Toward quantitative DNA tracer tests: development and validation of a novel capture device for groundwater flow characterization in karst and carbonate aquifers

• Novel device enables passive DNA tracer capture for groundwater studies. • DNA breakthrough curves align with salt and dye tracers in lab and field-scale tests. • Agarose beads with streptavidin ensure selective and stable DNA binding. • Passive devices allow high-sensitivity, low-impact tracer recovery. • Device supports low-impact, scalable hydrogeological investigations. Synthetic DNA tracers represent an innovative solution to overcome the limitations of conventional hydrological tracers, particularly in complex and large-scale hydrogeological systems. However, prolonged tracer tests still rely on manual sampling, which often provides sparse temporal coverage, increasing the risk of missing concentration peaks and biasing breakthrough curve interpretation. In addition, repeated site visits entail significant logistical effort and cost. To address these constraints, this study presents the development and validation of a novel passive device that selectively captures biotinylated synthetic DNA tracers, enabling time-integrated, semi-quantitative assessment of groundwater flow and pollutant migration. The device integrates streptavidin-functionalized superparamagnetic beads (1.0 µm; binding capacity ∼ 500pmol mg –1 ) encapsulated in 0.6% agarose hydrogel pearls and housed in a sealed hybridization chamber. Laboratory column tests (1.65 × 10 –4 L s –1 ; hydraulic gradient 1.4%) and a field-scale experiment in a natural stream (∼6 L s –1 ; 200 m reach), used as an analog for fracture flow or conduit flow in karst systems, benchmarked DNA behavior against conventional tracers (KCl, NaCl, fluorescein). Quantification by qPCR showed 100.2% efficiency and R 2 = 0.997, with sensitivity < 10 DNA molecules per reaction. Breakthrough curves from manual sampling and the passive device closely matched conservative tracer responses. The passive device produced slightly broader BTCs, consistent with time-integrated capture and detection of delayed or retained fractions. This approach provides a reproducible method for tracing hydrological pathways in karst and carbonate aquifers, thereby facilitating broader application of DNA tracers in groundwater studies.