Advancing in situ single-cell microbiological analysis through a microwell droplet array with a gradual open sidewall

The utilization of microfluidic analysis technology has resulted in the advancement of fast pathogenic bacteria detection, which can accurately provide information on biochemical reactions in a single cell and enhance detection efficiency. Nevertheless, the achievement of rapid and effective in situ detection of single-bacteria arrays remains a challenge due to the complexity of bacterial populations and low Reynolds coefficient fluid, resulting in insufficient diffusion. We develop microwell droplet array chips from the lateral hydrodynamic wetting approach to address this issue. The sidewall of the microwell gradually opens which aids in advancing the liquid-air interface and facilitates the impregnation of the solid microwells, preserving the Wenzel state and assisting in resisting the liquid force to separation from the drop. The feasibility of preparing cell arrays and identifying them inside the microwells was demonstrated through the simulated streamlined distribution of gradual and traditional microwells with different sizes. The water-based ink diffusion experiment examined the relationship between diffusion efficiency and flow velocity, as well as the position of the microwell relative to the channel. It showed that the smaller gradual microwell still has a good diffusion efficiency rate at a flow velocity of 2.1 μL min^-1 and that the infiltration state is easier to adjust. With this platform, we successfully isolated a mixed population containing E. coli and S. aureus, obtained single-bacteria arrays, and performed Gram assays after in situ propagation. After 20 hours of culture, single bacteria reproduced demonstrating the capability of this platform to isolate, cultivate, and detect pathogenic bacteria.