| Summary: | In recent years, Teflon-on-glass microwells have been successfully implemented in bead-based digital bioassays for the sensitive detection of single target molecules. Their hydrophilic-in-hydrophobic (HIH) nature enables the isolation and analysis of individual beads, carrying the target molecules, which can be further manipulated accurately through optical tweezer (OT) setups. However, these Teflon HIH-microwell platforms are conventionally fabricated through a complex, time-consuming and labor-intensive dry lift-off procedure which involves a series of major steps, limiting the up-scaling potential of these platforms. Alternative Teflon-based microwell fabrication methods have been extensively explored in literature but they preclude the generation of hydrophobic wells with hydrophilic bottom, thereby hampering the bioassay performance. Here, we present a new Teflon-on-glass molding method for the high throughput fabrication of hydrophilic-in-hydrophobic (HIH) microwell arrays, able to empower bead-based digital bioassays. Microwells 2.95 μm in depth and 3.86 μm in diameter were obtained to host individual beads. In these microwell arrays, sealing of reagents was demonstrated with an efficiency of 100% and seeding of superparamagnetic beads was achieved with an efficiency of 99.6%. The proposed method requires half as many steps when compared to the traditional dry lift-off process, is freely scalable and has the potential to be implemented in different bead-based bioassay applications.
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