Determination of Glucose in Submicroliter Samples by CE−LIF Using Precolumn or On-Column Enzymatic Reactions

Abstract

Two enzymatic reactions combined with capillary electrophoresis (CE) are used to determine glucose contained in sample volumes of ≤500 nL. In the first enzymatic reaction, glucose is oxidized in the presence of glucose oxidase producing hydrogen peroxide, which reacts quantitatively with the fluorogenic compound homovanillic acid catalyzed by the enzyme peroxidase. The second reaction generates a fluorescent species that is proportional to the glucose concentration. The reaction product is determined by CE using laser-induced fluorescence (LIF) as the detection mode. The overall reaction scheme is faster than commonly used precolumn derivatization procedures and can be performed using very small sample quantities. Alternatively, the enzymatic reactions can be performed on-column, similar to the electrophoretically mediated microanalysis approach, accommodating sample quantities in the nanoliter range. The on-column reaction is a simple and practical approach for the determination of glucose contained in low-volume samples by CE-LIF, where samples are injected directly into the capillary column without any pretreatment. However, sample handling and detectability of the precolumn approach proved to be superior. Determination of glucose using the precolumn and on-column reactions showed detection limits of 50 and 800 nM, respectively. The methods were shown to be linear in the range tested, 1-100 μM and 100 nM-30 μM, for the on-column and precolumn reactions, respectively. The reproducibility for each scheme was <5% RSD. To determine the possibility of using a noninvasive procedure for glucose monitoring, we used the CE-LIF methods to analyze human tear samples for glucose. The tear fluid samples were contained in a volume of ∼200 nL. The concentration of glucose in the human tear samples analyzed using the precolumn and on-column procedures was ∼138 μM.