Digital analysis with droplet-based microfluidic for the ultrasensitive detection of β-gal and AFP

In this study, we presented a digital analysis with droplet-based microfluidic, which was applied for the detection of β-galactosidase (β-Gal) and AFP. The β-Gal was quantified according to the Poisson Distribution in digital analysis with droplets containing β-Gal and fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG). In our method, the lowest concentration of β-Gal we could accurately detect was 5 fM. We found that the digital detection could be applied in quantifying protein biomarkers, take AFP for model sample. The AFP was detected through indirect detection with excess streptavidin-conjugated β-galactosidase (SA-β-gal) as signal tag. To be specific, firstly SA-β-gal was conjugated to biotin-labeled AFP antibody, and then the unbounded signal tag in the reaction solution was taken out for digital detection with droplet-based microfluidic. As a result, the AFP could be also indirectly quantified at an fM level through subtraction. This experimental result demonstrated that the indirect detection with droplet-based digital analysis was able to detect biomarkers at a low level with a few samples on an ordinary flow-focusing microfluidic chip.