Integration of a Precolumn Fluorogenic Reaction, Separation, and Detection of Reduced Glutathione

Abstract

Reduced glutathione (GSH) has been determined by fluorescence detection after derivatization together with a variety of separations. The reactions between GSH and fluorescent reagents usually carry out during the sample pretreatment and require minutes to hours for complete reactions. For continuous monitoring of GSH, it would be very convenient to have an integrated microdevice that could perform online precolumn derivatization, separation, and detection. Heretofore, thiol-specific fluorogenic reagents require fairly long reaction times, preventing effective online precolumn derivatization. We demonstrate here that the fluorogenic, thiol-specific reagent, ThioGlo-1, reacts rapidly enough for efficient precolumn derivatization. The second order rate constant for the reaction of GSH and reagent (pH 7.5, room temperature) is 2.1 × 104 M−1s−1. The microchip integrates this precolumn derivatization, continuous flow gated sampling, separation and detection on a single device. We have validated this device for monitoring GSH concentration continuously by studying the kinetics of glutathione reductase (EC 1.8.1.7), an enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to GSH in the presence of β-NADPH (β-Nicotinamide adenine dinucleotide phosphate, reduced form) as a reducing cofactor. During the experiment, GSH being generated in the enzymatic reaction was labeled with ThioGlo-1 as it passed through a mixing channel on the microfluidic chip. Derivatization reaction products were introduced into the analysis channel every 10 s using flow gated injections of 0.1 s. Baseline separation of the internal standard, ThioGlo-1, and the fluorescently labeled GSH was successfully achieved within 4.5 s in a 9 mm separation channel. Relative standard deviations of the peak area, peak height, and full width at half maximum (FWHM) for the internal standard were 2.5%, 2.0%, 1.0%, respectively with migration time reproducibility for the internal standard of less than 0.1% RSD in any experiment. The GSH concentration and mass detection limit were 4.2 nM and ~10−18 mol, respectively. The Michaelis constants (Km) for GSSG and β-NADPH were found to be 40 ± 11 μM and 4.4 ± 0.6 μM, respectively, comparable with those obtained from UV/Vis spectrophotometric measurements. These results show that this system is capable of integrating derivatization, injection, separation and detection for continuous GSH determinations.

Integrated Electroosmotic Perfusion of Tissue with Online Microfluidic Analysis to Track the Metabolism of Cystamine, Pantethine, and Coenzyme A

“…20, 29 solutes from the tissue arrive at the chip after a short delay time. Peak heights increase over a short time and reach a steady state.…”

“…20 It was fabricated using standard lithographic techniques as described earlier 29 with a 30 μm mask width for all channels. The etching depth is 20 μm and the channel length (solid line) is drawn to scale.…”

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

“…To study this and other problems involving extracellular processing of thiols, we built an “all electric” microfluidic system for EO sampling coupled to a fluorogenic reaction, electrophoretic separation, and laser-induced-fluorescence detection [106–108]. As illustrated in Fig.…”

Characterization of a novel particle into liquid sampler for analysis of single fluorescent aerosol particles through capillary electrophoresis

“…Higher applied potentials also offer simultaneous improvement in analysis times. Given these advantages CE methods have been developed to provide functional analysis times on the order of seconds, and in certain exemplary instances in s to ms time frames [29][30][31][32][33][34][35][36][37][38]. CE has been employed previously for aerosol analysis [39][40][41] however to best of our knowledge not in a single particle format.…”