Multi-mode droplet splitting on active-matrix digital microfluidics: quantitative boundaries and optimal sequential generation

Precise generation of microdroplets at picoliters to microliters scale is critical for advancing microfluidics technologies and precision life sciences research. Digital microfluidics enables programable individual droplet, however, there still lacks comprehensive characterization and analysis on optimal splitting modes, hindering its further application requiring extreme volume accuracy and splitting. Here, we report a systematic quantitative investigation of four droplet splitting strategies: symmetric splitting, asymmetric splitting, deformative splitting, squeezing, leveraging the high programmability advantage of large-scale active-matrix digital microfluidics. Droplet splitting is experimentally tested across varying droplet sizes, shapes, ratios and sub-droplet motion modes. Based on extensive experimental results, quantitative analysis is conducted to comprehensively characterize the splitting accuracy and effective ratio ranges. From these statistical results and optimal splitting modes, we establish an optimal sequential splitting decision framework. Aiming at precise generation for ultra-low-ratio sub-droplet through sequential splitting, the proposed framework can efficiently screen reasonable splitting paths from the combinatorial solution space. Ultra-low-ratio droplet generation at 0.78125% is realized, which is unattainable by any single-step method, with a cumulative accuracy of 1.05868 upon a target droplet of 12 nL. This work clarifies the quantitative performance boundaries of multi-mode droplet splitting strategies and demonstrates the capability of standardized optimal splitting sequence generation. These findings provide a theoretical and technical basis for customized multi-step droplet preparation and high-stability microfluidic manipulation.