Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells

Abstract

A microfluidic system to determine hydrogen peroxide (H₂O₂) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H₂O₂ in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H₂O₂, were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39–610 amol (R² = 0.9994). The detection limit was 0.55 amol or 9.0 × 10⁻¹⁰ M (S/N =3). The relative standard deviations (RSDs, n = 6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H₂O₂ in single HepG2 cells was found to be 16.09 ± 9.84 amol (n = 15). Separation efficiencies in excess of 17 000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H₂O₂ at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells.