Carbon-dot-based ratiometric fluorescence glucose biosensor

A ratiometric fluorescence glucose biosensor based on carbon dots (CDs) and rhodamine 6G (Rh6G) was developed both as an aqueous solution and as a crosslinked poly(acrylic acid) solid-state film. A ratiometric fluorescence color change was realized by fluorescence quenching due to the bienzymatic reaction of glucose oxidase (GOx) and horseradish peroxidase (HRP) with glucose. When excited at 360 nm, the blue fluorescence emission of the CDs, prepared by a solvothermal method with citric acid and ethylene diamine, was quenched by the bienzymatic reaction with glucose, whereas the fluorescence of Rh6G was inert to glucose. Thus, a ratiometric fluorescence color change from blue to green was observed as the glucose concentration increased. The optimized CD/Rh6G/GOx/HRP aqueous solution showed a linear range of 0.1–500 μM with a limit of detection (LOD) of 0.04 μM, good selectivity for glucose over the major ingredients in human blood and could be used with human blood serum. A stable solid-state biosensor film was fabricated by immobilization of CD/Rh6G/GOx/HRP in the hydrogel film prepared by ultraviolet curing of a mixture of acrylic acid and diacrylated poly(ethylene glycol) (70:30, w/w). Compared to the CD/Rh6G/GOx/HRP aqueous solution, the hydrogel film showed a similar ratiometric fluorescence color change, sensitivity (linear range of 0.5–500 μM with an LOD of 0.08 μM), and selectivity. Further, the solid-state glucose biosensor film was inherently stable and could be used whenever needed, overcoming the instability of the aqueous solution (owing to aggregation, enzyme denaturation, etc.). These ratiometric biosensors increase the ability to detect glucose using the naked eye compared to simple turn-on or turn-off modes. Thus, this approach expands the potential applications of CDs in biosensors to provide more convenient and practical detection methods.