Fully Dried Two-Dimensional Paper Network for Enzymatically Enhanced Detection of Nucleic Acid Amplicons

Two-dimensional paper networks (2DPNs) have enabled the use of paper-based platforms to perform multistep immunoassays for detection of pathogenic diseases at the point-of-care. To date, however, detection has required the user to provide multiple signal enhancement solutions and been limited to protein targets. We solve these challenges by using mathematical equations to guide the device design of a novel 2DPN, which leverages multiple fluidic inputs to apply fully dried solutions of hydrogen peroxide, diaminobenzidine, and horseradish peroxidase signal enhancement reagents to enhance the limit-of-detection of numerous nucleic acid products. Upon rehydration in our unique 2DPN design, the dried signal enhancement solution reduces the limit-of-detection (LOD) of the device to 5 × 10¹¹ nucleic acid copies/mL without increasing false positive detection. Our easy-to-use device retains activity after 28 days of dry storage and produces reliable signal enhancement 40 min after sample application. The fully integrated device demonstrated versatility in its ability to detect double-stranded and single-stranded DNA samples, as well as peptide nucleic acids.