DNA origami: thinking ‘outside the fold’ for direct integrity testing of membranes for virus removal in potable reuse applications†

Increasing water scarcity and water quality impairment have led to broader implementation of potable reuse throughout the world. High pressure membranes, including nanofiltration (NF) and reverse osmosis (RO), play a critical role in many potable reuse treatment trains because they are robust barriers against chemical and microbiological constituents. Despite achieving high pathogen log reduction values (LRVs) in practice (e.g., LRV > 5), high pressure membranes are often credited for only a fraction of observed LRVs (e.g., LRV < 3), which results in an LRV ‘gap’. This is because commonly used bulk water quality surrogates, namely total organic carbon (TOC) and electrical conductivity (EC), lack the resolution or analytical dynamic range to justify higher credit. The industry is now evaluating alternative surrogates (e.g., sucralose, sulfate, and strontium) that are both discrete and abundant in wastewater to narrow this regulatory ‘gap’. DNA origami technology can synthesize DNA nanostructures that mimic the size and morphology of viruses, potentially offering another novel surrogate for direct integrity testing. This study simultaneously evaluated pilot-scale NF and RO rejection of spiked MS2 bacteriophage (culture and qPCR), spiked DNA nanostructures (qPCR), and the aforementioned water quality surrogates. RO and NF achieved LRVs of ∼5 for culturable MS2 and censored LRVs of >4 for MS2 RNA. For RO, DNA nanostructure LRVs (up to ∼3) were comparable to the more advanced surrogates (e.g., sucralose, sulfate, and strontium), while DNA nanostructure LRVs for the NF membranes were generally <1 and consistent with EC and strontium. This study demonstrates that DNA nanostructures may have future value for potable reuse as they can be directly quantified via qPCR (without nucleic extraction) and can provide tailored structures that target various pathogens of interest. However, this study also highlights knowledge gaps that require further study, including the potential adsorption of DNA nanostructures to membrane surfaces and their ability to retain three-dimensional morphology in non-ideal wastewater matrices. Beyond the potential use of DNA origami technology, this study also highlights the value of rapid molecular methods in complementing, or even replacing, traditional culture methods when quantifying targets in membrane challenge tests.