Single Cell Enzyme Analysis by Using Continuous Flow Microfluidics

Precision medicine refers to giving the right therapeutics, to the right patient, at the right time. In the context of cancer, successful implementation of precision medicine, requires treatment individualization not only taking into account patient and tumor factors, but also tumor heterogeneity and tumor evolution over time. In this study, a continuous flow microfluidic device was developed as a functional flow cytometer (m-FACS) to detect secreted multiplexed protease activities at single cell resolution. The individual cells from patient samples are encapsulated within water-in-oil droplets for single cell multiplexed protease assay. We modified FRET (fluorescence resonance energy transfer)-based substrates to accommodate different fluorescent pairs with distinct excitation and emission wavelengths to obtain multiple signals from droplets containing single cells. Four substrate-protease reactions in a droplet were simultaneously monitored at three distinct pairs of fluorescent excitation (UV: 400nm, B: 470nm, G: 546nm, R: 635nm) and emission (B: 520nm, G: 580nm, R: 670nm) wavelengths. To infer a quantitative profile of multiple proteolytic activities from single cells, we applied the computational method Proteolytic Activity Matrix Analysis (PrAMA). The capability to determine multiple protease activities at single cell resolution has the potential to characterize tumor progress of individual patients.