Ultra-sensitive Fluorescence-Activated Droplet Single-cell Sorting based on Tetramer-HCR-EvaGreen Amplification

Abstract The current single-cell analysis technologies such as fluorescence-activated cell sorting (FACS) and fluorescence-activated droplet sorting (FADS) could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability. Here, an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen (THE) fluorescence signal amplification. The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification. Upon application to target MCF-7 cells, the platform exhibited high efficacy and selectivity while maintaining more than 95% cell viability. The THE-FADS achieved sorting efficiencies of 55.5% and 50.3% with purities of 91% and 85% for MCF-7 cells in PBS solutions and simulated serum samples, respectively. The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells. The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.