Valved Microwell Array Platforms for Stepwise Liquid Dispensing.

The fabrication of microarray chips and the precise dispensing of nanoliter to microliter liquids are fundamental for high-throughput parallel biochemical testing. Conventional microwells, typically featuring a uniform cross section, fill completely in a single operation, complicating the introduction of multiple reagents for stepwise and combinatorial analyses. To overcome this limitation, we developed an innovative valved microwell array. Using ultraviolet (UV)-curing resin three-dimensional (3D) printing, these multilayer configurations can be rapidly fabricated through direct template printing and polydimethylsiloxane (PDMS) casting. Each microwell incorporates a microvalve structure, truncating fluids within the upper metering well and allowing transfer to the bottom reservoir well under centrifugal force. Sequential operations enable the introduction of multiple reagents, facilitating orthogonal combinations for complex assays. We explored four types of valving methods: DeepWell, Expansion, Bottleneck, and Membrane valve, each offering varying degrees of design complexity, operational efficiency, robustness, and precision. These methods constitute a versatile toolkit to accommodate a broad spectrum of analytical requirements. Our innovative approach redefines microwell architecture, direct manufacturing techniques, and stepwise fluid dispensation in microarrays.