A microfluidic device was constructed for on-site determination of fish freshness by using adenosine-5′-triphosphate (ATP) concentration as an indicator of freshness. In the device, extracts from fish and reagent solutions were processed in the form of droplets separated by air. The volumes of the droplets of the extract solution, a solution containing enzyme substrates, and a washing buffer solution were measured in branched flow channels. To simplify the structure and procedure for mixing solutions, a wider portion was formed in one flow channel to merge the droplets. A row of droplets was ejected from each of three branched flow channels, merged in the wider portion, and transported to a sensing region located downstream. ATP was detected using two enzymatic reactions involving glycerol kinase (GK) and glycerol-3-phosphate oxidase (G3PO). A linear relationship between the generated current and ATP concentration was confirmed, and ATP detection from jack mackerel extracts was carried out. A distinct change in the current was observed with the fresh extracts as in the case of standard solutions. The obtained data correlated well with those obtained by high-performance liquid chromatography (HPLC).
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