Droplet reactors with bioluminescent enzymes for real time water pollution monitoring

It was shown that bioluminescent enzymatic reactions can be carried out in droplet reactors for biological measurements of water quality. Early detection of pollutants in wastewater, water treatment facilities, drinking water, and water for agricultural needs is a challenging problem. Effective water quality monitoring requires development of new methods for express detection of pollutants. Enzymes from bioluminescent bacteria can be used for the development of new express enzyme-based bioassay systems. This work demonstrates for the first time a microfluidic generator of emulsion droplets containing two enzymes of the bacterial bioluminescent system (luciferase and NAD(P)H:FMN-oxidoreductase) with reaction substrates. The developed chip generated “water-in-oil” emulsion droplets with a volume of 0.1 μL and a frequency of up to 12 droplets per second. Portable PMT was used to measure the bioluminescent signal in each individual droplet, the signal-to-noise ratio was 3000/1. To ensure the mixing of the system components in the emulsion droplets, the geometry of the microchannels of the chip was numerically simulated. The intensity of luminescence in droplets is depending on the concentration of copper sulfate and benzoquinone inhibitors. The limit of detection (LOD) for copper sulfate and benzoquinone was 1 and 0.2 mg/L, respectively. The studies carried out are applicable for the development of early warning systems at wastewater discharge sites of industrial enterprises or at water treatment plants in addition to the existing certified methods for the registration of water-soluble pollutants. Our results showed new potential of microfluidic polyenzymatic sensing for the biological metrology applications.