Manipulation of single cells inside nanoliter water droplets using acoustic forces

Droplet microfluidics enables high-throughput screening of single cells and is particularly valuable for applications, where the secreted compounds are analyzed. Typically, optical methods are employed for analysis, which are limited in their applicability as labeling protocols are required. Alternative label-free methods such as mass spectrometry would broaden the range of assays but are harmful to the cells, which is detrimental for some applications such as directed evolution. In this context, separation of cells from supernatant is beneficial prior to the analysis to retain viable cells. In this work, we propose an in-droplet separation method based on contactless and label-free acoustic particle manipulation. In a microfluidic chip, nanoliter droplets containing particles are produced at a T-junction. The particles are trapped in the tip of the droplet by the interplay of acoustic forces in two dimensions and internal flow fields. The droplets are subsequently split at a second T-junction into two daughter droplets-one containing the supernatant and the other containing the corresponding particles. The separation efficiency is measured in detail for polystyrene (PS) beads as a function of droplet speed, size, split ratio, and particle concentration. Further, single-bead (PS) and single-cell (yeast) experiments were carried out. At a throughput of 114 droplets/min, a separation efficiency of 100% ± 0% was achieved for more than 150 droplets. Finally, mammalian cells and bacteria were introduced into the system to test its versatility. This work demonstrates a robust, non-invasive strategy to perform single yeast cell-supernatant sampling in nanoliter volumes.