Molecularly Imprinted Polymers as Biomimetic Test Zones in Paper-Based Nucleic Acid Assays—Comparing Vertical and Lateral Flow Formats

The development of rapid and sensitive point-of-care nucleic acid tests benefits from robust synthetic recognition elements. Here, a biotin-specific molecularly imprinted polymer (MIP) was synthesized using an optimized protocol and integrated as a biomimetic test zone into two paper-based formats: nucleic acid vertical flow (NAVF) and nucleic acid lateral flow (NALF). Both platforms were evaluated for the detection of double-tagged PCR amplicons from <i>Escherichia coli</i>. NAVF enabled a 3 min visual readout with an LOD of 1.00 × 10^-2 ng mL^-1. NALF provided a total assay time of <15 min and achieved a visual LOD of 3.17 × 10^-2 ng mL^-1. Overall, the results demonstrate the versatility of biotin-MIPs as stable synthetic receptors for rapid, low-cost paper-based nucleic acid assays, with NAVF prioritizing speed and design flexibility and NALF prioritizing higher analytical sensitivity.