Sequential enzymatic derivatization coupled with online microdialysis sampling for simultaneous profiling of mouse tumor extracellular hydrogen peroxide, lactate, and glucose.

Probing tumor extracellular metabolites is a vitally important issue in current cancer biology. In this study an analytical system was constructed for the in vivo monitoring of mouse tumor extracellular hydrogen peroxide (H2 O2 ), lactate, and glucose by means of microdialysis (MD) sampling and fluorescence determination in conjunction with a smart sequential enzymatic derivatization scheme-involving a loading sequence of fluorogenic reagent/horseradish peroxidase, microdialysate, lactate oxidase, pyruvate, and glucose oxidase-for step-by-step determination of sampled H2 O2 , lactate, and glucose in mouse tumor microdialysate. After optimization of the overall experimental parameters, the system's detection limit reached as low as 0.002 mM for H2 O2 , 0.058 mM for lactate, and 0.055 mM for glucose, based on 3 μL of microdialysate, suggesting great potential for determining tumor extracellular concentrations of lactate and glucose. Spike analyses of offline-collected mouse tumor microdialysate and monitoring of the basal concentrations of mouse tumor extracellular H2 O2 , lactate, and glucose, as well as those after imparting metabolic disturbance through intra-tumor administration of a glucose solution through a prior-implanted cannula, were conducted to demonstrate the system's applicability. Our results evidently indicate that hyphenation of an MD sampling device with an optimized sequential enzymatic derivatization scheme and a fluorescence spectrometer can be used successfully for multi-analyte monitoring of tumor extracellular metabolites in living animals.