Abstract
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.
Highlights
Polytetrafluoroethylene (PTFE), commonly known under the brand name Teflon-AF®, is an amorphous fluoroplastics which possesses excellent properties including high optical clarity, low surface energy, high thermal stability and superior chemical and biological inertness
In such a bead-based digital bioassay, single target molecules are captured on magnetic beads, which are subsequently compartmentalized in femtoliter-sized microwells
Different molds with pillars of a particular height were fabricated to evaluate the influence of the the Teflon solution while molding
Summary
Polytetrafluoroethylene (PTFE), commonly known under the brand name Teflon-AF® , is an amorphous fluoroplastics which possesses excellent properties including high optical clarity, low surface energy, high thermal stability and superior chemical and biological inertness. It is applied in a variety of devices and fields, ranging from kitchen tools to lab-on-a-chip systems. Labeling of the target molecules with an enzyme and co-encapsulation of fluorogenic substrate in the microwells enables the generation of fluorescent product in the confined volume of the microwell This signal, which indicates the presence of a target molecule on the bead, can be detected using fluorescence microscopy or other optical detection strategies.
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