Paper Matters: Technical Evaluation of Paper-Based Substrates for Enhanced Preconcentration of Biomolecules in Liquid Biopsy Diagnostics

Early detection of disease biomarkers remains a major challenge due to their low abundance in biological fluids. Preconcentration strategies can address this issue, but conventional approaches often require additional time, costly materials, or complex instrumentation. This work explores the use of paper-based substrates as a sustainable and cost-effective alternative for analyte preconcentration, highlighting how the choice of paper type critically influences sensitivity enhancement in analytical methods without compromising simplicity. Three cellulose-based papers (Whatman grade 1, Whatman grade 4, and a commercial laboratory filter paper) were systematically characterized and configured as 3D-origami-inspired, capillary-driven platforms. Their structural and fluidic properties were evaluated to determine how substrate selection affects analyte accumulation, transport, and detection efficiency. The devices were combined with three detection assays: a naked-eye colorimetric assay with smartphone integration, an electrochemical assay for miRNA, and a spectrophotometric Bradford assay for protein quantification. The paper platforms demonstrated rapid and efficient preconcentration across multiple biomarker classes. Up to a 20-fold enhancement was achieved for miRNA detection in human serum within 30 s, approximately 10-fold for short and double-stranded DNA in buffer, and about 2-fold for larger proteins. These results illustrate the importance of substrate selection and the versatility of paper-based preconcentration in handling analytes of different molecular sizes and assay formats. This study shows that cost-effective, customizable paper substrates can bridge the gap between high-performance biomolecular analysis and accessible diagnostics, enabling early disease detection and real-time monitoring in both laboratory and point-of-care settings.