Wax transfer patterning of porous paper for lab-on-a-chip

Paper-based microfluidics has emerged as a promising platform for low-cost analytical and biological assays. However, fabrication of hydrophobic barriers on porous substrates remains largely dependent on direct wax printing, which limits patterning flexibility, requires specialized equipment, and constrains scalability. Here, we present a simple and versatile wax transfer technique that enables reliable replication of wax micropatterns from one porous substrate to another. The method leverages controlled wettability, thermal activation, and applied pressure to achieve efficient and reproducible pattern transfer. The resulting patterned substrates display well-defined hydrophobic–hydrophilic regions suitable for microfluidic applications. We demonstrate the versatility of the approach through applications in cell culture and biochemical assays, including the growth and spatial confinement of cervical and breast cancer cells, as well as Folin–Lowry protein quantification in saliva samples. Notably, wax-transferred patterns provide spatial confinement of assay signals, while maintaining biocompatibility, suitability for biological applications, and structural integrity of the substrate. The simplicity of the process, compatibility with diverse porous materials, and long-term stability of donor patterns highlight its potential for scalable and distributed fabrication. Overall, this work establishes a porous-to-porous transfer strategy that expands the design space of paper-based microfluidics and provides a practical pathway toward accessible, manufacturable, and widely deployable lab-on-paper technologies.