Miniaturized Droplet‐Based Adaptation of the Ames Test for High‐Throughput Mutagenicity Assessment

This work presents the first demonstration of a tube-based droplet microfluidic implementation of the Ames test, bridging single-droplet resolution with regulatory genotoxicity testing. The Ames test is a cornerstone assay for detecting mutagenicity, but conventional plate- and well-based formats suffer from high reagent consumption, low throughput, and limited automation. We report a droplet-based microfluidic Ames test assay using Salmonella typhimurium TA98, combining nanoliter compartmentalization with multiparameter optical detection. Cell density screening identified an optimal inoculum range of 10⁶-10⁷ cells/mL that maximized sensitivity while limiting spontaneous revertants. Dose-response analysis with the reference mutagen 4-nitro-o-phenylenediamine (4-NOPD) revealed clear increases in the fraction of droplets with growth of revertants, followed by a cytotoxic suppression at ≥ 8 μg/mL. A threshold-based evaluation enabled robust quantification of stochastic mutation events at single-droplet resolution. Compared with the classical fluctuation assay, the microfluidic format reduced reagent consumption by > 90%, generated statistically powerful datasets within 48 h, and eliminated subjective scoring. This study establishes segmented-flow microfluidics as a scalable, sensitive, and resource-efficient platform for mutagenicity testing, with applications in regulatory toxicology, environmental monitoring, and high-throughput chemical screening.