Sequential enzymatic derivatization coupled with online microdialysis sampling for simultaneous profiling of mouse...

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 (H₂O₂), 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 H₂O₂, 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 H₂O₂, 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 H₂O₂, 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.