Permeable Hydrogel Microreactors for On-Chip Analysis of Diatom Growth Dynamics at Single-Cell Resolution

Diatoms are environmentally responsive photosynthetic microorganisms whose growth dynamics and biosilicification processes are tightly regulated by external physicochemical conditions. However, conventional bulk cultivation and existing microfluidic platforms often fail to provide stable three-dimensional confinement together with dynamic environmental control, limiting long-term quantitative analysis at single-cell resolution. Here, we present a permeable hydrogel microreactor system integrated with microfluidic perfusion and a neural network-based image analysis workflow for on-chip investigation of diatom growth dynamics. Monodisperse alginate/carboxymethyl chitosan hydrogel microspheres were engineered to stably confine individual Cyclotella cryptica cells while permitting efficient molecular exchange. The microreactors were immobilized within a perfused microfluidic device, enabling long-term cultivation and real-time imaging under dynamically regulated conditions. Coupled with this neural network-based approach for segmentation and contour extraction, we quantitatively reconstructed single-cell growth trajectories and division events, achieving a specific growth rate of 1.874 d –1 under perfusion, which represents a 5.5-fold increase over batch controls. Furthermore, dynamic copper exposure enabled concentration-dependent stress profiling, yielding EC 50 values of 8.53 μM (growth inhibition) and 7.25 μM (proliferation inhibition) at single-cell resolution. This platform offers a versatile analytical framework for resolving cellular heterogeneity and environmental responses in photosynthetic microorganisms under precisely controlled microenvironments.