Immobilized Droplet Arrays in Thermosetting Oil for Dynamic Proteolytic Assays of Single Cells

Matrix metalloproteinases (MMPs) play an important role in tumor progression. The study of dynamic MMPs activity at the single-cell level can dissect tumor heterogeneity in the time domain and facilitate finding out more efficient clinical solutions for tumor treatment. Due to the fluidity of the carrier oil, the existing droplet-based methods for single-cell MMP analysis rarely have the capability to track proteolytic assays in droplets continuously. Therefore, we describe a thermosetting oil for real-time monitoring of MMP assays in droplets, which can immobilize droplets by transforming into solid after droplet generation. The solidification of this oil can be accomplished in 33 min at 37 °C, basing on the hydrosilation of vinyl silicone oil and hydrosilicone oil without other inducers (e.g. UV, Ca²⁺). Through monitoring the MMP assays of single cells, the reaction rates can be calculated according to real-time fluorescent curves, showing significant cell heterogeneity in MMP activity. Moreover, the dynamic MMP activity reveals that some of the A549 cells transiently secrete MMP. In conclusion, the thermosetting oil enables immobilize droplets to achieve real-time monitoring of single-cell proteolytic activity without impairing the flexibility of droplet microfluidics and has a potential in other cell-based assays for providing dynamic information at high resolutions.