Ratiometric bioluminescent detection of Cu(II) ion based on differences in enzymatic reaction kinetics of two...

Heavy metal contamination in water bodies has raised global concerns due to its significant threats to both public health and ecosystem. Copper (Cu), one of the most widely used metals, is also an essential trace element in physiological systems. Excessive intake of Cu from water can cause toxicity, potentially resulting in serious health risks. Ensuring water safety necessitates the critical detection of copper ion (Cu²⁺). Here, we report a ratiometric bioluminescent detection method for Cu²⁺, DERK-Cu(II), which is based on the Distinct Enzymatic Reaction Kinetics of two luciferase variants. In DERK-Cu(II), the blue luminescent luciferase exhibits lower catalytic efficiency than the green luminescent luciferase, thus it is less inhibited by Cu²⁺. Consequently, the luminescence color of their mixture is dependent on the Cu²⁺ concentrations, enabling us to find an optimal mixing ratio at which luminescence color changes evidently from green to blue. Building on this, we established a quantitative detection method for Cu²⁺ using a smartphone and successfully measured the Cu²⁺ concentrations in several water samples. The method we developed, using the difference in reaction kinetics of two enzymes with the same substrate specificity, would become a versatile approach applicable to the development of other enzyme-based indicators.