High-Throughput Characterization of Individual Microdroplets Unveils Ultrasound-Enhanced Radical Production

Reactive oxygen species (ROS) formed in microdroplets have attracted significant attention due to their importance in environmental chemistry, mechanochemistry, and various biological oxidative processes. However, quantifying and characterizing ROS in microenvironments remain a challenge, as most current approaches rely on bulk-phase measurements that lack droplet-level resolution or suffer from limited low-throughput. This not only limits mechanistic interpretations due to variabilities in individual droplets but also makes it nearly impossible to evaluate a large set of conditions for desired ROS productions. Here, we report a high-throughput strategy to characterize and quantify H₂O₂ production in hundreds of thousands of individual droplets simultaneously by horseradish peroxidase (HRP)-catalyzed fluorogenic reactions. To increase the signal-to-noise level, we carried out a ratiometric signal measurement by using Rhodamine 6G (R6G) as an internal control in each droplet. Our results confirm that smaller droplets produce more ROS per unit volume, consistent with current interfacial chemistry hypotheses. In contrast, we unveiled for the first time that ROS generation was enhanced in larger airborne droplets (>5 μm) when treated with ultrasound using this high-throughput platform. This approach offers a powerful framework for facilitated generation of ROS and mechanistic understanding of ROS dynamics through rapid and parallel characterization of a large ensemble of individual microdroplets.