An Affordable paper based platform for eDNA Filtration, Concentration, and nucleic acid extraction

• Triple-layer chitosan–silica in borosilicate pad enhances DNA binding efficiency. • High-molecular-weight chitosan–silica captures more DNA than the low-molecular-weight form. • Triple-layer chitosan–silica binding pads provide DNA recovery rate of 86% • Low-cost chitosan-silica pads achieve DNA extraction performance comparable to silica beads. Environmental DNA (eDNA) analysis is a non-invasive method for discovering and identifying rare and endangered species in a variety of ecosystems including aquatic environments. However, conventional eDNA analysis tends to be time consuming and labor-intensive, which limits their efficiency. In this study, a novel eDNA capture system is made from stacked layers of membranes with a binding pad of borosilicate glass paper embedded with a triple layer of high molecular weight chitosan and silica nanoparticles. The system is designed to enhance DNA capture efficiency and is part of a portable system for on-site eDNA extraction that measures approx. 22 x 70 mm using sample volumes up to 3 mL and enables DNA extraction under 10 min. The DNA recovery rate of 86% is comparable to silica bead-based laboratory methods at a significantly lower cost, and the Ct values from the nucleic acid amplification step are reached within 18 to 36 min. Real water samples were tested, yielding results comparable to those obtained with the silica bead-based laboratory methods. Thus, enables downstream applications, such as species detection through a portable system that has the potential to improve the accessibility of eDNA analysis for effective environmental monitoring and conservation practices.