Using copper ions to amplify ROS-mediated fluorescence for continuous online monitoring of extracellular glucose in living rat brain

In this study we developed a facile and sensitive method for continuous monitoring of extracellular glucose concentration in living rat brain through microdialysis (MD) sampling in conjunction with (i) online sample derivatization using glucose oxidase to generate H2O2, which converted a reactive oxygen species-responsive fluorescent dye, 2′,7′-dichlorodihydrofluorescein (DCFH), into fluorescent species, and (ii) a novel non-immobilized enzyme-based fluorescence assay strategy, featuring copper ion (Cu2+)-facilitated amplification of the fluorescence intensity. After evaluating the experimental conditions for glucose oxidation and fluorescence generation, the introduction of Cu2+ ions to this system resulted in an additional 51-fold amplification of the net fluorescence intensity. By sequentially loading brain microdialysate into the dual sample collection loops, the sampling frequency was 7.5 h−1. Based on a 40-μL sample volume, the system's detection limit reached as low as 0.18 mM, sufficiently accurate to determine the extracellular glucose concentrations in living rat brains. To demonstrate the proposed system's practical performance and applicability, we conducted (i) spike analyses of biomolecule-rich fetal bovine serum sample, confirming that the analytical reliability was similar to that of a commercial glucose kit, and (ii) in vivo dynamic monitoring of the extracellular glucose concentrations in living rat brains after inducing neural depolarization by perfusing a high-K+ medium from the MD probe.