Quantitative RNA imaging in single live cells reveals age-dependent asymmetric inheritance.

Asymmetric inheritance of cellular content through cell division plays an important role in cell viability and fitness. The dynamics of RNA segregation are so far largely unaddressed. This is partly due to a lack of approaches to follow RNAs over multiple cellular divisions. Here, we establish an approach to quantify RNA dynamics in single cells across several generations in a microfluidics device by tagging RNAs with the diSpinach aptamer. Using S. cerevisiae as a model, we quantitatively characterize intracellular RNA transport from mothers into their buds. Our results suggest that, at cytokinesis, ENO2 diSpinach RNA is preferentially distributed to daughters. This asymmetric RNA segregation depends on the lifespan regulator Sir2 and decreases with increasing replicative age of mothers but does not result from increasing cell size during aging. Overall, our approach opens more opportunities to study RNA dynamics and inheritance in live budding yeast at the single-cell level.

Droplet Microfluidic Device for Chemoenzymatic Sensing.

The rapid detection of pollutants in water can be performed with enzymatic probes, the catalytic light-emitting activity of which decreases in the presence of many types of pollutants. Herein, we present a microfluidic system for continuous chemoenzymatic biosensing that generates emulsion droplets containing two enzymes of the bacterial bioluminescent system (luciferase and NAD(P)H:FMN-oxidoreductase) with substrates required for the reaction. The developed chip generates "water-in-oil" emulsion droplets with a volume of 0.1 µL and a frequency of up to 12 drops per minute as well as provides the efficient mixing of reagents in droplets and their distancing. The bioluminescent signal from each individual droplet was measured by a photomultiplier tube with a signal-to-noise ratio of up to 3000/1. The intensity of the luminescence depended on the concentration of the copper sulfate with the limit of its detection of 5 µM. It was shown that bioluminescent enzymatic reactions could be carried out in droplet reactors in dispersed streams. The parameters and limitations required for the bioluminescent reaction to proceed were also studied. Hereby, chemoenzymatic sensing capabilities powered by a droplet microfluidics manipulation technique may serve as the basis for early-warning online water pollution systems.