Real-Time Fluorescence Measurement for Droplet Generation and Signal Detection in a Cylindrical Tube

This study provides an approach for generating droplets in a cylindrical tube. This creative design utilizes a single tube to generate droplets for the emulsion polymerase chain reaction (PCR). We fabricated the multi-layered tube and detected samples in 1 mm microchannel length for the minimum disposable material used for droplet signal analysis. In this research, we verify the validity of the non-planar droplet chip with a single driving pump by experimentally analyzing the light intensity of the liquids during droplet processing in real time. Experimental observation shows that droplet diameter from 150 to 285 μm was obtained by the cylindrical tube with different dispersed liquid and gas pressure settings. Average size of droplets decreased by approximately 37% as the dispersed phase liquids change at the same flow pressure of 50 mbar. In this study, the effects of the laser site, 6-carboxyfluorescein (6-FAM) dye buffer, DNA reagent, and driving pressure are analyzed directly by the signal recorded during droplet generation in the cylindrical tube. Finally, we have developed a real-time detection method to count exon 19 deletion mutations of epidermal growth factor receptor (EGFR) in a droplet using a cylindrical tube. Two digital droplet PCR methods were compared in measuring the copy numbers of EGFR with the same target DNA concentration of 10⁵ copies/μL.