biostudies-literatureJeong HNational Human Genome Research Institute of the National Institutes of HealthNHGRI NIH HHSe2504687https://www.ebi.ac.uk/biostudies/studies/S-EPMC12366287biostudies-literatureUnknown21(32)Studies on the dynamics of single cell phenotyping have been hampered by the lack of quantitative high-throughput metabolism assays. Extracellular acidification, a prominent phenotype, yields significant insights into cellular metabolism, including tumorigenicity. Here, it is developed a versatile microfluidic system for single cell optical pH analysis (SCO-pH), which compartmentalizes single cells in 140-pL droplets and immobilizes ≈40,000 droplets in a 2D array for temporal extracellular pH analysis. SCO-pH distinguishes cells undergoing hyperglycolysis induced by oligomycin A from untreated cells by monitoring their extracellular acidification. To facilitate pH sensing in each droplet, a cell-impermeable pH probe is encapsulated and its fluorescence intensities are quantified. Using this approach, hyperglycolytic cells can be differentiated, and single-cell heterogeneity in extracellular acidification dynamics can be concurrently observed. This high-throughput system will be useful in applications that require dynamic phenotyping of single cells with significant heterogeneity.Small (Weinheim an der Bergstrasse, Germany)SCO-ph: Microfluidic Dynamic Phenotyping Platform for High-Throughput Screening of Single Cell Acidification.PMC12366287RM1HG010023Kuang DSul JYLeyes Porello EALim BJeong HHan SHRosario JGKim JLee DfalseSCO-ph: Microfluidic Dynamic Phenotyping Platform for High-Throughput Screening of Single Cell Acidification.Studies on the dynamics of single cell phenotyping have been hampered by the lack of quantitative high-throughput metabolism assays. Extracellular acidification, a prominent phenotype, yields significant insights into cellular metabolism, including tumorigenicity. Here, it is developed a versatile microfluidic system for single cell optical pH analysis (SCO-pH), which compartmentalizes single cells in 140-pL droplets and immobilizes ≈40,000 droplets in a 2D array for temporal extracellular pH analysis. SCO-pH distinguishes cells undergoing hyperglycolysis induced by oligomycin A from untreated cells by monitoring their extracellular acidification. To facilitate pH sensing in each droplet, a cell-impermeable pH probe is encapsulated and its fluorescence intensities are quantified. Using this approach, hyperglycolytic cells can be differentiated, and single-cell heterogeneity in extracellular acidification dynamics can be concurrently observed. This high-throughput system will be useful in applications that require dynamic phenotyping of single cells with significant heterogeneity.2025-01-01T00:00:00Z2025 Aug2026-05-05T12:39:31.607Z2026-04-26T03:06:15.269ZS-EPMC123662874051905810.1002/smll.202504687