In-Oleo Microgasometry of Nanoliter-Scale Gas Volumes with Image-Based Detection

Gasometric assays involve measurements of the amounts of gases that are released during physical or chemical processes. The available instrumentation for gasometric analysis is generally difficult to use and requires large sample volumes. In some cases, toxic materials (mercury) are involved in the analysis process. Here, we propose a microscale gasometric assay using silicone oil as matrix. Microliter-volume (∼2.5 μL) aqueous droplets, containing sample and reagent and/or catalyst, are introduced to the oil matrix and merged. Nanoliters of gaseous products are released to the surrounding oil matrix forming tiny spherical bubbles. Due to the huge differences between refractive indices of the released gas and the surrounding liquids (aqueous assay solution, oil), the gas bubbles are clearly visible from the top, when the assay reservoir is illuminated from the bottom with light-emitting diodes. The released gas bubbles are documented by recording videos of the assay reservoir. Individual frames within these videos are then analyzed by a graphical software to obtain diameters of every gas bubble at each time point. Following a fixed period of time (typically, 5-90 s) after the sample/reagent droplet merger, the volume of the released gas scales with the amount of the substrate (analyte) present in the sample droplet. For example, hydrogen peroxide can be decomposed to oxygen by 0.44 U catalase enzyme and semiquantified in the range up to ∼1.0 μmol. Glutathione can be detected in a two-step procedure ((1) oxidation of glutathione by hydrogen peroxide; (2) decomposition of the hydrogen peroxide residue by catalase).