform successfully quantified the concentration-dependent chemotactic response of Chlamydomonas reinhardtii to sodium bicarbonate, revealing peak sensitivity at 10−3M (p < 0.001) while capturing population heterogeneity. Additionally, parallel evaluations with the morphologically distinct Euglena gracilis demonstrated the platform’s architectural adaptability to diverse cell sizes and shapes. These evaluations confirmed the assay’s efficacy in isolating active chemotaxis from passive fluid drift and context-dependent motility. Ultimately, this accessible, rapid-prototyping platform provides a highly customizable tool for investigating complex microbial behavioral dynamics.