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

ABSTRACT 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 10 6 –10 7 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.