Quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity.

Abstract

This study presented a quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity. Droplets encapsulating single cells and even distribution of fluorescein labeled antibodies removed from targeted cells deformed through the constriction microchannel where the excited fluorescent signals were sampled and interpreted into numbers of proteins based on volume equivalence in measurement of droplets and calibration of fluorescence. To improve the detection reliability, a comprehensive analysis and comparison of multiple stripping agents such as proteinase K, guanidine hydrochloride, and urea was conducted. To improve the detection sensitivity, light modulation was used to address electrical noises and quartz microchannels were fabricated to address optical noises. As a demonstration, based on this quantitative flow cytometry of droplet microfluidics, (1) mutant p53 expressions of single cells were quantified as 1.95 ± 0.60 × 105 (ncell =2918 of A431) and 1.30 ± 0.70 × 105 (ncell =3954 of T47D); (2) single-cell expressions of Ras, c-Myc, and β-tubulin were quantified as 1.90 ± 0.59 × 105 , 4.39 ± 1.44 × 105 , and 2.97 ± 0.81 × 105 (ncell =3298 of CAL 27), 1.83 ± 0.58 × 105 , 2.08 ± 0.13 × 106 , and 1.96 ± 0.74 × 105 (ncell =5459 of WSU-HN6). As a microfluidic tool capable of quantitatively estimating single-cell protein expressions, this methodology may provide a new quantitative perspective for the field of flow cytometry.