3D Printing as a New Solution for Paper-Based Device Manufacturing: On-Demand Wax 3D Printing for Integrated Microfluidic Reagent Delivery, Sample Treatment, and Biosensing.

Abstract

Since George Whitesides introduced the first paper-based device in the early 2000s, many scientists have faced challenges because wax printers are no longer produced. Although diverse strategies have been developed, there is still a need for cheap, scalable, and easy-to-use alternatives. Here, we introduce 3D printing of wax-based filaments as a direct, programmable strategy for fabricating microfluidic paper-based architectures and devices. Commercial wax filaments originally developed for investment casting and jewelry manufacturing were exploited owing to their low melting temperature, ultralow melt viscosity, and intrinsic hydrophobicity. Similarly to wax printing, here the wax filament is 3D-printed into the cellulose network, producing continuous through-thickness hydrophobic barriers and reliable fluid confinement. Using a low-cost commercial 3D printer, we established a library of customizable two- and three-dimensional architectures, including single-channel devices, multichannel configurations for fluid splitting and mixing, reaction networks, and miniaturized enzyme-linked immunosorbent assay (ELISA)-like well arrays. The structures were evaluated for dimensional fidelity, barrier integrity, fluid transport, flow time, reproducibility, batch consistency, dimensional retention, scalability, and nozzle-dependent spatial resolution. As a proof of concept, an integrated multistep paper-based electrochemical biosensor was fabricated incorporating dry-stored duplex-specific nuclease, a methylene blue-labeled DNA probe, sequential reagent reconstitution, and downstream electrochemical detection for microRNA analysis. Control and interference experiments further supported the analytical response under the investigated conditions. This work demonstrates how digital additive manufacturing can extend microfluidic paper-based analytical devices (μPADs) beyond conventional patterning, providing a rapid, customizable, accessible, and scalable route toward multifunctional analytical devices for decentralized and point-of-care applications.

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