Hierarchical alginate-bentonite beads enable instrument-free pre-analytic enrichment of liter-scale wastewater

Wastewater-based surveillance (WBS) offers a test-independent window into public community health, but its fidelity depends critically on pre-analytic enrichment that must convert liter-scale, inhibitor-rich influent into microliter analytical inputs without compromising low-titer targets. Here we introduce hierarchical alginate-sulfuric acid-activated bentonite beads (Alg@SAB) that recast pre-analytic enrichment as an interfacial mass-transfer problem. A diffusion-optimized macro-mesoporous scaffold, amine-functionalized surfaces (-NH₃⁺), and Ba²⁺ crosslinks collectively enable dual-mode capture-electrostatic adsorption and Ba²⁺↔Mⁿ⁺ cation-exchange-to retain free pathogens and pathogen-metal complexes under passive, pump-free operation, while batch-friendly fabrication from commodity precursors delivers reproducibility with per-test consumables of ∼US$0.06. Alg@SAB retains the two-fold-one-cycle linearity in input viral titer-Ct value while extending instrument-free concentration to the liter scale: 1 L inputs remain measurable where a vacuum-membrane kit fails at ≥ 500 mL; at 100 mL Alg@SAB achieves higher apparent recovery (56%, +27% versus kit). Validation used real municipal wastewater rather than synthetic surrogates, ensuring field realism in matrix complexity and inhibitor profiles. A single-tube, pH-dependent reversible crosslinking chemistry releases nucleic acids in situ while maintaining agreement and collapsing handoffs, enabling low-hardware deployment. Method-dependent microbiome profiling on these raw influent wastewater reveals mechanistically consistent selectivity without systematic inflation of alpha diversity, preserving ecological interpretability under real wastewater plant conditions. By integrating scalable materials engineering with practical deployment, Alg@SAB provides a simple and tunable route to decentralized, interpretable WBS, lowering technical barriers for real-time pathogen monitoring in diverse settings.